Anish Wadhwa

Interaction between Pyrin and the Apoptotic Speck Protein (ASC) Modulates ASC-induced Apoptosis

Patients with familial Mediterranean fever suffer sporadic inflammatory attacks characterized by fever and intense pain (in joints, abdomen, or chest). Pyrin, the product of theMEFV locus, is a cytosolic protein whose function is unknown. Using pyrin as a “bait” to probe a yeast two-hybrid library made from neutrophil cDNA, we isolatedapoptotic speck protein containing a caspase recruitment domain (CARD) (ASC), a proapoptotic protein that induces the formation of large cytosolic “specks” in transfected cells. We found that when HeLa cells are transfected with ASC, specks are formed. After co-transfection of cells with ASC plus wild type pyrin, an increase in speck-positive cells is found, and speck-positive cells show increased survival. Immunofluorescence studies show that pyrin co-localizes with ASC in specks. Speck localization requires exon 1 of pyrin, but exon 1 alone of pyrin does not result in an increase in the number of specks. Exon 1 of pyrin and exon 1 of ASC show 42% sequence similarity and resemble death domain-related structures in modeling studies. These findings link pyrin to apoptosis pathways and suggest that the modulation of cell survival may be a component of the pathophysiology of familial Mediterranean fever.

Episodic evolution of pyrin in primates: human mutations recapitulate ancestral amino acid states

Familial Mediterranean fever (FMF; MIM 249100) is an autosomal recessive disease characterized by recurrent attacks of fever with synovial, pleural or peritoneal inflammation. The disease is caused by mutations in the gene encoding the pyrin protein. Human population studies have revealed extremely high allele frequencies for several different pyrin mutations, leading to the conclusion that the mutant alleles confer a selective advantage. Here we examine the ret finger protein (rfp) domain (which contains most of the disease-causing mutations) of pyrin during primate evolution. Amino acids that cause human disease are often present as wild type in other species. This is true at positions 653 (a novel mutation), 680, 681, 726, 744 and 761. For several of these human mutations, the mutant represents the reappearance of an ancestral amino acid state. Examination of lineage-specific dN/dS ratios revealed a pattern consistent with the signature of episodic positive selection. Our data, together with previous human population studies, indicate that selective pressures may have caused functional evolution of pyrin in humans and other primates.

Mesenchymal Stromal Cells in Bronchoalveolar Lavage as Predictors of Bronchiolitis Obliterans Syndrome

RATIONALE Bronchoalveolar lavage fluid (BAL) from human lung allografts demonstrates the presence of a multipotent mesenchymal stromal cell population. However, the clinical relevance of this novel cellular component of BAL and its association with bronchiolitis obliterans syndrome (BOS), a disease marked by progressive airflow limitation secondary to fibrotic obliteration of the small airways, remains to be determined. OBJECTIVES In this study we investigate the association of number of mesenchymal stromal cells in BAL with development of BOS in human lung transplant recipients. METHODS Mesenchymal colony-forming units (CFUs) were quantitated in a cohort of 405 BAL samples obtained from 162 lung transplant recipients. Poisson generalized estimating equations were used to determine the predictors of BAL mesenchymal CFU count. MEASUREMENTS AND MAIN RESULTS Higher CFU counts were noted early post-transplantation; time from transplant to BAL of greater than 3 months predicted 0.4-fold lower CFU counts (P = 0.0001). BOS diagnosis less than or equal to 365 days before BAL was associated with a 2.11-fold higher CFU count (P = 0.02). There were 2.62- and 2.70-fold higher CFU counts noted in the presence of histologic diagnosis of bronchiolitis obliterans (P = 0.05) and organizing pneumonia (0.0003), respectively. In BAL samples obtained from BOS-free patients greater than 6 months post-transplantation (n = 173), higher mesenchymal CFU counts (≥10) significantly predicted BOS onset in both univariate (hazard ratio, 5.61; 95% CI, 3.03-10.38; P < 0.0001) and multivariate (hazard ratio, 5.02; 95% CI, 2.40-10.51; P < 0.0001) Cox regression analysis. CONCLUSIONS Measurement of mesenchymal CFUs in the BAL provides predictive information regarding future BOS onset.

Prostaglandin E2 As an Inhibitory Modulator of Fibrogenesis in Human Lung Allografts

RATIONALE Donor mesenchymal stromal/stem cell (MSC) expansion and fibrotic differentiation is associated with development of bronchiolitis obliterans syndrome (BOS) in human lung allografts. However, the regulators of fibrotic differentiation of these resident mesenchymal cells are not well understood. OBJECTIVES This study examines the role of endogenous and exogenous prostaglandin (PG)E2 as a modulator of fibrotic differentiation of human lung allograft-derived MSCs. METHODS Effect of PGE2 on proliferation, collagen secretion, and α-smooth muscle actin (α-SMA) expression was assessed in lung-resident MSCs (LR-MSCs) derived from patients with and without BOS. The response pathway involved was elucidated by use of specific agonists and antagonists. MEASUREMENT AND MAIN RESULTS PGE2 treatment of LR-MSCs derived from normal lung allografts significantly inhibited their proliferation, collagen secretion, and α-SMA expression. On the basis of pharmacologic and small-interfering RNA approaches, a PGE2/E prostanoid (EP)2/adenylate cyclase pathway was implicated in these suppressive effects. Stimulation of endogenous PGE2 secretion by IL-1β was associated with amelioration of their myofibroblast differentiation in vitro, whereas its inhibition by indomethacin augmented α-SMA expression. LR-MSCs from patients with BOS secreted significantly less PGE2 than non-BOS LR-MSCs. Furthermore, BOS LR-MSCs were found to be defective in their ability to induce cyclooxygenase-2, and therefore unable to up-regulate PGE2 synthesis in response to IL-1β. BOS LR-MSCs also demonstrated resistance to the inhibitory actions of PGE2 in association with a reduction in the EP2/EP1 ratio. CONCLUSIONS These data identify the PGE2 axis as an important autocrine-paracrine brake on fibrotic differentiation of LR-MSCs, a failure of which is associated with BOS.

The Keys to Making a Confident Diagnosis of IPF

Diffuse parenchymal lung diseases (DPLDs) are characterized by injury primarily to the interstitium of the lung, but may involve alveolar spaces, airways, and vessels (American Thoracic Society/European Respiratory Society. Am J Respir Crit Care Med 165(2):277–304, 2002). Many DPLDs are idiopathic (referred to as idiopathic interstitial pneumonias, or IIPs), but DPLD can develop secondary to other factors, including connective tissue disease (CTD), environmental exposures, and drugs/toxins (American Thoracic Society/European Respiratory Society. Am J Respir Crit Care Med 165(2):277–304, 2002). The IIPs include idiopathic pulmonary fibrosis (IPF), nonspecific interstitial pneumonia (NSIP), cryptogenic organizing pneumonia (COP), respiratory bronchiolitis interstitial lung disease (RB-ILD), acute interstitial pneumonia (AIP), lymphocytic interstitial pneumonia (LIP), and desquamative interstitial pneumonia (DIP) (American Thoracic Society/European Respiratory Society. Am J Respir Crit Care Med 165(2):277–304, 2002). There is significant overlap in the clinical features of the IIPs, including chronic dyspnea, interstitial changes on imaging studies, reduction in lung volumes, and impairment in diffusion capacity (DLCO) (American Thoracic Society/European Respiratory Society. Am J Respir Crit Care Med 165(2):277–304, 2002). Distinct radiographic and histopathological features can distinguish between the clinical entities, and establishing an accurate diagnosis is critical to determining treatment and understanding prognosis (Flaherty et al. Eur Respir J 19(2):275–83, 2002; Bjoraker et al. Am J Respir Crit Care Med 157(1):199–203, 1998).

Mechanisms of Fibrogenesis in Post-transplant Bronchiolitis Obliterans

Bronchiolitis obliterans (BO) post-lung transplant is a fibroproliferative disease of the small airways. Among all fibrotic diseases, BO is unique in that we have the opportunity to follow the evolution of airway remodeling and fibrosis via serial lung samplings. Although there is a definite role of immune and nonimmune injury in the pathogenesis of this disease, the physiological impairment in BO is related to fibrogenesis. Hence, understanding the pathogenesis of fibroproliferation in BO is critical for identifying targets for future therapeutic interventions and the timing of such therapy. This review updates the understanding of the cellular and molecular participants in fibrotic remodeling of the lung allograft.