Analysis of expression of MHC class I molecules and TAP genes in malignant human cell lines.

TAP proteins (transporters associated with antigen processing) take part in the transport of oligopeptides created in proteasomes from cytoplasm into endoplasmic reticulum. In the endoplasmic reticulum those oligopeptides are bound to MHC class I molecules and transported to the cell surface. TAP proteins consist of two subunits: TAP1 and TAP2. It has been previously shown that TAP protein expression can be decreased in malignant cells, followed by reduced protein expression or complete lack of MHC class I antigens on the cell surface. The aim of the study was to characterize of MHC class I protein expression and TAP mRNA synthesis in twenty human malignant tumor cell lines. MHC class I protein expression was examined by immunohistochemistry and flow cytometry. Expression of TAP genes was studied using RT-PCR and real-time PCR. All tested cell lines expressed MHC class I molecules. Flow cytometry showed different expression of MHC class I protein in tested cell lines. Molecular analysis revealed the presence of TAP1 and TAP2 gene transcripts in all cell lines examined. Quantitative real time PCR analysis showed differences of gene expression among cell lines tested

Immunoexpression of IgA receptors (CD89, CD71) in dermatitis herpetiformis

Introduction. The role of IgA receptors in dermatitis herpetiformis (DH) pathogenesis is still unknown. CD89 and CD71 may be associated with immune response during DH development. The purpose of this study was to perform semiquantitative analysis of simultaneous immunoexpression of CD89 and CD71 in DH and IgA/neutrophil-mediated non-DH dermatoses (IgAN) in relation to specific IgA autoantibodies/antibodies (tissue and epidermal transglutaminases, nonapeptides of gliadin — eTG/tTG/npG) as well neutrophil activation via the release of neutrophil elastase (NE). Material and methods. In total, 48 patients were studied. The study was conducted on skin lesions and sera obtained from DH and IgAN patients. DH and IgAN served as mutually positive control groups. We used immunohistochemical technique with semiquantitative digital morphometry and ELISA to measure serum levels of anti-eTG/tTG/npG IgA. Results. CD89 showed a significantly higher expression in DH than in IgAN. CD71 was overexpressed in DH and IgAN. CD89 immunoexpression correlated negatively with CD71 in IgAN. A positive correlation was revealed between CD89 immunoexpression and anti-npG IgA in DH. No statistically significant correlations were found in DH between the CD89/CD71 and NE immunoexpression, between CD71 immunoexpression and anti-tTG/eTG/npG IgA, or between CD89 immunoexpression and anti-eTG/tTG IgA serum levels. Conclusions. CD89 is probably a key IgA Fc receptor in DH development, where it is associated with immune response to gluten. CD71 may be linked with inflammation in DH and IgAN. We suggest that interaction between CD89 and CD71 can modulate the inflammation in IgAN

Polysaccharides and mucin 5AC (MUC5AC) expression in gallbladder mucosa of young patients with gallstones as evaluated by spatial visualization and quantification

The study aimed at examination of tissue expression of polysaccharides and secretory mucin 5AC (MUC5AC)in young patients (up to 25 years of age) with a symptomatic gallstones. For comparison, patients most frequently subjectedto cholecystectomy were studied, i.e. patients of approximately 50 years of age with the same diagnosis. In quantitativestudies on tissue expression of both mucus components, the modern technique of spatial visualization was applied for thefirst time. Application of the technique permitted to demonstrate significant positive relationships between expression ofglycoproteins (immunocytochemical ABC technique for detection of MUC5AC) and expression of sugar components inmucus (PAS technique) and to confirm suitability of the technique for quantitative appraisal of both histochemical andimmunocytochemical reactions. An even higher expression of polysaccharides in the entire mucosa and of MUC5AC wasdetected in gallbladder epithelium of 50-year-old patients, as compared to young patients with symptomatic gallstones. Inthe young patients, expression of polysaccharides correlated with inflammatory activity (grading), width of gallbladder walland PLT level in peripheral blood. A significantly higher expression of polysaccharides in gallbladder epithelium wasdemonstrated in young patients admitted in the emergency mode to the hospital. These correlations in young patients maysuggest a role of both mucus components in pathogenesis of cholelithiasis in this age group. A quantitative appraisal ofmucus component expression in the two parts of gallbladder mucosa (epithelium vs. entire mucosa) using spatial visualizationtechnique permitted to more accurately compare production of glycoproteins and of polysaccharides in patients withcholelithiasis and to demonstrate additional correlations of a potential clinical significance

Analysis of expression of MHC class I molecules and TAP genes in malignant human cell lines.

TAP proteins (transporters associated with antigen processing) take part in the transport of oligopeptides created in proteasomes from cytoplasm into endoplasmic reticulum. In the endoplasmic reticulum those oligopeptides are bound to MHC class I molecules and transported to the cell surface. TAP proteins consist of two subunits: TAP1 and TAP2. It has been previously shown that TAP protein expression can be decreased in malignant cells, followed by reduced protein expression or complete lack of MHC class I antigens on the cell surface. The aim of the study was to characterize of MHC class I protein expression and TAP mRNA synthesis in twenty human malignant tumor cell lines. MHC class I protein expression was examined by immunohistochemistry and flow cytometry. Expression of TAP genes was studied using RT-PCR and real-time PCR. All tested cell lines expressed MHC class I molecules. Flow cytometry showed different expression of MHC class I protein in tested cell lines. Molecular analysis revealed the presence of TAP1 and TAP2 gene transcripts in all cell lines examined. Quantitative real time PCR analysis showed differences of gene expression among cell lines tested

Adenosine A1 Receptors Promote Vasa Vasorum Endothelial Cell Barrier Integrity via Gi_{i} and Akt-Dependent Actin Cytoskeleton Remodeling

Background: In a neonatal model of hypoxic pulmonary hypertension, a dramatic pulmonary artery adventitial thickening, accumulation of inflammatory cells in the adventitial compartment, and angiogenic expansion of the vasa vasorum microcirculatory network are observed. These pathophysiological responses suggest that rapidly proliferating vasa vasorum endothelial cells (VVEC) may exhibit increased permeability for circulating blood cells and macromolecules. However, the molecular mechanisms underlying these observations remain unexplored. Some reports implicated extracellular adenosine in the regulation of vascular permeability under hypoxic and inflammatory conditions. Thus, we aimed to determine the role of adenosine in barrier regulation of VVEC isolated from the pulmonary arteries of normoxic (VVEC-Co) or chronically hypoxic (VVEC-Hyp) neonatal calves. Principal Findings We demonstrate via a transendothelial electrical resistance measurement that exogenous adenosine significantly enhanced the barrier function in VVEC-Co and, to a lesser extent, in VVEC-Hyp. Our data from a quantitative reverse transcription polymerase chain reaction show that both VVEC-Co and VVEC-Hyp express all four adenosine receptors (A1, A2A, A2B, and A3), with the highest expression level of A1 receptors (A1Rs). However, A1R expression was significantly lower in VVEC-Hyp compared to VVEC-Co. By using an A1R-specific agonist/antagonist and siRNA, we demonstrate that A1Rs are mostly responsible for adenosine-induced enhancement in barrier function. Adenosine-induced barrier integrity enhancement was attenuated by pretreatment of VVEC with pertussis toxin and GSK690693 or LY294002, suggesting the involvement of G$_{i}$ proteins and the PI3K-Akt pathway. Moreover, we reveal a critical role of actin cytoskeleton in VVEC barrier regulation by using specific inhibitors of actin and microtubule polymerization. Further, we show that adenosine pretreatment blocked the tumor necrosis factor alpha (TNF-α)-induced permeability in VVEC-Co, validating its anti-inflammatory effects. Conclusions: We demonstrate for the first time that stimulation of A1Rs enhances the barrier function in VVEC by activation of the G$_{i}$/PI3K/Akt pathway and remodeling of actin microfilament

Macrophages sense and kill bacteria through carbon monoxide-dependent inflammasome activation

Microbial clearance by eukaryotes relies on complex and coordinated processes that remain poorly understood. The gasotransmitter carbon monoxide (CO) is generated by the stress-responsive enzyme heme oxygenase-1 (HO-1, encoded by Hmox1), which is highly induced in macrophages in response to bacterial infection. HO-1 deficiency results in inadequate pathogen clearance, exaggerated tissue damage, and increased mortality. Here, we determined that macrophage-generated CO promotes ATP production and release by bacteria, which then activates the Nacht, LRR, and PYD domains-containing protein 3 (NALP3) inflammasome, intensifying bacterial killing. Bacterial killing defects in HO-1-deficient murine macrophages were restored by administration of CO. Moreover, increased CO levels enhanced the bacterial clearance capacity of human macrophages and WT murine macrophages. CO-dependent bacterial clearance required the NALP3 inflammasome, as CO did not increase bacterial killing in macrophages isolated from NALP3-deficient or caspase-1-deficient mice. IL-1β cleavage and secretion were impaired in HO-1-deficient macrophages, and CO-dependent processing of IL-1β required the presence of bacteria-derived ATP. We found that bacteria remained viable to generate and release ATP in response to CO. The ATP then bound to macrophage nucleotide P2 receptors, resulting in activation of the NALP3/IL-1β inflammasome to amplify bacterial phagocytosis by macrophages. Taken together, our results indicate that macrophage-derived CO permits efficient and coordinated regulation of the host innate response to invading microbes.NIH grants: (HL-071797, HL-076167, HL-106227), American Heart Association grants: (10SDG2640091 and NIH R21CA169904-01), Julie Henry Fund, Transplant Center of the BIDMC, FCT grants: (SFRH/BPD/25436/2005, PTDC/BIO/70815/2006, PTDC/BIA-BCM/101311/2008, PTDC/SAU-FCF/100762/2008), the European Community, 6th Framework grant LSH-2005-1.2.5-1 and ERC-2011-AdG, Howard Hughes Medical Institute