Effects of Sildenafil and Tadalafil on Edema and Reactive Oxygen Species Production in an Experimental Model of Lung Ischemia-Reperfusion Injury.

BACKGROUND: Lung ischemia-reperfusion injury is characterized by formation of reactive oxygen species and cellular swelling leading to pulmonary edema and primary graft dysfunction. Phosphodiesterase 5 inhibitors could ameliorate lung ischemia-reperfusion injury by interfering in many molecular pathways. The aim of this work was to evaluate and compare the effects of sildenafil and tadalafil on edema and reactive oxygen species formation in an ex vivo nonhuman animal model of lung ischemia-reperfusion injury. METHODS: Thirty-two Wistar rats were distributed, treated, perfused and the cardiopulmonary blocks were managed as follows: control group: immediate excision and reperfusion without pretreatment; ischemia reperfusion group: treatment with dimethylsulfoxide 0.9% and excision 1 hour later; sildenafil group: treatment with sildenafil (0.7 mg/kg) and excision 1 hour later; and tadalafil group: treatment with tadalafil (0.15 mg/kg) and excision 2 hours later. All cardiopulmonary blocks except control group were preserved for 8 hours and then reperfused. Pulmonary arterial pressure, pulmonary venous pressure, and capillary filtration coefficient were measured. Reactive oxygen species were measured. RESULTS: Edema was similar between control and sildenafil groups, but significantly greater in the ischemia-reperfusion (P ≤ .04) and tadalafil (P ≤ .003) groups compared with the sildenafil group. The malondialdehyde levels were significantly lower in the sildenafil (P ≤ .001) and tadalafil (P ≤ .001) groups than the ischemia-reperfusion group. CONCLUSIONS: Administration of sildenafil, but not tadalafil, decreased edema in lung ischemia-reperfusion injury. Both drugs decreased reactive oxygen species formation in a lung ischemia-reperfusion injury model.

Achalasia--An Autoimmune Inflammatory Disease: A Cross-Sectional Study.

Idiopathic achalasia is a disease of unknown etiology. The loss of myenteric plexus associated with inflammatory infiltrates and autoantibodies support the hypothesis of an autoimmune mechanism. Thirty-two patients diagnosed by high-resolution manometry with achalasia were included. Twenty-six specimens from lower esophageal sphincter muscle were compared with 5 esophagectomy biopsies (control). Immunohistochemical (biopsies) and flow cytometry (peripheral blood) analyses were performed. Circulating anti-myenteric autoantibodies were evaluated by indirect immunofluorescence. Herpes simplex virus-1 (HSV-1) infection was determined by in situ hybridization, RT-PCR, and immunohistochemistry. Histopathological analysis showed capillaritis (51%), plexitis (23%), nerve hypertrophy (16%), venulitis (7%), and fibrosis (3%). Achalasia tissue exhibited an increase in the expression of proteins involved in extracellular matrix turnover, apoptosis, proinflammatory and profibrogenic cytokines, and Tregs and Bregs versus controls (P < 0.001). Circulating Th22/Th17/Th2/Th1 percentage showed a significant increase versus healthy donors (P < 0.01). Type III achalasia patients exhibited the highest inflammatory response versus types I and II. Prevalence of both anti-myenteric antibodies and HSV-1 infection in achalasia patients was 100% versus 0% in controls. Our results suggest that achalasia is a disease with an important local and systemic inflammatory autoimmune component, associated with the presence of specific anti-myenteric autoantibodies, as well as HSV-1 infection.

Kinetics and cellular sources of cathelicidin during the course of experimental latent tuberculous infection and progressive pulmonary tuberculosis.

In spite of advances in immunology on mycobacterial infection, there are few studies on the role of anti-microbial peptides in tuberculosis. The cathelin-related anti-microbial peptide (CRAMP) is the only cathelicidin isolated from mice. In this work we investigated the cellular sources and the production kinetics of this molecule during experimental tuberculosis, using two well-characterized models of latent or chronic infection and progressive disease. The lung of non-infected control mice expressed CRAMP at very low levels. In both models of experimental tuberculosis the main cells immunolabelled for CRAMP were bronchial epithelial cells, macrophages and pneumocytes types II and I. After intratracheal infection with a high bacilli dose (H37Rv strain) in Balb/c mice to produce progressive disease, a high CRAMP gene expression was induced showing three peaks: very early after 1 day of infection, at day 21 when the peak of protective immunity in this model is raised, and at day 28 when the progressive phase starts and the immunoelectronmicroscopy study showed intense immunolabelling in the cell wall and cytoplasm of intracellular bacilli, as well as in cytoplasmic vacuoles. Interestingly, at day 60 post-infection, when advanced progressive disease is well established, characterized by high bacillary loads and extensive tissue damage, CRAMP gene expression decreased but strong CRAMP immunostaining was detected in vacuolated macrophages filled with bacilli. Thus, cathelicidin is highly produced during experimental pulmonary tuberculosis from diverse cellular sources and could have significant participation in its pathogenesis.

Mycobacterium bovis with different genotypes and from different hosts induce dissimilar immunopathological lesions in a mouse model of tuberculosis.

With the hypothesis that genetic variability of Mycobacterium bovis could influence virulence and immunopathology, five M. bovis strains were selected from an epidemiological study in Argentina on the basis of their prevalence in cattle and occurrence in other species. We then determined the virulence and the immunopathology evoked by these strains in a well-characterized mouse model of progressive pulmonary tuberculosis. The reference strain AN5 was used as a control. BALB/c mice infected with this M. bovis reference strain showed 50% survival after 4 months of infection, with moderate bacillary counts in the lung. Two weeks after inoculation, it induced a strong inflammatory response with numerous granulomas and progressive pneumonia. In contrast, strain 04-303, isolated from a wild boar, was the most lethal and its most striking feature was sudden pneumonia with extensive necrosis. Strain 04-302, also isolated from wild boar but with a different spoligotype, induced similar pathology but to a lesser extent. In contrast, strains 534, V2 (both from cattle) and 02-2B (from human) were less virulent, permitting higher survival after 4 months of infection and limited tissue damage. Strain AN5 and the cattle and human isolates induced rapid, high and stable expression of interferon (IFN)-gamma and inducible nitric oxide synthase (iNOS). In contrast, the more virulent strains induced lower expression of IFN-gamma, tumour necrosis factor-alpha and iNOS. Interestingly, these more virulent strains induced very low expression of murine beta defensin 4 (mBD-4); whereas, the control strain AN5 induced progressive expression of this anti-microbial peptide, peaking at day 120. The less virulent strains induced high mBD-4 expression during early infection. Thus, as reported with clinical isolates of M. tuberculosis, M. bovis also showed variable virulence. This variability can be attributed to the induction of a different pattern of immune response.

A combination of a transforming growth factor-beta antagonist and an inhibitor of cyclooxygenase is an effective treatment for murine pulmonary tuberculosis.

Transforming growth factor-beta (TGF-beta) and prostaglandins (PG) regulate the cell-mediated immune response, so it has been proposed that they affect the progression of pulmonary tuberculosis. Here we report that the administration of soluble betaglycan, a potent TGF-beta antagonist, and niflumic acid, a PG synthesis inhibitor, during the chronic phase of experimental murine tuberculosis enhanced Th1 and decreased Th2 cytokines, increased the expression of iNOS and reduced pulmonary inflammation, fibrosis and bacillary load. This immunotherapeutic approach resulted in significant control of the disease comparable to that achieved by anti-microbial treatment alone. Importantly, the combination of immunotherapy and anti-microbials resulted in an accelerated clearance of bacilli from the lung. These results confirm that TGF-beta and PG have a central pathophysiological role in the progression of pulmonary tuberculosis in the mouse and suggest that the addition of immunotherapy to conventional anti-microbial drugs might result in improved treatment of the disease.

Anti-avian antibodies and rheumatoid factor in pigeon hypersensitivity pneumonitis.

BACKGROUND: Although several immunological abnormalities may be present in pigeon hypersensitivity pneumonitis (HP), few specific hallmarks have been described. OBJECTIVE: To determine whether the presence of rheumatoid factor (RF) could be useful to discriminate pigeon HP from asymptomatic breeders (AB) and other interstitial lung diseases. METHODS: Fifty-three patients with pigeon HP, 47 AB, 31 idiopathic pulmonary fibrosis (IPF) patients and a rheumatoid arthritis (RA) group were studied. IgM RF was determined through enzyme-linked immunosorbent assay (ELISA) and western blot using human IgG and IgG Fc fragment as antigens. IgG and IgA anti-avian antibodies (AA) against pigeon serum antigen were also measured. The use of F(ab')2 fraction of peroxidase-labelled anti-human immunoglobulins prevented endogenous interferences. Possible cross-binding of RF with avian antigens and the reactivity against human IgG by AA were studied. RESULTS: RF tests were frequently positive in HP (52.8%) in comparison to AB (4.2%) and IPF (12.9%; P = 2.6 x 10-10 and 4.1 x 10-5). Therefore, the presence of RF in pigeon HP showed a sensitivity of 52% and was highly specific considering the results of AB and IPF (95 and 87%, respectively). The RA group revealed positive RF but negative AA tests. RF activity was confirmed through western blot using purified IgG Fc fragment. Overlapping levels of IgG and IgA AA were found in HP and AB. The frequency of AA was low in IPF. The cross-reaction of RF with avian antigens was excluded, and no reactivity against human IgG by AA was detected. Other endogenous interferences were ruled out. CONCLUSION: No single immunological test may definitively distinguish pigeon HP from AB and other interstitial lung disorders; however, positive RF, together with high AA levels, seems to be useful in differentiating the diagnosis.

IgM antiavian antibodies in sera from patients with pigeon breeder's disease.

The authors' objective was to study the presence of IgM antiavian antibodies in sera from patients with pigeon breeder's disease. We studied 93 patients with interstitial lung disease admitted for the assessment of pigeon breeder's disease. Eighty sera from healthy donors with no history of bird contact and 47 asymptomatic pigeon breeders were included as controls. The presence of IgM, IgG, and IgA antiavian antibodies was detected by ELISA and Western blot using avian-pooled serum antigen. Fifty-three patients were classified as having definite pigeon breeder's disease, whereas 40 did not fulfill these diagnostic criteria. The levels of IgM antiavian-antibodies in pigeon breeder's disease by ELISA exceeded both the values of healthy subjects with no history of avian contact (P = 2.5 x 10(-8)) and the results of asymptomatic breeders (P = 0. 03). Positive IgA antiavian antibodies were the most frequent abnormalities in pigeon breeder's disease showing values over the reference levels of control groups that reach significant statistical differences. Both precipitin-positive and -negative samples demonstrated IgM reactivity. IgM antiavian antibodies were confirmed by Western blot. A relationship of IgM positive tests with a recent history of avian antigen exposure and acute disease was found. Additionally, the positive IgM group included patients having subacute and chronic lung disease. Antiavian antibodies have previously been considered of minor significance in hypersensitivity pneumonitis; nevertheless, recent studies support their use in clinical diagnosis. Although no specific laboratory tests can confirm the diagnosis in pigeon breeder's disease, IgM antiavian antibodies may be useful for detecting recent antigen exposure and the acute stage of the disease.

Inflammatory cytokine production by immunological and foreign body multinucleated giant cells.

Multinucleated giant cells (MGC) are a common feature of granulomas. The mechanism of their formation has been studied extensively, but their function has not been completely characterized. A new method for the in vivo production of MGC was developed involving subcutaneous injection of microscopic nitrocellulose particles with adsorbed mycobacterial antigens into the footpads of sensitized BALB/c mice (immune [I]-MGC), or by nitrocellulose administration to non-sensitized mice (foreign body [FB]-MGC). The development of granulomas with a highly enriched MGC population was observed 2 weeks after the nitrocellulose injection. MGC were larger with a greater number of nuclei in I-MGC than in FB-MGC. From days 7-28 after nitrocellulose administration, the production of interleukin-1alpha (IL-1alpha) and tumour necrosis factor-alpha (TNF-alpha) was demonstrated in both MGC types by in situ reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. After 2 months, the MGC had ceased production of IL-1alpha and TNF-alpha, but the expression of transforming growth factor-beta (TGF-beta) was very high, occurring together with extensive fibrosis. These results suggest that MGC are an active source of inflammatory cytokines, which can contribute to the initiation, maintenance and down-regulation of granulomatous inflammation induced by immunological and inert substances.

Anticardiolipin antibodies in serum and cerebrospinal fluid from patients with systemic lupus erythematosus.

Anticardiolipin antibodies were studied in serum and cerebrospinal fluid from 32 consecutive patients with systemic lupus erythematosus, admitted for the assessment of neuropsychiatric disease. Ten of the 16 patients with active neuropsychiatric complaints showed positive anticardiolipin antibodies in cerebrospinal fluid, including eight with the simultaneous presence of antibodies in their sera. By contrast, only 2 of the 16 patients with headaches, lacking further data of neurological disease, revealed anticardiolipin antibodies in their cerebrospinal fluid. The assessment of Q-albumin index showed abnormal values in a subset of patients with active neuropsychiatric changes who showed positive cerebrospinal anticardiolipin antibodies, suggesting that an impairment of the blood brain barrier function may lead to a leakage of intrathecal antiphospholipid antibodies from systemic circulation. Additionally, few patients revealed normal Q-albumin values with high IgG-cerebrospinal fluid index suggesting increased intrathecal synthesis of autoantibodies. The study of anticardiolipin antibodies in cerebrospinal fluid was useful to detect active neuropsychiatric disease in systemic lupus erythematosus.

Anti-dsDNA and Sm autoantibodies in systemic lupus erythematosus.

During the present study the coincidence of anti-dsDNA and Sm antibodies was detected in 16 percent of 51 consecutive SLE patients. These antibodies were detected by the standard indirect immunofluorescence and Ouchterlony tests. All patients with anti-dsDNA and Sm antibodies showed disease activity, including renal, CNS and pulmonary disease. We excluded a cross reactivity of these antibodies by ELISA, using competitive experiments with dsDNA and Sm antigens. The results support the presence of multiple autoantibody production during SLE activity, and suggest that different mechanisms may underlie the induction and regulation of both autoantibodies.