Beta1-integrins shedding in a guinea-pig model of chronic asthma with remodelled airways.

BACKGROUND: A hallmark of airway remodelling in asthma is subepithelial fibrosis, but its relation with airway dysfunction is still controversial. OBJECTIVE: To describe airway functional abnormalities and subepithelial remodelling induced by repetitive antigenic challenges. METHODS: Nine inhaled antigenic challenges were applied every 10 days to guinea-pigs sensitized to ovalbumin (OVA). Antigen-induced airway hyperresponsiveness (AI-AHR) to histamine and its immunohistopathological relationship was evaluated at the first, third and ninth OVA challenges. RESULTS: From the first challenge on, OVA induced acute transient bronchoobstruction followed by development of AI-AHR. A progressive rise in baseline Penh (a bronchoobstruction index) and granulocyte airway infiltration was also observed. After the ninth OVA challenge, the amount of extracellular matrix in the subepithelial region (SER) of bronchi and bronchioles was increased. Immunohistochemistry analysis showed that this SER fibrosis was associated to beta1-integrin subunit overexpression, even in acellular areas. Immunoelectronmicroscopy corroborated the location of beta1-integrin in extracellular matrix, essentially in types l and II collagen fibres. Presence of alpha1- and alpha2-integrin subunits in these areas was also corroborated. AI-AHR was correlated with degree of SER increment, cell infiltration, and beta1-integrin expression. CONCLUSION: Our data suggested that beta1-integrin shedding produced by repetitive allergen challenges in guinea-pigs was associated with collagen deposition in SER of bronchi and bronchioles, along with inflammatory cells infiltration and AI-AHR development.

A/G Gln20Arg (exon 1) and G/A Val156Met (exon 5) polymorphisms of the human orosomucoid 1 gene in Mexico.

The human orosomucoid 1 gene (ORM1) codes an alpha-1-acid glycoprotein that has been classified as an acute-phase reactive protein, and a major drug-binding serum component, as well as an immunomodulatory protein with genetic polymorphisms. Evaluation of ORM variation through isoelectric focusing and immunobloting has revealed a world-wide distribution of the ORM1 F and ORM1 S alleles. We evaluated and examined the genetic characteristics of two Mexican populations that have different anthropological and cultural antecedents, examining two ORM1 genotypes (exon 1 - A/G (Gln20Arg) and exon 5 G/A (Val156Met)) in 145 individuals, using nested polymerase chain reaction, sequencing, and restricted fragment length polymorphism. Mexican Mestizos had higher frequencies of the exon 1 A allele (P = 0.020) and AA genotype (P = 0.018) and lower frequency of the G allele (P = 0.020) when compared to Teenek Amerindians. When we examined exon 5 G/A (Val156Met) polymorphisms, we found significantly higher frequencies of the G allele (P = 0.0007) and the GG genotype (P = 0.0003) in the Mexican Mestizo population. The Teenek population had a significantly higher frequency of the A allele than has been reported for Chinese and African (P < 0.05) populations, and the G/A genotype was more frequently found in this Mexican population than in Chinese, African and European populations (P < 0.05).

A/G Gln20Arg (exon 1) and G/A Val156Met (exon 5) polymorphisms of the human orosomucoid 1 gene in Mexico

The human orosomucoid 1 gene (ORM1) codes an alpha-1-acid glycoprotein that has been classified as an acute-phase reactive protein, and a major drug-binding serum component, as well as an immunomodulatory protein with genetic polymorphisms. Evaluation of ORM variation through isoelectric focusing and immunobloting has revealed a world-wide distribution of the ORM1 F and ORM1 S alleles. We evaluated and examined the genetic characteristicsof two Mexican populations that have different anthropological and cultural antecedents, examining two ORM1 genotypes (exon 1 - A/G (Gln20Arg) and exon 5 G/A (Val156Met)) in 145 individuals, using nested polymerase chain reaction, sequencing, and restrited fragment length polymorphism. Mexican Mestizos had higher frequencies of the exon 1 A allele (P = 0.020) and AA genotype(P = 0.018) and lower frequency of the G allele (P = 0.020) when compared to Teenek Amerindians. When we examined exon 5 G/A (Val156Met) polymorphisms, we found significantly higher frequencies of the G allele (P = 0.0007) and the GG genotype (P = 0.0003) in the Mexican Mestizo population. The Teenek population had a significantly higher frequency of the A allele than has been reported for Chinese and African (P < 0.05) populations, and the G/A genotype was more frequently found in this Mexican population than in Chinese, African and European populations (P < 0.05).

Alpha-1-acid glycoprotein, its local production and immunopathological participation in experimental pulmonary tuberculosis.

Alpha-1-acid glycoprotein (AGP) is one of the major acute-phase proteins (APPs). Hepatic production and serum concentrations increase in response to systemic injury, inflammation, or infection. We reported previously that expression of the AGP gene is induced in the liver during experimental pulmonary tuberculosis. Since AGP may also be produced at the infection site and has some immunomodulatory properties, we used a model of progressive pulmonary tuberculosis in Balb/c mice to study the kinetics of AGP production in the lung and its influence on immunopathology. We found that AGP was produced in the lung during experimental tuberculosis. Alveolar macrophages and type II pneumocytes were the most important cellular sources during early infection (days 1-14). From day 21 postinfection, during the progressive phase of the infection, foamy macrophages located in pneumonic areas were the most important source of AGP and 10-fold higher concentrations were found on day 60. In a second part of the study, AGP was inactivated during the progressive phase by the administration of specific blocking antibodies. In comparison with control infected animals, tuberculous mice treated with blocking AGP antibodies showed higher expression of interferon gamma (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), and inducible nitric oxide synthase (iNOS) in association with significantly reduced bacillary loads and tissue damage. Thus, AGP is produced in the lung during experimental pulmonary tuberculosis and it has immunomodulatory activities, suppressing cell-mediated immunity and facilitating growth of bacilli and disease progression.

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.

Interference by rheumatoid factor activity in the detection of antiavian antibodies in pigeon breeders disease.

The assessment of antiavian antibodies is relevant for the study of pigeon breeder's disease; nevertheless, different factors may hamper their accurate detection. The objective of this study was to determine whether an endogenous interfering effect in pigeon breeder's disease might explain the simultaneous presence of IgM, IgG, and IgA antiavian antibodies in high titers as assessed by ELISA. Fifty-nine patients with pigeon breeder's disease, 80 healthy controls, and 47 asymptomatic breeders were studied. To assess possible interfering effects by endogenous immunoglobulins, serum IgG was separated through protein A-Sepharose CL-4B chromatography. Antiavian antibodies were measured in whole and separated samples by ELISA. Since a decline of IgM antiavian antibodies following IgG removal was consistent with a false-positive effect, the causes were studied. High values of IgM, IgG, and IgA antiavian antibodies were found in 47.4% [corrected] of patients with pigeon breeder's disease. An IgM rheumatoid factor activity against IgG was found through ELISA in sera with false-positive IgM antiavian antibodies. Rheumatoid factor binding was confirmed by Western blot. Experimental addition of purified rheumatoid factor to sera with IgG antiavian antibodies replicated the interfering effect. A control group of rheumatoid arthritis with high rheumatoid factor values did not show positive antiavian antibodies tests. No IgG with anti-IgM or anti-IgA activity was found, and the detection of IgA against IgM and IgG was negative. In conclusion, the study of antiavian antibodies might be affected by different immunoassay conditions. An endogenous rheumatoid factor activity produced false-positive IgM results. Other similar interferences warrant a careful evaluation during the serological assessment of pigeon breeder's disease.

Association of polymyositis with rheumatoid arthritis.

The association of polymyositis (PM) and rheumatoid arthritis (RA) is described in a 40-year-old female Mexican patient who was studied for a long period of time. The characteristic changes of PM that preceded the onset of RA for 7 years included proximal symmetrical muscle weakness, increased creatine kinase activity, and distinctive electromyography and muscle biopsy results. The occurrence of RA during the final 4 years of the 11-year period was characterized by long-lasting deforming and symmetric polyarthritis, high positive rheumatoid factor, subcutaneous nodules, and erosive joint changes. Through observation, myopathic changes other than those from PM were excluded. Joint changes other than from RA were also ruled out. A review of the literature revealed few specific reports of the coexistence of both diseases.

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.

Evaluation of disease activity by laboratory tests in juvenile rheumatoid arthritis.

We studied 29 juvenile rheumatoid arthritis patients with polyarticular onset in order to detect the presence of disease activity by laboratory tests. Laboratory studies included hemoglobin, hematocrit, erythrocyte sedimentation rate (ESR), white blood cell (WBC) and platelet counts. We also determined the levels of IgG, IgA, IgM, C3, C4 and C reactive protein, as well as the presence of rheumatoid factor and antinuclear antibodies. There were 13 patients in the group with polyarticular disease activity, and 16 in the group with asymptomatic juvenile rheumatoid arthritis. Low hematocrit and hemoglobin values, abnormal ESR and elevated WBC and platelet counts occurred in polyarticular juvenile rheumatoid arthritis patients with active disease. Increased levels of IgA, C3 and C4 were also found in the exacerbation stage, as compared to the asymptomatic control group. No differences were found in rheumatoid factor and antinuclear antibodies between the active and inactive juvenile arthritis patients. Our data agreed with previous studies in that a single laboratory test cannot necessarily confirm juvenile arthritis activity, and they suggest that two or more abnormal parameters would be useful in assessing the flare-up of the disease.

Pulmonary function abnormalities in patients with primary Sjögren's syndrome.

Thirty-two patients with primary Sjögren's syndrome (SS) were studied in order to determine the frequency of pulmonary disease using respiratory function tests. Eleven of the 32 patients revealed positive symptoms compatible with lung disease, including dyspnea, dry or productive cough and pleuritic pain. Seven of these 11 patients also showed a positive physical examination, and 5 showed abnormal chest roentgenograms. Restrictive lung disease was detected in 28.1% of the patients, obstructive disease in 21.8%, and 25% showed a combination of these features (mixed disease). Only 8 patients had completely normal function. There was no significant relationship between respiratory changes and age, smoking habits or glandular and extraglandular manifestations. Positive antinuclear antibodies (p < 0.03) detected by the indirect immunofluorescence test, and anti-SS-A(Ro) and anti-SS-B(La) antibodies (p < 0.009) detected by Ouchterlony occurred more frequently in those patients with primary SS and restrictive pulmonary disease. The patients with anti-SS-A(Ro) and anti-SS-B(La) antibodies also showed significantly lower values for total lung capacity (TLC) (p < 0.0001) and forced vital capacity (FVC) (p < 0.0005) than patients without these serum abnormalities. The study of primary SS may help to elucidate the prevalence of lung abnormalities and their relationship with serum autoantibodies.

Rheumatoid arthritis associated with ankylosing spondylitis defined by scintigraphic and CT abnormalities.

Rheumatoid arthritis and ankylosing spondylitis were detected in the same patient after a long period of observation of the disease. X-ray studies demonstrated the characteristic rheumatoid arthritis changes in peripheral joints. By contrast, few X-ray changes of ankylosing spondylitis were detected, during follow-up. Diagnostic approach through scintigraphic studies disclosed a symmetric uptake of the radionuclide in sacroiliac joints, and computed tomography revealed bilateral ankylosis. The combination of these tests was useful to define the presence of axial disease. This patient was both HLA B27 and DR4 positive. Rheumatoid arthritis occurred before ankylosing spondylitis, that interestingly was defined as a late onset disease.

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.

Rheumatoid factor activity in serum and bronchoalveolar lavage fluid from patients with acute hypersensitivity pneumonitis.

The presence of rheumatoid factor was detected in both serum and bronchoalveolar lavage fluid (BALF) from patients with acute pigeon breeder's disease. IgM rheumatoid factor was positive in 14 of 20 serum samples and 6 of 20 BALF samples by ELISA. In contrast, negative results were found in 20 healthy subjects with a history of avian antigen exposure, as well as in the serum and BALF from 10 normal subjects. The simultaneous presence of rheumatoid factor in serum and BALF occurred in five patients, and four of them revealed high levels of rheumatoid factor in BALF in comparison with serum determinations. These abnormalities may play an important role during the acute inflammatory reaction in hypersensitivity pneumonitis.

[Thrombocytopenic purpura and hemolytic anemia in children with generalized lupus erythematosus]. / Púrpura trombocitopénica y anemia hemolítica en niños con lupus eritematoso generalizado.

We studied 30 children with systemic lupus erythematosus in order to detect the clinical, pathological and serological findings associated with the development of thrombocytopenic purpura and hemolytic anemia. A group of 13 of the 30 children revealed hematological manifestations as the most prominent changes of SLE. Thrombocytopenic purpura and hemolytic anemia were the beginning manifestation of SLE in 11 children. Different causes, for these hematological changes such as hypersplenism, renal microangiopathy or drugs reactions were excluded by history and examination. Interestingly, other immunohematological manifestations including splenomegaly and lymphadenopathy were more frequent in children with thrombocytopenic purpura and hemolytic anemia, than in those patients without these hematological complications. Positive antiplatelet antibodies were found in 4/6 children with thrombocytopenia and 2/5 with hemolytic anemia. A relation of antiplatelet with anticardiolipin antibodies occurred in 4 patients; 3 of them in children with thrombocytopenic purpura and one with hemolytic anemia. Anti-dsDNA and anti-Sm antibodies were positive in almost all patients. Four children shown a transition from anti-dsDNA to anti-Sm antibodies or viceversa and all of them revealed a significant variation in the titer of these antibodies by ELISA, in relation with disease activity. The presence of hematological manifestations associated with anti-platelet and anti-cardiolipin antibodies in children with SLE support that different mechanisms triggers autoimmunity in childhood.