Martha Montaño

Surfactant Protein Genetic Marker Alleles Identify a Subgroup of Tuberculosis in a Mexican Population

Pulmonary surfactant and its components are essential for normal lung function and are involved in local host defense. Surfactant protein (SP)-A and SP-D bind to and modulate phagocytosis of Mycobacterium tuberculosis by macrophages. Frequency comparisons of SP marker alleles in tuberculosis patients and healthy control subjects (tuberculin-skin test positive or general population) were performed. Regression analyses of the tuberculosis and the tuberculin-skin test positive groups revealed, on the basis of odds ratios, tuberculosis susceptibility (DA11_C and GATA_3) and protective (AAGG_2) marker alleles. Similarly, between tuberculosis patients and general population control subjects, susceptibility 1A(3), 6A(4), and B1013_A and protective AAGG_1, and AAGG_7 marker alleles were observed. Moreover, interactions were seen between alleles 6A(2) and 1A(3) (P=.0064) and between 1A(3) and B1013_A (P=. 036). The findings indicate a possible involvement of SP alleles in tuberculosis pathogenesis.

Surfactant protein gene A, B, and D marker alleles in chronic obstructive pulmonary disease of a Mexican population

Chronic obstructive pulmonary disease (COPD) is characterized by chronic inflammation. It is most likely the result of complex interactions of environmental and genetic factors. Because pulmonary surfactant components play important roles in normal lung function, innate host defence, and inflammation in the lung, this study investigated the hypothesis that the surfactant protein genes are involved in certain cases of COPD. Genotype analysis of surfactant protein (SP)-A, SP-B, SP-B-linked microsatellite, and SP-D marker alleles was performed in patients with COPD (n=97) and smoker (n=82) or nonsmoker (n=99) controls. Univariate and multiple logistic regression analyses were performed. The regression analysis results between COPD and smokers revealed several COPD susceptibility alleles (AA62_A, B1580_C, D2S388_5), based on an odds ratio (OR >2.5). The predictive ability of this model for developing COPD is good (c=0.926). Allele-allele (B1580_C and D2S388_5) and allele-environment (i.e. smoking) interactions were detected. When smoker controls were compared to nonsmoker controls, marker D2S388 5 appeared to be smoking-independent (p=0.874), whereas marker alleles AA62_A (p=0.045) and B1580_5 (p=0.007) were smoking-dependent. Males were at higher risk (OR=6.05, p=0.001), and smoking (>50 packs x yr(-1)) increased risk (OR=5.38, p=0.007). Males and alleles of loci flanking SP-B were associated with more severe cases (forced expiratory volume in one second/forced vital capacity < or = 40%). The present results indicate that the surfactant protein alleles may be useful in chronic obstructive pulmonary disease by either predicting the disease in a subgroup and/or by identifying disease subgroups that may be used for therapeutic intervention. These observations should now be confirmed in a larger study, designed according to strict epidemiological criteria.

FGF-1 reverts epithelial-mesenchymal transition induced by TGF-β1 through MAPK/ERK kinase pathway

Idiopathic pulmonary fibrosis (IPF) is a progressive and lethal lung disease characterized by the expansion of the fibroblast/myofibroblast population and aberrant remodeling. However, the origin of mesenchymal cells in this disorder is still under debate. Recent evidence indicates that epithelial-mesenchymal transition (EMT) induced primarily by TGF-beta1 plays an important role; however, studies regarding the opposite process, mesenchymal-epithelial transition, are scanty. We have previously shown that fibroblast growth factor-1 (FGF-1) inhibits several profibrogenic effects of TGF-beta1. In this study, we examined the effects of FGF-1 on TGF-beta1-induced EMT. A549 and RLE-6TN (human and rat) alveolar epithelial-like cell lines were stimulated with TGF-beta1 for 72 h, and then, in the presence of TGF-beta1, were cultured with FGF-1 plus heparin for an additional 48 h. After TGF-beta1 treatment, epithelial cells acquired a spindle-like mesenchymal phenotype with a substantial reduction of E-cadherin and cytokeratins and concurrent induction of alpha-smooth muscle actin measured by real-time PCR, Western blotting, and immunocytochemistry. FGF-1 plus heparin reversed these morphological changes and returned the epithelial and mesenchymal markers to control levels. Signaling pathways analyzed by selective pharmacological inhibitors showed that TGF-beta1 induces EMT through Smad pathway, while reversion by FGF-1 occurs through MAPK/ERK kinase pathway, resulting in ERK-1 phosphorylation and Smad2 dephosphorylation. These findings indicate that TGF-beta1-induced EMT is reversed by FGF-1 and suggest therapeutic approaches to target this process in IPF.

Changes in matrix metalloproteinases during the evolution of interstitial renal fibrosis in a rat experimental model.

The aim of the present study was to analyze the matrix metalloproteinase (MMP) activity during the evolution of interstitial renal fibrosis in a rat experimental model of unilateral ureteral obstruction. The interstitial type I collagenase and the gelatinolytic activities were analyzed by radiolabeled substrate degradation. Interstitial collagenase activity was low at all times while gelatinolytic activity increased on day 6 of evolution, with a decrease in activity from this point. The use of organomercurials revealed the presence of latent enzyme in all cases. Normal kidney samples contained MMP-9 in both active and proenzyme forms as revealed by zymography. On day 3 MMP-9 dimers appeared, and increased activity was observed until day 6. A decrease in the gelatinolytic activity was detected from days 9–15 of evolution. This observation was confirmed by Western blot analysis that revealed the presence of proMMP-9 mainly from days 6–12. Tissue inhibitor of metalloproteinase-1 (TIMP-1) was also detected alone and in combination with proMMP-1 and MMP-1, particularly from days 6–15 of evolution. The presence of MMP-9 and MMP-1 was detected in the cytoplasm of cortical tubular cells by immunohistochemistry, with no difference between the experimental and the normal kidneys. There was also an increase in collagen concentration from day 3 after surgery that increased during the entire evolution of the experimental model. This work reveals that the decrease in the MMP-9 and MMP-1 enzymatic activity, due to their interaction with TIMP-1 and to the lack of activation of the latent forms, may participate in the excessive collagen deposit during the evolution of experimental interstitial renal fibrosis.

Transporter associated with antigen processing (TAP) 1 gene polymorphisms in patients with hypersensitivity pneumonitis.

Hypersensitivity pneumonitis (HP) is a lung inflammatory disease caused by the inhalation of a variety of antigens. Previous studies support the role of the major histocompatibility complex (MHC) class II genes in the susceptibility to develop HP. However, the putative role of other MHC loci has not been elucidated. Transporters associated with antigen processing (TAP) genes are located within the MHC class II region and play an important role transporting peptides across the endoplasmic reticulum membrane for MHC class I molecules assembly. The distribution of single nucleotide polymorphisms (SNPs) in TAP1 genes was analyzed in 73 hypersensitivity pneumonitis (HP) patients and 58 normal subjects. We found a significant association of the allele Gly-637 (GGC) (p=0.00004, OR=27.30, CI=3.87-548.04) and the genotypes Asp-637/Gly-637 (p=0.01, OR=16.0, CI=2.19-631.21), Pro-661/Pro-661 (p=0.006, OR=11.30, CI=2.28-75.77) with HP. A significant decrease in the frequency of the allele Pro-661 (CCA) (p=0.008, OR=0.06, CI=0-0.45), the genotype Asp-637/Asp-637 (p=0.01, OR=0.17, 95% CI=0.05-0.58) and the haplotype [Val-333 (GTC), Val-458 (GTG), Gly-637 (GGC), Pro-661 (CCA)] was detected in HP patients compared with controls (p=0.002, OR=0.07, CI=0.0-0.57). These findings suggest that TAP1 gene polymorphisms are related to HP risk, and highlight the importance of the MHC in the development of this disease.

PSMB8 (LMP7) but not PSMB9 (LMP2) gene polymorphisms are associated to pigeon breeder's hypersensitivity pneumonitis

Hypersensitivity Pneumonitis (HP) is a lung inflammatory disorder caused by inhalation of organic particles by a susceptible host. However, only a small proportion of individuals exposed to HP-associated antigens develop the disease, suggesting that additional host/environmental factors may play a role. We have previously found that genetic susceptibility associated to the major histocompatibility complex (MHC) plays an important role in this disease. The low molecular weight proteosome (LMP, currently named PSMB) genes code for subunits of the proteosome, a multimeric enzymatic complex that degrades proteins into peptides in order to be presented in the MHC class I pathway. We hypothesized that polymorphisms in PSMB8 or PSMB9 genes could be involved in the susceptibility to HP. Thus, in this study we analyzed the polymorphic site at amino acid position 60 (Arg/His) of the fourth exon in the PSMB9 gene and the amino acid position 49 (Gln/Lys) in the second exon of PSMB8 gene in 50 Mexican patients with HP and 50 healthy ethnically matched controls. PSMB typing was performed using polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP). Our results demonstrated that HP patients had a significant increase of the PSMB8 KQ genotype frequency (OR = 7.25, CI = 2.61-21.3; p = 0.000034). No differences were found in the distribution of PSMB9 alleles/genotypes. However, PSMB9-RH/PSMB8 KQ haplotype was significantly increased in HP patients (OR = 6.77, CI = 1.34-65.31, p < 0.02). These findings suggest that PSMB8 KQ genotype could increase the risk to develop hypersensitivity pneumonitis.

Malondialdehyde and superoxide dismutase correlate with FEV1 in patients with COPD associated with wood smoke exposure and tobacco smoking

Tobacco smoking is the primary risk factor for chronic obstructive pulmonary disease (COPD). However, recent epidemiological studies have established domestic exposure to wood smoke and other biomass fuels as additional important risk factors, characteristic in developing countries. Oxidative stress is one of the mechanisms concerned with pathogenesis of COPD. However, the molecular mechanisms involved in the onset and progress of COPD associated with biomass and specifically that derived from wood smoke exposure remain unknown. We analyzed the relationship between forced expiratory volume in first second (FEV1) with plasma malondialdehyde (MDA) concentration and activities of superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR), and glutathione-S-transferase (GST) in COPD patients associated with wood smoke (WSG; n = 30), tobacco smoking (TSG; n = 30), and healthy control subjects (HCG; n = 30). Differences between FEV1 from WSG and TSG (58 ± 22% and 51 ± 24%, respectively) with HCG (100 ± 6%) were observed (P < 0.01). Plasma MDA concentration was higher in both WSG and TSG (1.87 ± 0.81 and 1.68 ± 0.82 nmol/mL, respectively) compared with HCG (0.42 ± 0.17 nmol/mL; P < 0.01). SOD activity showed a significant increase in both WSG and TSG (0.36 ± 0.12 and 0.37 ± 0.13 U/mL) compared with HCG (0.19 ± 0.04 U/mL; P < 0.01). No differences were shown regarding GPx, GR, and GST activities between COPD and control groups. Inverse correlations were founded between MDA and SOD with FEV1 in both COPD patients and control subjects (P < 0.001). These results indicate a role for oxidative stress in COPD associated with wood smoke similar to that observed with tobacco smoking in subjects who ceased at least 10 years previous to this study.

Experimental pulmonary fibrosis induced by paraquat plus oxygen in rats: A morphologic and biochemical sequential study

Changes in lung structure and collagen metabolism were studied at 1, 2, 3, 4, 6, and 8 weeks in a model of pulmonary fibrosis induced in rats with paraquat plus hyperoxia. Morphologic examination of the lungs revealed that the earliest lesions consisted of severe and irreversible endothelial and alveolar epithelial cell damage. Afterward, an inflammatory process took place, initially dominated by polymorphonuclear leukocytes and then by mononuclear cells, but with the constant presence of granulocytes. From the fourth week on there were fibroblast proliferation and a moderate increase of mast cells. In the early stages alveolitis was focal, but from the second week the lungs were diffusely affected with severe distortion of the architecture. Collagen content was moderately increased in the first 2 weeks and then showed a progressive increment until the end of the experiment. Collagen synthesis was significantly elevated from the fourth week, coinciding with interstitial fibroblast proliferation, although there were some animals that showed increased collagen production from the first week. Collagenolytic activity occurred in 3 stages: at 2 weeks there was increased collagen degradation, at 3, 4, and 6 weeks the values showed a trimodal behavior, and at 8 weeks almost all experimental rats presented an important decrease of collagenolysis. Thus, the development of lung fibrosis was associated first with increased rates of collagen synthesis and later with a decrease of collagen degradation.

Correlation between pulmonary fibrosis and the lung pressure-volume curve

The severity of pulmonary fibrosis is the main prognostic factor for survival of patients with interstitial lung diseases (ILD). Unfortunately, lung biopsy, which is the best method to assess fibrosis quantitatively, is done only once during the evolution of the disease. In this study we analyzed the relationship between the degree of fibrosis and the exponential constant k, derived from the lung pressure-volume curve (LPVC) in 33 patients with chronic ILD, 19 with pigeon breeders disease (PBD), and 14 with idiopathic pulmonary fibrosis (IPF). Pulmonary function tests, including the LPVC, were obtained before biopsy. A semiquantitative histologic assessment of the severity of fibrosis was performed on lung tissues. All patients showed a decrease of total lung capacity, residual volume, compliance, and PaO2. The mean value of the constant k was 0.08 ± 0.06. When expressed as a percent of normal values, 25 patients exhibited values of k lower than 70% of predicted; of the remaining 8 patients whose values were above 70% of predicted, 7 had PBD and only one IPF. On morphologic analysis, 19 patients displayed more than 50% fibrosis. No significant correlations were found between the extent of the lesion or severity of lung fibrosis and the conventional pulmonary function tests. By contrast, a moderate but significant correlation was found between k and the severity of lung fibrosis (r = −0.38, p < 0.05). These findings show that the shape of the LPVC, represented by the constant k, predicts the degree of lung fibrosis and could be useful in the clinical assessment and follow-up of patients with ILD.

Increase of Matrix Metalloproteinases in Woodsmoke-Induced Lung Emphysema in Guinea Pigs

Elastolysis, collagenolysis and gelatinolysis are essential in the pathogenesis of tobacco smoke-induced emphysema; however, these activities have been scantily studied in emphysema secondary to woodsmoke. The aim of this study was to analyze elastolysis, collagenolysis and gelatinolysis, MMP-1, MMP-2, and MMP-9 expression, and apoptosis in guinea pigs exposed to smoke produced by 60 g/day of pine wood, 5 days/week, from 1 to 7 months. Histological analysis after 4 to 7 months in smoke exposed guinea pigs showed alveolar mononuclear phagocyte and lymphocytic peribronchiolar inflammation, epithelial and smooth muscle hyperplasia, and pulmonary arterial hypertension. Mild to moderate emphysematous lesions were observed in woodsmoke-exposed animals at 4 to 7 months by increase of mean linear intercepts. A higher percentage of whole blood carboxyhemoglobin (COHb) and elastolytic activity in bronchoalveolar lavage macrophages and lung tissue homogenates was observed at all times. Collagenolysis was increased after 4 to 7 months in woodsmoke-exposed animals, although collagen concentration did not change. Zymography revealed increase in lysis bands of the active MMP-2 and MMP-9 at 4 and 7 months in bronchoalveolar lavage fluid and lung tissue homogenate. Positive immunostaining for MMP-1 and MMP-9 was observed in epithelial cells and macrophages in wood exposed animals at 4 to 7 months. Real-time PCR showed MMP-2 and MMP-9 expression at 3 to 7 months in exposed animals. Furthermore, apoptosis was increased at all times in bronchoalveolar lavage macrophages and lung tissue from exposed animals. Results support a role of metalloproteinases and apoptosis in emphysema secondary to woodsmoke exposure.