Cytokine gene polymorphisms and serum cytokine levels in patients with idiopathic pulmonary fibrosis

BACKGROUND: Studies have demonstrated associations between cytokine gene polymorphisms and the risk of idiopathic pulmonary fibrosis (IPF). We therefore examined polymorphisms in the genes encoding interleukin (IL)-6, IL-10, interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), and transforming growth factor-beta 1 (TGF-β(1)), and compared the serum levels of these cytokines in IPF patients and healthy controls. Furthermore, we examined the association of the studied genotypes and serum cytokine levels with physiological parameters and the extent of parenchymal involvement determined by high-resolution computed tomography (HRCT). METHODS: Sixty patients with IPF and 150 healthy controls were included. Cytokine genotyping was performed using the polymerase chain reaction sequence specific primer (PCR-SSP) method. In a subset of patients and controls, serum cytokine levels were determined by enzyme-linked immunosorbent assay. RESULTS: There was no difference between IPF patients and controls in the genotype and allele distributions of polymorphisms in TNF-α, IFN-γ, IL-6, IL-10, and TGF-β(1) (all p > 0.05). The TNF-α (−308) GG, IL-6 (−174) GG and CG, and IL-10 (−1082, -819, -592) ACC ATA genotypes were significantly associated with HRCT scores (all p < 0.05). IL-10 (−1082, -819, -592) ACC haplotype was associated with the diffusion capacity of the lung for carbon monoxide, and ATA haplotype was associated with the partial pressure of oxygen (PaO(2)) (all p < 0.05). The TGF-β(1) (codons 10 and 25) TC GG, TC GC, CC GG and CC GC genotypes were significantly associated with the PaO(2) and HRCT scores (p < 0.05). The TGF-β(1) (codons 10 and 25) CC GG genotype (5 patients) was significantly associated with higher PaO(2) value and less parenchymal involvement (i.e., a lower total extent score) compared to the other TGF-β(1) genotypes (81.5 ± 11.8 mm Hg vs. 67.4 ± 11.1 mm Hg, p = 0.009 and 5.60 ± 1.3 vs. 8.51 ± 2.9, p = 0.037, respectively). Significant differences were noted between patients (n = 38) and controls (n = 36) in the serum levels of IL-6 and IL-10 (both, p < 0.0001), but not in the levels of TNF-α and TGF-β(1) (both, p > 0.05). CONCLUSION: The studied genotypes and alleles do not predispose to the development of IPF but appear to play an important role in disease severity. Our results suggest that the TGF-β(1) (codons 10 and 25) CC GG genotype could be a useful genetic marker for identifying a subset of IPF patients with a favorable prognosis; however, validation in a larger sample is required

Structural and Functional Consequences of Cleavage of Human Secretory and Human Serum Immunoglobulin A1 by Proteinases from Proteus mirabilis and Neisseria meningitidis

The cleavage of human serum monomeric immunoglobulin A1 (IgA1) and human secretory IgA1 (S-IgA1) by IgA1 proteinase of Neisseria meningitidis and cleavage by the proteinase from Proteus mirabilis have been compared. For serum IgA1, both proteinases cleaved only the α chain. N. meningitidis proteinase cleaved only in the hinge. P. mirabilis proteinase sequentially removed the tailpiece, the CH3 domain, and the CH2 domain. The cleavage of S-IgA1 by N. meningitidis proteinase occurred only in the hinge and was as rapid as that of serum IgA1. P. mirabilis proteinase predominantly cleaved the secretory component (SC) of S-IgA1. The SC of S-IgA1, whether cleaved or not, appeared to protect the α1 chain. Purified Fc fragment derived from the cleavage of serum IgA1 by N. meningitidis proteinase stimulated a respiratory burst in neutrophils through Fcα receptors, whereas the (Fcα1)(2)-SC fragment from digested S-IgA1 did not. The loss of the tailpiece from serum IgA1 treated with P. mirabilis proteinase had little effect, but the loss of the CH3 domain was concurrent with a rapid loss in the ability to bind to Fcα receptors. S-IgA1 treated with P. mirabilis proteinase under the same conditions retained the ability to bind to Fcα receptors. The results are consistent with the Fcα receptor binding site being at the CH2-CH3 interface. These data shed further light on the structure of S-IgA1 and indicate that the binding site for the Fcα receptor in S-IgA is protected by SC, thus prolonging its ability to activate phagocytic cells at the mucosal surface

Rapid detection of circulating fibrocytes by flowcytometry in idiopathic pulmonary fibrosis

Background: Current protocols for detection of circulating fibrocytes (CFs) in peripheral blood described in various pulmonary and nonpulmonary disorders involve complex and time consuming, non standardized techniques. Objective: Testing a method to rapidly detect and quantify CFs using whole blood lysis flow cytometry-based assay in patients with idiopathic pulmonary fibrosis (IPF) and healthy controls. Methods: One milliliter of venous blood sample in ethylenediaminetetraacetic acid (EDTA) from 33 IPF patients and 35 healthy control subjects was collected. Using whole blood lysis method peripheral blood leukocytes were labeled with monoclonal antibodies for cell surface (CD34 and CD45) and intracellular markers (collagen-1) for flow cytometric analysis. CFs were defined as CD45 + cells coexpressing collagen-I and CD34 molecules. Results: In 29 (87.8%) IPF patients and 10 (28.5%) control subjects, a well-defined highly granular CD45 + cell population was detected in dot plots generated by side scatter properties of CD45 + cells. These CD45 + cells were identified as CFs on the basis of coexpression of collagen-I and CD34; none of the other cell types in the peripheral blood were labeled with these monoclonal antibodies. In IPF patients the percentage of CFs was significantly higher compared to healthy controls (median (range): 1.37% (0.52-5.65) and 1.04% (0.1-1.84), respectively; P = 0.03). Conclusions: Whole blood lysis method combined with fluorescence-activated cell sorting (FACS) allows detecting a well-defined homogeneous population of CFs. This method is simple, reproducible, and provides an accurate and rapid estimation of CFs

Rapid detection of circulating fibrocytes by flowcytometry in idiopathic pulmonary fibrosis

Background: Current protocols for detection of circulating fibrocytes (CFs) in peripheral blood described in various pulmonary and nonpulmonary disorders involve complex and time consuming, non standardized techniques. Objective: Testing a method to rapidly detect and quantify CFs using whole blood lysis flow cytometry-based assay in patients with idiopathic pulmonary fibrosis (IPF) and healthy controls. Methods: One milliliter of venous blood sample in ethylenediaminetetraacetic acid (EDTA) from 33 IPF patients and 35 healthy control subjects was collected. Using whole blood lysis method peripheral blood leukocytes were labeled with monoclonal antibodies for cell surface (CD34 and CD45) and intracellular markers (collagen-1) for flow cytometric analysis. CFs were defined as CD45 + cells coexpressing collagen-I and CD34 molecules. Results: In 29 (87.8%) IPF patients and 10 (28.5%) control subjects, a well-defined highly granular CD45 + cell population was detected in dot plots generated by side scatter properties of CD45 + cells. These CD45 + cells were identified as CFs on the basis of coexpression of collagen-I and CD34; none of the other cell types in the peripheral blood were labeled with these monoclonal antibodies. In IPF patients the percentage of CFs was significantly higher compared to healthy controls (median (range): 1.37% (0.52-5.65) and 1.04% (0.1-1.84), respectively; P = 0.03). Conclusions: Whole blood lysis method combined with fluorescence-activated cell sorting (FACS) allows detecting a well-defined homogeneous population of CFs. This method is simple, reproducible, and provides an accurate and rapid estimation of CFs

Development, Characterization, and Immunotherapeutic Use of Peptide Mimics of the Thomsen-Friedenreich Carbohydrate Antigen

The tumor-associated carbohydrate Thomsen-Friedenreich antigen (TF-Ag; Galß1-3GalNAcα-O-Ser/Thr) is overexpressed on the cell surface of several types of tumor cells, contributing to cancer cell adhesion and metastasis to sites containing TF-Ag-binding lectins. A highly specific immunoglobulin G3 monoclonal antibody (Ab) developed to TF-Ag (JAA-F11) impedes TF-Ag binding to vascular endothelium, blocking a primary metastatic step and providing a survival advantage. In addition, in patients, even low levels of antibodies to TF-Ag seem to improve prognosis; thus, it is expected that vaccines generating antibodies toward TF-Ag would be clinically valuable. Unfortunately, vaccinations with protein conjugates of carbohydrate tumor-associated Ags have induced clinically inadequate immune responses. However, immunization using peptides that mimic carbohydrate Ags such as Lewis has resulted in both Ab and T-cell responses. Here, we tested the hypothesis that vaccinations with unique TF-Ag peptide mimics may generate immune responses to TF-Ag epitopes on tumor cells, useful for active immunotherapy against relevant cancers. Peptide mimics of TF-Ag were selected by phage display biopanning using JAA-F11 and rabbit anti-TF-Ag Ab and were analyzed in vitro to confirm TF-Ag peptide mimicry. In vitro, TF-Ag peptide mimics bound to TF-Ag-specific peanut agglutinin and blocked TF-Ag-mediated rolling and stable adhesion of cancer cells to vascular endothelium. In vivo, the immunization with TF-Ag-mimicking multiple antigenic peptides induced TFAg- reactive Ab production. We propose that this novel active immunotherapy approach could decrease tumor burden in cancer patients by specifically targeting TF-Ag-positive cancer cells and blocking metastasis