ABSTRACT
Recent reports have indicated different effects of immunostimulatory sequences containing CpG-Oligodeoxynucleotides [ODN] on various immune cells. However, the exact role of CpG-ODN in the human gut is unclear. In the present study, we assessed potential effects of CpG-ODN on non lymphoid cell [intestinal epithelial cell line HT-29] on a dose-response and time-course basis. Intestinal epithelial cell line HT-29 was treated with CpG-ODN [CpG 2006] and lipopolysaccharide [LPS] at 5, 10, 25, 50 micro g/ml and 1, 5, 10 micro g/ml concentrations, respectively. Following treatments, dose-response and time-course cytotoxicity using a colorimetric method, Metaloproteinase-2 [MMP-2] activity [using gelatin zymography] and apoptosis [using annexin-v flowcytometry method] assays were performed. Chloroquine treatment was also used for its inhibitory effect on endosomal acidification process to verify specific CpG-ODN and Toll Like Receptor 9 [TLR9] interactions. Cytotoxicity analysis of CpG-ODN showed that CpG-ODN increased significantly the proliferation of CpG-ODN treated cells, as compared to untreated cells, at concentrations of 10-25 micro g/ml [p<0.05]. Overall MMP-2 activity analysis showed significant differences between treated and untreated cells. However, minimal changes were observed when MMP-2 activity was assessed per cell. Moreover, CpG-ODN treated cells demonstrated an increasing apoptosis rate of 0.8%, 6.46% and 14.21% at concentrations of 5, 10, 25 micro g/ml, respectively. Collectively, our data indicated that intestinal epithelial cell line HT-29 is highly responsive to CpG effect in vitro and exhibits modified activities. The direct CpG-ODN and TLR-9 interactions in HT-29 cells could provide new approaches in malignant tumor therapeutic strategies
ABSTRACT
There are emerging data on novel tumor markers such as matrix metalloproteinase-2 [MMP-2 or gelatinase-A], which play a key role in tissue invasion and metastasis. We designed an investigation to assess the usefulness of MMP-2 activity as compared to prostate specific antigen [PSA] in cancer staging process. We have analyzed the circulating form of MMP-2 in serum samples of patients suffering from either benign prostate hyperplasia [BPH, n = 54] or prostate cancer [PC, n = 26], and prostatitis [n = 4] as compared to control normal individual [n = 26], respectively. Protein-content adjusted samples were separated by gelatin-embedded polyacrylamide gel electrophoresis then were subjected to densitometric analysis. Total PSA [tPSA] and free PSA [fPSA] were quantified using a standard ELISA technique. Correlation coefficient [r] between tPSA and MMP-2 activity in patient group was +0.938 and in controls group was 0.799 [P<0.01]. In addition, [r] between tPSA and MMP-2 activity in PC patients was 0.940 and in BPH patients was 0.962 [P<0.01]. [r] between fPSA and MMP-2 activity in PC patients was 0.913, in BPH patients was 0.644, and in prostatitis patients was -0.994 [P<0.01]. These results demonstrate that MMP-2, compared to PSA, might be considered as a better tumor marker in monitoring and screening patients with PC
Subject(s)
Humans , Male , Prostatic Neoplasms/diagnosis , Prostate-Specific Antigen/blood , Matrix Metalloproteinase 2/bloodABSTRACT
Vitiligo is an acquired skin disorder that selectively destroys melanocytes in epidermis with an unknown etiology. To investigate the exon 1 A49G polymorphism of cytotoxic T lymphocyte antigen-4 [ctla-4] gene in vitiligo patients. The A49G polymorphism was detected by Po-lymerase Chain Reaction-Restriction Fragment Length Polymorphism [PCR-RFLP] method in 101 patients and 208 normal healthy age/ethnicity matched individuals. The frequencies of heterozygote genotypes in patients and controls were found to be 42 [41.6%] of 101 and 85 [40.9%] of 208, respectively. The frequencies of homozygote A and G genotypes were 49 [48.5%] and 10 [9.9%] in 101 patients, whereas, these frequencies in 208 control individuals were 103 [49.5%] and 20 [9.6%], respectively. There was no significant difference between the genotype [P = 0.98] and allele [P = 0.86] frequencies of A49G polymorphism in patients and normal healthy individuals. Our results indicate that in contrast to several immune mediated disorders, there is no asso-ciation between ctla-4 A49G gene polymorphism and vitiligo
ABSTRACT
This study was conducted to examine if allergic contact dermatitis [ACD] alters the expression ofMMPs in human dermal fibroblasts. Fibroblasts are the primary source for MMP and matrix production in skin. MMPs are known to involve in a number of physiological and pathological processes. Some published data indicated a gelatinaselike activity in acute and chronic phases of allergic contact dermatitis. However, no exact source of gelatinase activity was demonstrated. Moreover, little is known about the role of MMPs in immune responses. To study and predict the pathophysiological effects of [MMP-2] in allergic contact dermatitic [ACD] patients, we established an in vitro tissue culture survey based on fibroblast explanted from ACD wounds and normal tissues respectively. We also employed a precise proliferation assay [i.e. MTT; 3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide] to analyze and compare three ACD vs. three normal cell strains Parallel to MTT assay, we assessed the activity as well as the kinetics of gelatinase [MMP-2] in conditioned media using a zymogeraphy analysis. There was a significant difference in proliferation capacity between mean ACD fibroblast strains vs. mean normal cells, particularly in days 6 to 8 post explantation, 492.5 +/- 6.6 vs. 361.75 +/- 8.25 respectively. Zymoanalyses indicated significant differences betweenACD cells and normal fibroblasts both in time-course and MMP-2 activity per cell fashions, 163.7 +/- 16.21 for meanACD fibroblasts vs. 130 +/- 9.09 for normal cells respectively. These data suggest that fibroblasts overproliferated in the process ofACD. Moreover, simultaneous overexpression of MMPs observed inACD fibroblasts vs. normal strains, is indicative of altered fibroblast functionality in the process of allergic contact dermatitis. The activity per cell analysis showed that MMP-2 expression in ACD fibroblasts is independent of cell number, suggesting that either intra- or inter-cellular control signals are also altered and that ACD fibroblasts exhibit hyper-responsiveness to mitogenic or fibrogenic stimulants. Altogether, these data address the chronocity and non-healing tendency of ACD wounds. However, more studies are required to examine possible MMPs inhibition and differential expression of mytogenic, fibrogenic and antifibrogenic cytokines in ACD wound beds. In particular, MMP-2 is postulated to be an aim for further gene therapy protocols