uPAR peptide antagonist alters regulation of MAP kinases and Bcl-2 family members in favor of apoptosis in MDA-MB-231 cell line.

Urokinase plasminogen activator receptor (uPAR) and its ligands play a major role in many tumors by mediating extracellular matrix degradation and signaling cascades leading to tumor growth, invasion and metastasis. Recently we introduced uPAR decapeptide antagonist with cytotoxic effect on MDA-MB-231 cell line. In this study we assessed the alteration in uPAR downstream signaling following treatment with the peptide antagonist. In this regard, extracellular-signal-regulated kinase (ERK) and p38 from mitogen-activated protein kinase family and Bcl-2, Bim and Bax from Bcl-2 protein family were investigated. Our data revealed that the peptide caused p38 activation and low ERK activation. On the other hand, the peptide induced down-regulation of Bcl-2 and up-regulation of Bim without Bax modulation. Changes in target protein expression/activation explain the apoptotic property of the peptide and highlight its potential to be used as a therapeutic agent in cancerous cells expressing high levels of uPAR.

Natural suppressor cells; past, present and future.

Myeloid Derived Suppressor Cells (MDSCs) are a mixed group of bone marrow-derived myeloid cells containing macrophages, granulocytes, immature DCs and early myeloid precursors that have immune suppressive activity. MDSCs infiltrate the BM, spleen and peripheral blood of tumors-bearing experimental animals and are found in the blood of cancer patients as a result of tumor-induced alterations in myelopoiesis. Evidence from murine model systems indicated that myeloid-derived cells with suppressor activity also accumulate in non-tumor bearing hosts in response to infection, chemotherapy, stress, and immune senescence. MDSCs are considered key negative regulators of immune responses. Their association with tumor-associated immune defects make MDSCs an attractive target for therapeutic intervention in cancer.

Production and characterization of liquid-stored and lyophilized reconstituted human infusible platelet membranes.

INTRODUCTION: During recent years, the need for platelet concentrate (PC) has increased. Infusible platelet membranes (IPM) have been developed as an alternative to standard PCs, with the additional advantage of long shelf-life and increased viral safety. In this study, IPM construction and the morphological and biological features of these microvesicles were surveyed to determine their binding capacities in vitro. METHODS: Thirty-five PC units prepared by the Iranian Blood Transfusion Organization were used to produce IPM. Platelets were lysed by repeated cycles of freezing and thawing, virally inactivated with wet heat in the presence of different concentrations of sodium octanoate as a heat stabilizer, and then sonicated. IPM were separated, kept at 4°C or lyophilized, and examined for binding to collagen and von Willebrand factor (VWF). RESULTS: IPM retained the binding capacity for collagen and VWF, and the extent of VWF binding was dependent on the concentration of the heat stabilizer. Additionally, a higher binding capacity was demonstrated for liquid-stored compared with lyophilized IPM. CONCLUSION: This study revealed the potential of IPM microvesicles to mimic the binding features of platelets in vitro.

Angiotensin II induces NF-κB, JNK and p38 MAPK activation in monocytic cells and increases matrix metalloproteinase-9 expression in a PKC- andRho kinase-dependent manner

Angiotensin II (ANG II), the main effector of the renin-angiotensin system, is implicated in endothelial permeability, recruitment and activation of the immune cells, and also vascular remodeling through induction of inflammatory genes. Matrix metalloproteinases (MMPs) are considered to be important inflammatory factors. Elucidation of ANG II signaling pathways and of possible cross-talks between their components is essential for the development of efficient inhibitory medications. The current study investigates the inflammatory signaling pathways activated by ANG II in cultures of human monocytic U-937 cells, and the effects of specific pharmacological inhibitors of signaling intermediates on MMP-9 gene (MMP-9) expression and activity. MMP-9 expression was determined by real-time PCR and supernatants were analyzed for MMP-9 activity by ELISA and zymography methods. A multi-target ELISA kit was employed to evaluate IκB, NF-κB, JNK, p38, and STAT3 activation following treatments. Stimulation with ANG II (100 nM) significantly increased MMP-9 expression and activity, and also activated NF-κB, JNK, and p38 by 3.8-, 2.8- and 2.2-fold, respectively (P < 0.01). ANG II-induced MMP-9 expression was significantly reduced by 75 and 67 percent, respectively, by co-incubation of the cells with a selective inhibitor of protein kinase C (GF109203X, 5 µM) or of rho kinase (Y-27632, 15 µM), but not with inhibitors of phosphoinositide 3-kinase (wortmannin, 200 nM), tyrosine kinases (genistein, 100 µM) or of reactive oxygen species (α-tocopherol, 100 µM). Thus, protein kinase C and Rho kinase are important components of the inflammatory signaling pathways activated by ANG II to increase MMP-9 expression in monocytic cells. Both signaling molecules may constitute potential targets for effective management of inflammation.

Angiotensin II induces NF-κB, JNK and p38 MAPK activation in monocytic cells and increases matrix metalloproteinase-9 expression in a PKC- andRho kinase-dependent manner.

Angiotensin II (ANG II), the main effector of the renin-angiotensin system, is implicated in endothelial permeability, recruitment and activation of the immune cells, and also vascular remodeling through induction of inflammatory genes. Matrix metalloproteinases (MMPs) are considered to be important inflammatory factors. Elucidation of ANG II signaling pathways and of possible cross-talks between their components is essential for the development of efficient inhibitory medications. The current study investigates the inflammatory signaling pathways activated by ANG II in cultures of human monocytic U-937 cells, and the effects of specific pharmacological inhibitors of signaling intermediates on MMP-9 gene (MMP-9) expression and activity. MMP-9 expression was determined by real-time PCR and supernatants were analyzed for MMP-9 activity by ELISA and zymography methods. A multi-target ELISA kit was employed to evaluate IκB, NF-κB, JNK, p38, and STAT3 activation following treatments. Stimulation with ANG II (100 nM) significantly increased MMP-9 expression and activity, and also activated NF-κB, JNK, and p38 by 3.8-, 2.8- and 2.2-fold, respectively (P < 0.01). ANG II-induced MMP-9 expression was significantly reduced by 75 and 67%, respectively, by co-incubation of the cells with a selective inhibitor of protein kinase C (GF109203X, 5 µM) or of rho kinase (Y-27632, 15 µM), but not with inhibitors of phosphoinositide 3-kinase (wortmannin, 200 nM), tyrosine kinases (genistein, 100 µM) or of reactive oxygen species (α-tocopherol, 100 µM). Thus, protein kinase C and Rho kinase are important components of the inflammatory signaling pathways activated by ANG II to increase MMP-9 expression in monocytic cells. Both signaling molecules may constitute potential targets for effective management of inflammation.

The prevalence of cagA and cagE genes in Helicobacter pylori strains isolated from different patient groups by polymerase chain reaction.

The aim of this study was to investigate the prevalence of cagA and cagE genes in H. pylori strains isolated from different patient groups with Non-Ulcer Dyspepsia (NUD), Duodenal Ulcer (DU), Gastric Ulcer (GU) and Gastric Cancer (GC). The patients admitted to the gastroenterology unit at Sharyati hospital in Tehran in 2006 were included in this study. Gastric biopsy specimens were obtained from the antrum of the stomach from each patient then cultured for detection of H. pylori. Identification of H. pylori was performed according to the standard bacteriological methods. Genomic DNA was extracted using a commercially available Qia gene kit. PCR was done using primers cagA-F, cagA-R and cagE-F, cagE-R to detect the target genes cagA and cagE, respectively. Amplified products of target genes were confirmed by sequencing. The cagA and cagE were detected among 85 and 86% of H. pylori isolates, respectively. Prevalence of cagA and cagE genes in the patients with NUD, DU, GU and GC were 22 (64.7%), 28 (100%), 18 (90%), 10 (100%) and 25 (73.5%), 27 (96.4%), 19 (95%), 7 (70%), respectively. The current study demonstrated a significant correlation between peptic ulceration and the presence of H. pylori isolates carrying cagE and cagA genes in Iranian patients.

Two matrix metalloproteinases inhibitors from Ferula persica var. persica.

Matrix metalloproteinases (MMPs) play a role in several physiologic and pathologic events. There is some evidence indicating the involvement of MMPs in tumor invasion and inflammatory diseases. Here we studied the chloroform extract of Ferula persica var. persica. The influence of these extracts vs. a reference drug, diclofenac sodium, on MMP production by the fibrosarcoma cell line was investigated using an in vitro cytotoxicity assay, sodium dodecyl sulfate-polyacrylamide, and gelatin zymography. The total extract of the roots was found to exhibit a selective inhibitory effect on tumor cell invasion. The bioactivity-guided fractionation of this extract led to the isolation of two compounds. These compounds showed highest MMP inhibitory effect at minimal toxic dose levels. Using conventional spectroscopy methods, the active fractions were identified as t-butyl 3-[(1-methylthiopropyl)dithio]-2-propenyl malonate (persicasulphide B) and umbelliprenin, previously isolated from F. persica var. latisecta. Since inhibition of MMP activity has been employed in modality therapy in diseases such as cancer, this compound might be promising in the preparation of anti-MMP therapeutic derivatives.

Occurrence of enteropathogenic bacteria in children under 5 years with diarrhoea in south Tehran.

This study was carried out on 1600 rectal swabs from children under 5 years of age admitted at the health centre in Islamshahr, Tehran province, Islamic Republic of Iran, during 1998-99. The specimens were examined for various bacterial pathogens. Isolation rates were: enteropathogenic Escherichia coli 6.8%, Shigella spp. 3.4%, Salmonella spp. 2.9%, Campylobacter spp. 0.9%, Yersinia spp. 0.7%. The isolation rate was highest in the summer, except for Yersinia spp., which was predominantly isolated in spring. The results of this study demonstrate the significance of Yersinia spp. and Campylobacter spp. in patients with diarrhoea.

Occurrence of enteropathogenic bacteria in children under 5 years with diarrhoea in south Tehran

This study was carried out on 1600 rectal swabs from children under 5 years of age admitted at the health centre in Islamshahr, Tehran province, Islamic Republic of Iran, during 1998-99. The specimens were examined for various bacterial pathogens. Isolation rates were: enteropathogenic Escherichia coli 6.8%, Shigella spp. 3.4%, Salmonella spp. 2.9%, Campylobacter spp. 0.9%, Yersinia spp. 0.7%. The isolation rate was highest in the summer, except for Yersinia spp., which was predominantly isolated in spring. The results of this study demonstrate the significance of Yersinia spp. And Campylobacter spp. in patients with diarrhoea

Sodium alginate as a novel therapeutic option in experimental colitis.

The potential therapeutic effect of low-viscosity sodium alginate (LVA) was studied in a rat model of acute colitis induced by intracolonic administration of acetic acid. This experimental model produced a significant ulcerative colitis. Induction of colitis also significantly enhanced the serum and colonic mucosal cytokine (IL-6 and TNF-alpha) and eicosanoid (LTB4 and PGE2) levels, which paralleled with the severity of colitis. LVA solution was administered orally as drinking water at concentration of 0.5% (W/V) for 1 week. The tolerability and inhibitory effect of LVA on matrix metalloproteinase-2 (MMP-2) were tested using WEHI-164 cell line and zymography method. The results showed that LVA therapy is able to significantly reduce colonic damage score, histological lesion, serum and colonic mucosal IL-6, TNF-alpha, LTB4 and PGE2 levels in treated group compared with nontreated controls. Moreover, in vitro examinations revealed that treatment with LVA could diminish MMP-2 activity. It is concluded that LVA is able to suppress acetic acid-induced colitis in rats. Some of the action of LVA may be associated with its inhibitory effects on cytokine and eicosanoid production and MMP-2 activity. Our data suggest that LVA could potentially be a novel therapeutic option for inflammatory bowel disease.

Effect of piroxicam on matrix metalloproteinase 2 and apoptosis.

We examined the effect of a nonsteroidal anti-inflammatory drug (NSAID), piroxicam, on apoptosis and matrix metalloproteinase 2 (MMP-2) activity compared with diclofenac and dexamethasone. The fibrosarcoma (WEHI-164) cell line was used to assess tolerability, MMP-2 activity and apoptosis. Piroxicam, dexamethasone and diclofenac were used at concentrations of 10-200 microg/ml in triplicate and 2-fold dilutions. MMP-2 activity was assessed using zymography. For assessment of apoptosis, the terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) method was used. The results of this study show that piroxicam is able to diminish MMP-2 activity and induce apoptosis under in vitro conditions. Piroxicam also showed high tolerability compared with diclofenac and dexamethasone. In conclusion, piroxicam is able to induce apoptosis and suppress MMP-2 activity.

Tolerability and anti-inflammatory effects of glucuronoxylomannan in collagen-induced arthritis.

This investigation was planned to assess the therapeutic efficacy of glucuronoxylomannan (GXM) in collagen-induced arthritis (CIA). GXM was isolated from culture filtrate of Cryptococcus neoformans var. gattii, serotype C. CIA was induced by the immunization of Dark Agouti rats with bovine type II collagen in incomplete Freund's adjuvant. GXM solution at two doses, 25 and 50 mg/kg, was administered intraperitoneally. Onset of i.p. injections of GXM to prevention and treatment groups was days 0 and 10 postimmunization, respectively. The WEHI-164 cell line was used for assaying tolerability, matrix metalloproteinase type 2 (MMP-2) activity and apoptosis. MMP-2 activity was assessed using zymography. For assessment of apoptosis, the terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labelling method was used. The results of this experiment showed that the treatment of CIA with GXM at a dose of 50 mg/kg could suppress disease development both prophylactically and therapeutically. This beneficial effect of GXM was associated with a significant decrease in the anti-CII antibody response compared with untreated rats. Moreover, GXM therapy could diminish MMP-2 activity, but it had no notable effect on apoptosis. GXM also showed a high tolerability compared with certain steroidal and non-steroidal anti-inflammatory drugs. We conclude that GXM suppresses the development of disease in CIA and it could be recommended as a new immunosuppressive and anti-inflammatory agent for further investigations.

Alteration in Matrix Metalloproteinases (MMPS) Activity in Fibroblast Cell Line by Dexamethasone: A Possible Mechanism in Corticosteroid-Induced Glaucoma.

Corticosteroids are often used as anti-inflammatory agents in a variety of inflammatory diseases. It is well established that long-term administration of corticosteroids predisposed the patients to develop glaucoma. Although the exact pathophysiology of steroid-induced glaucoma is unknown, it is assumed that Matrix metalloproteinases (MMPs) have a role in its pathogenesis. To study and estimate the pathophysiological effects of MMPs in glaucoma, we established an in vitro cell culture model. We also employed a precise proliferation assay to analyze cytotoxic effect of dexamethasone. The influence of dexamethasone on MMPs production was investigated using an in vitro gelatin Zymography. Cytotoxcity analysis of Dexamethasone revealed no significant cell death in low concentration. However, it caused 50% and 70% cell death at 80 and 100 mg/mL respectively. It also revealed an inhibitory effect on MMPs by dexamethasone in a dose dependent fashion. It may be concluded that an alteration in the level of MMPs expression by dexamethasone interferes with ocular fluid drainage and may contribute to the pathogenesis of glaucoma.

Aging differentially modulates the expression of collagen and collagenase in dermal fibroblasts.

This study was conducted to investigate the effects of aging on collagen and collagenase expression by human dermal fibroblasts. To evaluate this effect, the expression of these ECM was determined and compared between either fetal and adult fibroblasts or dermal fibroblasts at various passages. A total of 13 cell strains, 8 fetal foreskin and 5 adult dermal fibroblasts, were grown to 80-90% confluency and their rates of cell proliferation and expression of mRNA for collagenase (MMP-1) and pro alpha1(I) chain of type I collagen was determined and compared. Fetal cells had a significantly higher rate of proliferation relative to adult fibroblasts evaluated within 10 days of culture. Northern analysis was used to evaluate the steady state levels of mRNA in these cells. The result of these experiments revealed a significantly greater expression of mRNA for collagenase (58.6 +/- 7.7 vs. 9.9 +/- 1.5, p < 0.05) in strains of adult fibroblasts. This was consistent with collagenase activity of conditioned medium derived from adult cells relative to fetal fibroblasts. However the expression of pro alpha1 (I) chain of type I collagen mRNA was not significantly (56.2 +/- 5.2 vs. 58.5 +/- 3.5) different between adult and fetal fibroblasts. This finding was confirmed by measuring total collagen production present in conditioned medium of these cells using hydroxyproline as an index for collagen production. The cellular response to IGF-1 and IFN-alpha2b as representatives of fibrogenic and anti-fibrogenic factors were also evaluated. When expression of collagenase was used as an indication for cellular response, the degree of this response to IGF-1 but not IFN-alpha2b was significantly greater in fetal relative to adult cells. Serial passage was also used as an in vitro model for aging fibroblasts and found a gradual reduction in pro alpha1(I) chain of type I collagen mRNA and hydroxyproline formation due to passaging. In conclusion, a slower rate of proliferation, a greater collagenase activity and expression of collagenase mRNA by aging fibroblasts could be some of the main reasons for attenuation of wound healing in elderly patients.

IFN-alpha2b suppresses the fibrogenic effects of insulin-like growth factor-1 in dermal fibroblasts.

The interferon (IFN) proteins, including IFN-alpha2b have been used as antifibrogenic factors to modulate the expression of extracellular matrix (ECM) proteins associated with fibroproliferative disorders in skin. This study was conducted to determine if IFN-alpha2b can counteract the fibrogenic effects of insulin-like growth factor-1 (IGF-1), which is present in large quantity in fibrotic dermis. Human dermal fibroblasts were established in culture and treated with either vehicle (control), 2000 U/ml IFN-alpha2b alone, 100 ng/ml IGF-1 alone, or both IFN-alpha2b and IGF-1. The results showed that treatment with IFN-alpha2b inhibited the proliferation of dermal fibroblasts, reduced the steady-state levels of type I procollagen mRNA in the cells, and reduced the production of collagen as measured by hydroxyproline in conditioned medium. However, this treatment also increased levels of collagenase mRNA in the cells and collagenase activity in the medium. Cells treated with IGF-1 showed increased proliferation and collagen production and decreased collagenase. Cells treated with both IFN-alpha2b and IGF-1 exhibited a 44% reduction in hydroxyproline production (p < 0.05) and a 363% increase in collagenase activity over cells treated with IGF-1 alone (p < 0.01). These results indicate that when IGF-1 and IFN-alpha2b are used individually, they function as fibrogenic and antifibrogenic factors for dermal fibroblasts, respectively, and that fibrogenic effects of IGF-1 on cell proliferation, collagen, and collagenase expression can be counteracted by IFN-alpha2b. These findings support the potential use of IFN-alpha2b as a therapeutic agent for treatment of fibroproliferative disorders, such as postburn hypertrophic scarring.