Search

IgA plays a crucial role in establishment and maintenance of mucosal homeostasis between host cells and commensal bacteria. To this end, numerous IgA plasma cells are located in the intestinal lamina propria. Whether the (immediate) precursor cells for these plasma cells can expand locally is not completely known and was studied here. The total number of IgA plasma cells in human ileal biopsies was counted. Sequence analysis of IgA V(H) genes from human ileal biopsies revealed the occurrence of many clonally related sequences within a biopsy, but not between different biopsies. This observation strongly argues for local expansion of IgA precursor cells. By comparing the number of unique sequences with the number of clonally related sequences within a biopsy, we estimated that approximately 100-300 precursors were responsible for the 75,000 IgA-producing cells that were present per biopsy. These precursor cells must therefore have divided locally 9-10 times. Since all sequences contained mutations and most of the mutations present in clonally related sequences were shared, the IgA precursor cells must have arrived initially as mutated cells in the lamina propria. Our data show evidence for the existence of two waves of expansion for IgA-producing cells in human ileum. The first wave occurs during initial stimulation in germinal centers as evidenced by somatic hypermutations. A second wave of expansion of IgA-committed cells occurs locally within the lamina propria as evidenced by the high frequency of clonally related cells.

Receptor activator for nuclear factor-kappaB ligand (RANKL), a critical osteoclastogenic factor expressed in marrow stromal/preosteoblast cells is up-regulated in Paget's disease of bone (PDB). We previously demonstrated that heat-shock factor-2 (HSF-2) is a downstream target of fibroblast growth factor-2 (FGF-2) signaling to induce RANKL expression in bone marrow stromal/preosteoblast cells. In this study, we identified a 2.5-fold increase in serum FGF-2 levels in patients (n = 8) with PDB compared with normal subjects (n = 10). We showed that HSF-2 co-immunoprecipitates with heat-shock protein-27 (HSP-27) and that FGF-2 stimulation significantly increased phospho-HSP-27 levels in marrow stromal cells. Confocal microscopy revealed HSF-2 colocalization with HSP-27 in unstimulated cells and HSF-2 nuclear translocation upon FGF-2 stimulation. We further show that FGF-2 stimulation significantly increased the levels of phosphorylated signal transducers and activators of the transcription (p-STAT-1) in these cells. Western blot analysis confirmed that small interfering RNA suppression of STAT-1 significantly decreased (3.2-fold) RANKL expression and promoter activity in FGF-2-stimulated cells. Chromatin immunoprecipitation assay revealed STAT-1 binding to a putative motif located far upstream (-8 kb) in the hRANKL gene promoter region. These results suggest STAT-1 is a downstream effector of FGF-2 signaling and that elevated levels of FGF-2 stimulates RANKL expression in PDB.

Oral squamous cell carcinomas (OSCC) are malignant tumors with a potent activity of local bone invasion/osteolysis. The chemokine ligand, CXCL13, has been identified as a prognostic marker for OSCC development and progression. Here in, we show that recombinant hCXCL13 treatment of OSCC cells stimulates (5-fold) RANK ligand (RANKL), a critical bone resorbing osteoclastogenic factor expression. Anti-CXCR5 chemokine receptor antibody abrogates CXCL13-induced RANKL expression in these cells. Also, CXCL13 stimulated (3.0-fold) hRANKL gene promoter activity in SCC14a cells. SuperArray screening for transcription factors by real-time RT-PCR identified significant increase in the levels of c-Jun and NFATc3 mRNA expression in CXCL13-stimulated OSCC cells. CXCL13 treatment significantly increased (3.5-fold) phospho-c-Jun levels in these cells and a c-Jun-NH(2)-kinase inhibitor abolished CXCL13-stimulated RANKL expression. Furthermore, we show that CXCL13 stimulation induced nuclear translocation of NFATc3 in OSCC cells. Chromatin-immune precipitation assay confirmed NFATc3 binding to the RANKL promoter region. We also show that overexpression of NFATc3 stimulates RANKL expression/promoter activity and that siRNA suppression of NFATc3 abolished CXCL13-stimulated RANKL expression. Thus, our results suggest that NFATc3 is a downstream target of the CXCL13/CXCR5 axis to stimulate RANKL expression in OSCC cells and implicates CXCL13 as a potential therapeutic target to prevent OSCC bone invasion/osteolysis.

Background: Tamoxifen (TAM) a non-steroidal antiestrogen, is widely used in adjuvant therapy for all stages of breast carcinomas and in chemoprevention of high-risk group. TAM also has estrogenic activity on liver and endometrium causing severe oxidative stress with various biochemical derangements. Coenzyme Q(10), Riboflavin and Niacin (CoRN) are well-known potent antioxidants and protective agents against many diseases including cancer. In this context, this study was undertaken to find if co-administration of TAM along with CoRN could alleviate the sole TAM-induced biochemical derangements in postmenopausal women with breast cancer., Method: The vitamin supplementation with TAM was given for a period of 90 days. Blood samples were collected at the base line, 45th and 90th day during the course of treatment. Various blood chemistry profiles were assessed in 78 untreated, sole TAM treated and combinatorial treated group along with 46 age- and sex-matched controls., Results: A statistically significant alteration in various blood chemistry parameters, such as serum total bilirubin (S. BIL), serum glutamate oxaloacetate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT), gamma glutamyl transpeptidase (gamma-GT), uric acid (UA), lipoprotein lipase (LPL), lecithin: cholesterol acyl transferases (LCAT), potassium, calcium and Na(+), K(+)-ATPase in sole TAM-treated group, was favorably reverted back to near normal levels on combinatorial therapy with CoRN., Conclusion: TAM on co-administration with CoRN has a favorable impact on various blood chemistry profiles. However, large scale randomized studies over a longer time span are required to ascertain the safety and efficacy of co-administrating antioxidants with conventional chemotherapy.

The gastrointestinal tract is a complex niche and the main port of entry of many pathogens that trigger a wide range of diseases like inflammatory bowel disease (IBD) and colon cancer. Antibodies are effective for treating such diseases, but a system capable of local delivery at the site of the pathology, thus avoiding systemic side effects, is not yet available. Here we report a novel recombinant scFvSIgA1 protein produced by Lactococcus lactis, anchored to the bacterial membrane, which retains its full immuno-recognizing potential. This scFv fragment employed was specific for a colon cancer epitope, epithelial glycoprotein protein-2 (EGP-2). Accordingly L. lactis expressing this chimeric protein was capable of binding cells expressing this epitope. Expression of specific antibodies on bacteria may allow local delivery of anticancer agents produced by such bacteria in conjunction with the antibody and provides a new avenue in the quest for targeted drug delivery.

Background: Reactive oxygen species (ROS) such as superoxide anion, hydrogen peroxide (H(2)O(2)), hydroxyl radical have been implicated in pathogenesis of various diseases including cancer and metastasis. Tamoxifen (TAM) is a non-steroidal anti-estrogen drug most widely used as an adjuvant hormonal therapy in breast cancer. TAM also has estrogenic activity on liver and endometrium causing severe oxidative stress and hypertriglycerdemia. Coenzyme Q(10) (CoQ(10)), Niacin and Riboflavin are well-known potent antioxidants and protective agents against many diseases including cancer. In this context, this study was undertaken to find if co-administration of CoQ(10), Niacin and Riboflavin along with TAM could augment the antioxidant (AO) status in postmenopausal women with breast cancer., Methods: The vitamin supplementation with Tamoxifen was given for a period of 90 days. Blood samples were collected at the base line, 45th and 90th day during the course of treatment. Plasma lipids, lipid peroxides and various circulating enzymatic and non-enzymatic antioxidants were estimated in 78 untreated, sole TAM treated and combinatorial treated group along with 46 age- and sex-matched controls., Results: Enhanced oxidative stress as evidenced by increased lipids and lipid peroxides with decreased AO levels in untreated breast cancer patients was observed. Adjuvant TAM-treated group had a limited impact on the increased oxidative stress with decreased AO status. Severe hypertriglycerdemia was observed in TAM-treated group when compared to untreated and control subjects. Combinatorial therapy (CT) of CoQ(10), Niacin and Riboflavin along with TAM decreased the oxidative stress and increased the AO status., Conclusion: The antioxidant defense system is compromised in breast cancer patients. There is a shift in the oxidant / antioxidant balance in favor of lipid peroxidation (LPO), which could lead to tumour promotion observed in the disease. CT of CoQ(10), Niacin and Riboflavin along with TAM significantly increased the AO status, while decreasing lipid and lipid peroxides. The results suggest the necessity of therapeutic co-administration of antioxidants along with conventional drug to such patients. However, due to limited number of cases included in this study, more studies may be required to substantiate the results and arrive at a definitive conclusion, in terms of safety and efficacy of adding an AO therapy in treatment of breast cancer.

Tumour angiogenesis is a complex mechanism consisting of multi-step events including secretion or activation of angiogenic factors by tumour cells, activation of proteolytic enzymes, proliferation, migration and differentiation of endothelial cells. Both primary and metastatic tumours in the breast are dependent on angiogenesis. In the present study, 84 breast cancer patients were randomized to receive a daily supplement of CoQ(10) 100 mg, riboflavin 10 mg and niacin 50 mg (CoRN), one dosage per day along with tamoxifen (TAM) 10 mg twice a day. Serum pro-angiogenic levels were elevated in untreated breast cancer patients (Group II) and their levels were found to be reduced in breast cancer patients undergoing TAM therapy for more than 1 year (Group III). When these group III breast cancer patients were supplemented with CoRN for 45 days (Group IV) and 90 days (Group V) along with TAM, a further significant reduction in pro-angiogenic marker levels were observed. Supplementing CoRN to breast cancer patients has found to decrease the levels of pro-angiogenic factors and increase the levels of anti-angiogenic factors. A reduction in pro-angiogenic marker levels attributes to reduction in tumour burden and may suggest good prognosis and efficacy of the treatment, and might even offer protection from cancer metastases and recurrence.

Background: Src kinase plays a critical role in bone metabolism, particularly in osteoclasts. However, the ability of Src kinase to modulate the activity of other bone cells is less well understood. In this work, we examined the expression and activity of Src and low molecular weight protein tyrosine phosphatase (LMWPTP) during osteoblast differentiation and assessed the modulation of Src kinase by LMWPTP., Methods: Differentiation of MC3T3-E1 pre-osteoblasts was induced by incubation with ascorbic acid and beta-glycerophosphate for up to 28 days. Src phosphorylation and LMWPTP expression were analyzed by immunoblotting. Src dephosphorylation in vitro was assessed by incubating immunoprecipitated Src with LMWPTP followed by assay of the residual Src activity using Sam68 as substrate. The importance of LMWPTP in Src dephosphorylation was confirmed by silencing pre-osteoblasts with siRNA-LMWPTP and then assessing Src phosphorylation., Results: Pre-osteoblast differentiation was accompanied by a decrease in phosphorylation of the activator site of Src and an increase in phosphorylation of the inhibitory site. The expression of total Src was unaltered, indicating that post-translational modifications play a pivotal role in Src function. LMWPTP expression was higher in periods when the activator site of Src was dephosphorylated. LMWPTP dephosphorylated pY(527)-Src and pY(416)-Src in vitro, with greater specificity for pY(527)Src. Activation of LMWPTP produced strong activation of Src mediated by fast dephosphorylation of pY(527)-Src, followed by slower deactivation of this kinase via dephosphorylation of pY(416)Src., Conclusion: These results provide new insight into the mechanisms governing the dynamics of Src activity during osteoblast differentiation. A fuller understanding of these mechanisms will improve our knowledge of bone metabolism and of the regulation of Src in other types of cells., (Copyright 2008 S. Karger AG, Basel.)

Background: Both the role of inducible nitric oxide synthase (iNOS) in the development of inflammatory bowel disease (IBD) as well as the molecular details governing its mucosal induction remain unclear., Methods: In the present study we evaluated the role of the residing intestinal microflora in the induction of epithelial iNOS upon transfer of CD45RB(high) CD4(+) T cells to SCID mice. CB-17 SCID mice were reared with conventional flora (CNV) or germfree CB-17 SCID mice were monoassociated with Helicobacter muridarum, act A(-) mutant Listeria monocytogenes, segmented filamentous bacteria (SFB), or Ochrobactrum anthropi., Results: Within 2 weeks CNV SCID mice injected with CD45RB(high) CD4(+) T cells showed a focal, epithelial iNOS expression on the apical site of villi that preceded the infiltration of CD4(+) T cells and cytokine production followed by extension of this expression to the entire surface along the whole crypt axis as the colitis progressed. SCID mice monoassociated with H. muridarum developed a severe colitis and showed high epithelial iNOS expression. CNV-SCID mice without T cells and SCID mice monoassociated with SFB did not show any iNOS expression, whereas SCID mice monoassociated with act A(-) mutant L. monocytogenes and O. anthropi showed some scattered epithelial iNOS staining on the apical site of a few villi, but none of these mice developed colitis., Conclusions: These findings demonstrate that the expression of epithelial iNOS is highly bacterium-specific and correlates with the severity of disease, suggesting an important role for this enzyme in the development of IBD.

Objectives: Tamoxifen (TAM), a non-steroidal anti-estrogen that is widely used in adjuvant therapy for all stages of breast carcinomas and in chemoprevention of high-risk group. The hepatic estrogenic effect of TAM induces hypertriglyceridemia by reduced activity of lipolytic enzymes (LPL) on triglycerides. Coenzyme Q10 (Co Q10), riboflavin and niacin are proved to be potent antioxidant and protective agents against many diseases including cancer and cardiovascular diseases (CVD). In this context, the objective of the study is to find the effect of the combined modality of Co Q10 (100 mg), riboflavin (10 mg) and niacin (50 mg) with TAM (10 mg twice a day) on serum lipids and lipoprotein levels in postmenopausal women with breast cancer., Design and Methods: The vitamin supplementation with tamoxifen was given for a period of 90 days. Blood samples were collected at the base line, 45th and 90th day during the course of treatment. Plasma total cholesterol (TC), free cholesterol (FC), ester cholesterol (EC), phospholipids (PL), triglycerides (TGL), free fatty acids (FFA), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C) and very low density cholesterol (VLDL-C) were estimated in 78 untreated, only TAM-treated and combinatorialy treated group along with 46 age- and sex-matched controls., Results: Serum TGL and VLDL-C (p<0.001) were found to be significantly elevated and LDL-C (p<0.01), significantly reduced among TAM-treated patients as compared to the untreated breast cancer subjects. All the lipids and lipoprotein levels were found to be significantly altered in the untreated breast cancer patients when compared to their normal counterparts. All the lipid and lipoprotein abnormalities were reverted back to near normal levels on 90 days of treatment on combinatorial therapy., Conclusion: The study figures the altered lipid and lipoprotein levels in the untreated and TAM-treated breast cancer patients. On combination therapy with Co Q10, riboflavin and niacin, it counteracts the tamoxifen-induced hyperlipidemia to normal levels.

The prognostic significance of supplementing co-enzyme Q(10) (CoQ(10)), riboflavin and niacin (CoRN) along with tamoxifen to breast cancer patients was evaluated by measuring the serum cytokine levels of interleukin (IL)-1beta, IL-6, IL-8, tumour necrosis factor alpha (TNF-alpha) and vascular endothelial growth factor. In the present study, 84 breast cancer patients were randomized to receive a daily supplement of CoQ(10) 100 mg, riboflavin 10 mg and niacin 50 mg, one dosage per day along with tamoxifen 10 mg twice a day. Serum cytokine levels were elevated in untreated breast cancer patients (Group II) and significantly reduced after tamoxifen therapy for more than 1 year (Group III). When group III breast cancer patients were supplemented with CoRN for 45 days (Group IV) and 90 days (Group V) along with tamoxifen, a significant reduction in cytokine levels were observed (P < 0.05). Such a decrease in serum cytokine levels after CoRN supplementation in breast cancer patients may suggest good prognosis and efficacy of the treatment, and might even offer protection from metastases and recurrence of cancer.

In breast cancer patients, it is not the primary tumour, but its metastases at distant sites that are the main cause of death. Circulating breast cancer tumour markers such as carcinoembryonic antigen (CEA) and carbohydrate antigen 15-3 (CA 15-3) are reliable indicators of impending relapse, in which an increasing tumour marker level is associated with a very likelihood of developing recurrence. In the present study, 84 breast cancer patients were randomized to receive a daily supplement of 100 mg coenzyme Q10 (CoQ10), 10 mg riboflavin and 50 mg niacin (CoRN) one dosage per day along with 10 mg tamoxifen (TAM) twice a day. Serum CEA and CA 15-3 levels were elevated in untreated breast cancer patients (group II) and their tumour marker levels significantly reduced upon tamoxifen therapy for more than 1 year (group III). Group III patients supplemented with CoRN for 45 d (group IV) and 90 d (group V) along with tamoxifen significantly reduced CEA and CA 15-3 levels. This study suggests supplementing CoRN to breast cancer patients along with tamoxifen reduces the serum tumour marker level and thereby reduce the risk of cancer recurrence and metastases.

Diabetes mellitus of both type I (insulin-dependent) and type II (noninsulin-dependent) has adverse effects on male sexual and reproductive functions in adolescent boys and men, which include impairment of spermatogenesis, reduced sperm count, serum testosterone and seminal fluid volume, impotency, and loss of libido. Streptozotocin (STZ)-induced diabetes in rats provides a relevant model to study reproductive dysfunction under diabetic conditions, as they exhibit a number of deficits in reproductive function that resemble those seen in human diabetics. Therefore, the present investigation is aimed to understand the effects of STZ diabetes on the structure and development of ventral prostate during the critical period of sexual maturation in rats. Prepubertal (40-days-old) male Wistar rats were made diabetic by single injection of STZ (120 mg/kg body weight, intraperitoneally). Induction of diabetes was confirmed by serum insulin titer, hyperglycemia, and polyuria. To another set of STZ-diabetic rats, after 3 days of diabetes induction, insulin was replaced at a dose of 3 U/100 g body weight, subcutaneously in two equally divided doses at 8:00 AM and 6:00 PM. Diabetes caused regression of prostate, leading to a decrease in the absolute weight. Histologically, glandular epithelium has undergone shrinkage with transformation of acinar cells into low cuboidal type with less prominent secretory granules and blebs. Nevertheless, the secretory activity was not totally abolished. Interstitial space was increased due to shrinkage of glandular epithelium. Histomorphometric studies on the tubular diameter, volume and surface density of acinar epithelium, lumen, and stroma also support regressive changes in prostate. Insulin replacement prevented the detrimental effects of diabetes partially. These findings implicate the adverse effects of STZ diabetes on the differentiation of ventral prostate during sexual maturation., (Copyright (c) 2005 Wiley-Liss, Inc.)

Polychlorinated biphenyls (PCBs) are global pollutants of major concern to human and animal reproductive health. The present study has examined the impact of Aroclor 1254 exposure on oxidative stress and testicular Leydig cell function. Adult albino male rats of the Wistar strain were dosed for 30 days with daily intraperitoneal injections of 2 mg/kg Aroclor 1254 or vehicle (corn oil). One day after the last treatment, animals were euthanized and blood collected for the assay of serum testosterone and estradiol. Testes were removed and Leydig cells were isolated for the assay of luteinizing hormone (LH) receptors, steroidogenic enzymes cytochrome P450 side chain cleavage enzyme (P450 scc), 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and 17beta-hydroxysteroid dehydrogenase (17beta-HSD). Cellular antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), glutathione reductase (GR), and glutathione-S-transferase (GST) were also assayed. Lipid peroxidation (LPO) and reactive oxygen species (ROS) were quantified. Results showed that Aroclor 1254 exposure lowered serum testosterone and estradiol levels. Leydig cell LH receptor density, activities of the steroidogenic enzymes P450 scc, 3beta-HSD, 17beta-HSD, antioxidant enzymes SOD, CAT, GPX, GR, and GST were significantly diminished whereas, LPO and ROS significantly elevated. Taken together, these results suggest that inefficient LH receptors, steroidogenic enzymes and antioxidant enzymes are possible mechanisms by which Aroclor 1254 treatment disrupts Leydig cell steroidogenesis.

Hexavalent chromium detection in the presence of a high load of colourants without any false positive and in-procedure oxidation of Cr(III) is an important area of study. Colourants are a class of interfering substances in many spectroscopic analyses and chromatographic separations and detection. A purification method using an on-line dialysis technique for ion chromatography (IC) has been developed to remove water-soluble anionic dyes and particulate colourants and other substances to facilitate Cr(VI) quantification and the method is discussed. The dialysis was optimized with Cr(VI) standard solutions for quantification. The efficacy of the procedure for the removal of anionic dyes and detection of Cr(VI) was checked with a Cr(VI) spiked synthetic preparation containing a water-soluble dye and trivalent chromium. Soluble Cr(VI) extracted with organic dyes from environmental samples was analyzed. The method has a detection limit of 5 microg/l, recovery rate of 100% and analysis time less than 20 min.

The present study was designed to identify the effects of metyrapone-induced corticosterone deficiency on Leydig cell steroidogenesis in adult male rats. Adult Wistar rats (200-250 g body weight) were treated with metyrapone, an inhibitor of corticosterone synthesis (10 mg/100 g body weight, s.c., twice daily) for 10 days. Experimental animals were killed along with controls, blood was collected, and sera separated for testosterone and estradiol assays. Testes were removed and Leydig cells were isolated, purified and used for estimating the specific activity of 17beta-hydroxysteroid dehydrogenase (17beta-HSD) and 14C-glucose oxidation. Serum testosterone (p < 0.05), Leydig cellular 14C-glucose oxidation (p < 0.001) and the specific activity of 17beta-HSD (p < 0.01) were significantly decreased in metyrapone treated rats. However, serum estradiol was not markedly altered compared to control. In addition to this, a set of in vitro experiments were also performed to identify the effects of metyrapone-induced corticosterone deficiency on hCG and prolactin-induced Leydig cell testosterone production. Metyrapone treatment significantly (p < 0.05) decreased the Leydig cellular basal as well as hCG and its combination with prolactin stimulated testosterone production in vitro. It is concluded from the present study that the inhibitory effects of metyrapone-induced corticosterone deficiency on Leydig cell steroidogenesis are mediated through impaired glucose oxidation and 17beta-HSD activity. In vitro studies showed that corticosterone deficiency impairs not only hCG action but also the potentiating effect of prolactin on Leydig cell steroidogenesis.