Sialylation of beta 1 Integrins blocks cell adhesion to galectin-3 and protects cells against galectin-3-induced apoptosis

In previous studies, we determined that beta 1 integrins from human colon tumors have elevated levels of alpha 2-6 sialylation, a modification added by beta-galactosamide alpha-2,6-sialyltranferase I (ST6Gal-I). Intriguingly, the beta 1 integrin is thought to be a ligand for galectin-3 (gal-3), a tumor-associated lectin. The effects of gal-3 are complex; intracellular forms typically protect cells against apoptosis through carbohydrate-independent mechanisms, whereas secreted forms bind to cell surface oligosaccharides and induce apoptosis. In the current study, we tested whether alpha 2-6 sialylation of the beta 1 integrin modulates binding to extracellular gal-3. Herein we report that SW48 colonocytes lacking alpha 2-6 sialylation exhibit beta 1 integrin-dependent binding to gal-3-coated tissue culture plates; however, binding is attenuated upon forced expression of ST6Gal-I. Removal of alpha 2-6 sialic acids from ST6Gal-I expressors by neuraminidase treatment restores gal-3 binding. Additionally, using a blot overlay approach, we determined that gal-3 binds directly and preferentially to unsialylated, as compared with alpha 2-6-sialylated, beta 1 integrins. To understand the physiologic consequences of gal-3 binding, cells were treated with gal-3 and monitored for apoptosis. Galectin-3 was found to induce apoptosis in parental SW48 colonocytes ( unsialylated), whereas ST6Gal-I expressors were protected. Importantly, gal-3-induced apoptosis was inhibited by function blocking antibodies against the beta 1 subunit, suggesting that beta 1 integrins are critical transducers of gal-3-mediated effects on cell survival. Collectively, our results suggest that the coordinate up-regulation of gal-3 and ST6Gal-I, a feature that is characteristic of colon carcinoma, may confer tumor cells with a selective advantage by providing a mechanism for blockade of the pro-apoptotic effects of secreted gal-3.

Alterations in Cytokine Profile and Dendritic Cells Subsets in Peripheral Blood of Rheumatoid Arthritis Patients before and after Biologic Therapy

Rheumatoid arthritis (RA) is an autoimmune disorder characterized by chronic joint inflammation and continuous immune cell infiltration in the synovium. These changes are linked to inflammatory cytokine release, leading to eventual destruction of cartilage and bone. During the last decade new therapeutic modalities have improved the prognosis, with the introduction of novel biological response modifiers including anti-TNF alpha CTLA4Ig and, more recently, anti-IL6. In the present study we looked at the immunological effects of these three forms of therapy. Serum, obtained from patients with RA was analyzed for TNF alpha, IL6, IL10, IFN gamma, and VEGF, and in parallel, circulating plasmacytoid and myeloid dendritic cells (DC) were enumerated before and after three infusions of the respective biological treatments. After treatment with anti-IL6, we found a significant reduction of IL6 and TNF alpha levels and the percentage of both DC subsets decreased. Although the results did not reach statistical significance for anti-TNF alpha treatment, similar trends were observed. Meanwhile, CTLA4Ig therapy led to the reduction IFN gamma levels only. None of the treatments modified significantly VEGF or IL10 levels. These findings may explain why patients with RA improve more rapidly on IL-6 therapy than with the other two modalities.

Cholinergic Hyperresponsiveness of Peripheral Lung Parenchyma in Chronic Obstructive Pulmonary Disease

Background: Up to 60% of chronic obstructive pulmonary disease ( COPD) patients can present airway hyperresponsiveness. However, it is not known whether the peripheral lung tissue also shows an exaggerated response to agonists in COPD. Objectives: To investigate the in vitro mechanical behavior and the structural and inflammatory changes of peripheral lung tissue in COPD patients and compare to nonsmoking controls. Methods: We measured resistance and elastance at baseline and after acetylcholine (ACh) challenge of lung strips obtained from 10 COPD patients and 10 control subjects. We also assessed the alveolar tissue density of neutrophils, eosinophils, macrophages, mast cells and CD8+ and CD4+ cells, as well as the content of alpha-smooth muscle actin-positive cells and elastic and collagen fibers. We further investigated whether changes in in vitro parenchymal mechanics correlated to structural and inflammatory parameters and to in vivo pulmonary function. Results: Values of resistance after ACh treatment and the percent increase in tissue resistance (%R) were higher in the COPD group (p <= 0.03). There was a higher density of macrophages and CD8+ cells (p < 0.05) and a lower elastic content (p = 0.003) in the COPD group. We observed a positive correlation between %R and eosinophil and CD8+ cell density (r = 0.608, p = 0.002, and r = 0.581, p = 0.001, respectively) and a negative correlation between %R and the ratio of forced expiratory volume in 1 s to forced vital capacity (r = -0.451, p < 0.05). Conclusions: The cholinergic responsiveness of parenchymal lung strips is increased in COPD patients and seems to be related to alveolar tissue eosinophilic and CD8 lymphocytic inflammation and to the degree of airway obstruction on the pulmonary function test. Copyright (C) 2011 S. Karger AG, Basel

GB virus type C infection modulates T-cell activation independently of HIV-1 viral load

Background: Many clinical studies have suggested a beneficial effect of GB virus type C (GBV-C) on the course of HIV-1 infection, but the mechanisms involved in such amelioration are not clear. As recent evidence has implicated cellular activation in HIV-1 pathogenesis, we investigated the effect of GBV-C viremia on T-cell activation in early HIV-1 infection. Methods: Forty-eight recently infected HIV-1 patients (23 GBV-C viremic) were evaluated for T-cell counts, expanded immunophenotyping GBV-C RNA detection, and HIV-1 viral load. Nonparametric univariate and multivariate analyses were carried out to identify variables associated with cellular activation, including GBV-C status, HIV-1 viral load, T lymphocyte counts, and CD38 and chemokine (C-C motif) receptor 5 (CCR5) surface expression. Finding: We not only confirmed the positive correlation between HIV-1 viral load and the percentage of T cells positive for CD38(+)CD8(+) but also observed that GBV-C viremic patients had a lower percentage of T cells positive for CD38(+)CD4(+), CD38(+)CD8(+), CCR5(+)CD4(+), and CCR5(+)CD8(+) compared with HIV-1-infected patients who were not GBV-C viremic. In regression models, GBV-C RNA(+) status was associated with a reduction in the CD38 on CD4(+) or CD8(+) T cells and CCR5(+) on CD8(+) T cells, independent of the HIV-1 viral load or CD4(+) and CD8(+) T-cell counts. These results were also supported by the lower expression of CD69 and CD25 in GBV-C viremic patients. Interpretation: The association between GBV-C replication and lower T-cell activation may be a key mechanism involved in the protection conferred by this virus against HIV-1 disease progression to immunodeficiency in HIV-1-infected patients. (C) 2009 Wolters Kluwer Health | Lippincott Williams & Wilkins

Equal Numbers of Neuronal and Nonneuronal Cells Make the Human Brain an Isometrically Scaled-Up Primate Brain

The human brain is often considered to be the most cognitively capable among mammalian brains and to be much larger than expected for a mammal of our body size. Although the number of neurons is generally assumed to be a determinant of computational power, and despite the widespread quotes that the human brain contains 100 billion neurons and ten times more glial cells, the absolute number of neurons and glial cells in the human brain remains unknown. Here we determine these numbers by using the isotropic fractionator and compare them with the expected values for a human-sized primate. We find that the adult male human brain contains on average 86.1 +/- 8.1 billion NeuN-positive cells (""neurons"") and 84.6 +/- 9.8 billion NeuN-negative (""nonneuronal"") cells. With only 19% of all neurons located in the cerebral cortex, greater cortical size (representing 82% of total brain mass) in humans compared with other primates does not reflect an increased relative number of cortical neurons. The ratios between glial cells and neurons in the human brain structures are similar to those found in other primates, and their numbers of cells match those expected for a primate of human proportions. These findings challenge the common view that humans stand out from other primates in their brain composition and indicate that, with regard to numbers of neuronal and nonneuronal cells, the human brain is an isometrically scaled-up primate brain. J. Comp. Neurol. 513:532-541, 2009. (c) 2009 Wiley-Liss, Inc.

Integration pattern of HIV-1 based lentiviral vector carrying recombinant coagulation factor VIII in Sk-Hep and 293T cells

293T and Sk-Hep-1 cells were transduced with a replication-defective self-inactivating HIV-1 derived vector carrying FVIII cDNA. The genomic DNA was sequenced to reveal LTR/human genome junctions and integration sites. One hundred and thirty-two sequences matched human sequences, with an identity of at least 98%. The integration sites in 293T-FVIIIDB and in Sk-Hep-FVIIIDB cells were preferentially located in gene regions. The integrations in both cell lines were distant from the CpG islands and from the transcription start sites. A comparison between the two cell lines showed that the lentiviral-transduced DNA had the same preferred regions in the two different cell lines.

Immunolocalization of nitric oxide synthase isoforms in human archival and rat tissues, and cultured cells

Nitric oxide (NO) exerts important physiological and pathological roles in humans. The study of NO requires the immunolocalization of its synthesizing enzymes, neuronal, endothelial and inducible NO synthases (NOS). NOS are labile to formalin-fixation and paraffin-embedding, which are used to prepare human archival tissues. This lability has made NOS immunohistochemical studies difficult, and a detailed protocol is not yet available. We describe here a protocol for the immunolocalization of NOS isoforms in human archival cerebellum and non-nervous tissues, and in rat tissues and cultured cells. Neuronal NOS antigenicity in human archival and rat nervous tissue sections was microwave-retrieved in 50 mM Tris-HCl buffer, pH 9.5, for 20 min at 900W. Neuronal NOS was expressed in stellate, basket, Purkinje and granule cells in human and rat cerebellum. Archival and frozen human cerebellar sections showed the same neuronal NOS staining pattern. Archival cerebellar sections not subjected to antigen retrieval stained weakly. Antigenicity of inducible NOS in human lung was best retrieved in 10 mM sodium citrate buffer, pH 6.0, for 15 min at 900W. Inflammatory cells in a human lung tuberculoma were strongly stained by anti-inducible NOS antibody. Anti-endothelial NOS strongly stained kidney glomeruli. Cultured PC12 cells were strongly stained by anti-neuronal NOS without antigen retrieving. The present immunohistochemistry protocol is easy to perform, timeless, and suitable for the localization of NOS isoforms in nervous and non-nervous tissues, in human archival and rat tissues. It has been extensively used in our laboratory, and is also appropriate for other antigens. (C) 2011 Elsevier B.V. All rights reserved.

Coordinated regulation of follicle development by germ and somatic cells

The continuum of folliculogenesis begins in the fetal ovary with the differentiation of the oogonia and their isolation within the primordial follicles. Primordial follicle activation is an enigmatic process, whereby some follicles enter the growing pool to become primary follicles, thereby embarking on an irreversible progression towards ovulation or atresia. This process is under the coordinated regulation of factors from the oocyte itself, as well as from the somatic cells of the ovary, in particular the theca and granulosa cells, which are structural components of the follicle. These two influences provide the principal stimuli for the growth of the follicle to the late preantral or early antral stage of development. The endocrine effects of the gonadotrophins FSH and LH are essential to the continued progression of the follicle and most atresia can be attributed to the failure to receive or process the gonadotrophin signals. The peri-ovulatory state has received intensive investigation recently, demonstrating a coordinated role for gonadotrophins, steroids, epidermal growth factor family proteins and prostaglandins. Thus, a complex programme of coordinated interaction of governing elements from both germ and somatic cell sources is required for successful follicle development.

Oestrogen and Progesterone Receptor Gene Expression in Canine Oocytes and Cumulus Cells Throughout the Oestrous Cycle

The aim of this research was to analyze oestrogen receptor-alpha (ER alpha), ER beta and progesterone receptor (PR) gene expression in the canine oocyte and cumulus cells throughout the oestrous cycle. Ovaries from 38 bitches were recovered after ovariohysterectomy and sliced. The phase of the oestrous cycle was determined by vaginal cytology, vaginoscopy and serum hormonal measurements. Oocytes were mechanically denuded by repeated pipetting. For each phase of the cycle, a sample was composed by a pool of 50 oocytes (sample number: prooestrus = 3, oestrus = 8, dioestrus = 5 and anoestrus = 5) or a pool of cumulus cells (prooestrus = 4, oestrus = 7, dioestrus = 4 and anoestrus = 6). Oocyte and cumulus cells` total RNA was isolated and reverse transcription was conducted to perform real-time PCR. Oestrogen receptor-alpha was expressed throughout the cycle in the oocyte (33.33%, 25.0%, 20.0% and 60.0% for prooestrus, oestrus, dioestrus and anoestrus, respectively) and cumulus cells (50.0%, 47.14%, 25.0% and 66.67% for prooestrus, oestrus, dioestrus and anoestrus, respectively). In the oocyte, the ER beta was also expressed in all phases of the cycle (33.33%, 50.0%, 20.0% and 60.0% for prooestrus, oestrus, dioestrus and anoestrus, respectively), whereas in cumulus cells, ER beta was only expressed during prooestrus (50%) and oestrus (14.29%). Interestingly, while the oocyte PR was not detected in any phase of the cycle, this receptor was expressed during prooestrus (50%), oestrus (42.86%) and anoestrus (16.67%) in cumulus cells. In conclusion, canine oocytes express ER alpha and ER beta throughout the oestrous cycle, however, there is a lack of PR expression in all these phases. Moreover, in cumulus cells, only ER alpha was expressed throughout the oestrous cycle.

MMP-9 and CD68(+) cells are required for tissue remodeling in response to natural hydroxyapatite

Large bone defects represent major clinical problems in the practice of reconstructive orthopedic and craniofacial surgery. The aim of this study was to examine, through immunohistochemistry approach, the involvement of MMP-9 and CD68(+) cells during tissue remodeling in response to natural hydroxyapatite (HA) implanted in rat subcutaneous tissue. Before experimentation, forty animals were randomly distributed into two experimental groups: Group-I (Gen-Ox (TM) micro-granules) and Group-II (Gen-Ox (TM) macro-granules). Afterwards, the biopsies were collected after 10, 20, 30, and 60 days post-implantation. Our results showed that at 10 days, a low-renewal foreign body type granuloma formation was observed in most of the cases. Macrophage- and fibroblast-like cells were the predominant type of cells positively stained for MMP-9 in both groups. Once macrophage-like cells seemed to be the major source of MMP9, antibody against pan-CD68 epitope was used to correlate these findings. In agreement, MMP-9 and CD68(+) cells were distributed at the periphery and the central region of the granuloma in all experimental periods, however no staining was observed in cell contacting to material. Besides macrophages, the lysosomal glycoprotein epitope recognized by CD68 antibodies can be expressed by mast cell granules and sometimes by fibroblasts. Taken together, our results suggest that xenogenic HA promotes extracellular matrix remodeling through induction of MMP-9 activity and presence of CD68(+) cells.

The Impact of Vitamin A Supplementation on the Immune System of Vitamin A-deficient Children

Background & Aims: To investigate the effect of vitamin A supplementation on parameters of the immune system of vitamin A-deficient children. Methods: The study was carried out in four phases: 1) determination of serum retinol in 631 children from 36 to 83 months of age; 2) assessment of immunological markers [immunoglobulins and complement fractions, immunophenotyping of T and B lymphocytes, and natural killer (NK) cells], blood count, and serum ferritin of 52 vitamin A-deficient children (serum retinol <0.70 mu mol/L); 3) supplementation of the 52 deficient children with 200,000 IU of vitamin A; 4) determination of serum retinol and the immunological parameters 2 months after vitamin A supplementation. Results: Before vitamin A supplementation, 24.0% of the children were anemic and 4.3 %had reduced ferritin concentrations. There was no significant difference between mean values of retinol according to the presence/absence of anemia. The mean values of the humoral and cellular immunological parameters did not show a statistically significant difference before and after supplementation with vitamin A. Children with concomitant hypovitaminosis A and anemia presented a significant increase in absolute CD4 and CD8 T-cell counts after vitamin A supplementation (p < 0.05). Conclusion: Vitamin A had an effect on the recruitment of T and B lymphocytes to the circulation of children with hypovitaminosis A and anemia.

Separation and viability of gill and hepatopancreatic cells of a mangrove crab Ucides cordatus

The gills contain essential cells for respiration and osmoregulation, whereas the hepatopancreas is the site of digestion, absorption, and nutrients storage. The aim of this work was to separate and characterize gill and hepatopancreatic cells of the mangrove crab, Ucides cordatus. For gills, the methodology consisted of an enzymatic cellular dissociation using Trypsin at 0.5%, observation of cellular viability with Tripan Blue, and separation of cells using discontinuous sucrose gradient at concentrations of 10%, 20%, 30%, and 40%. The hepatopancreatic cells were dissociated by magnetic stirring, with posterior separation by sucrose gradient at the same concentrations above. For gills, a high cellular viability was observed (92.5 +/- 2.1%), with hemocyte cells in 10% sucrose layer (57.99 +/- 0.17%, *P < 0.05), principal cells in the 20% sucrose layer (57.33 +/- 0.18, *P < 0.05), and thick cells and pillar cells in the 30% and 40% sucrose layers, respectively (39.54 +/- 0.05%, *P < 0.05; and 41.81 +/- 0.04%, *P < 0.05). The hepatopancreatic cells also showed good viability (79.22 +/- 0.02%), with the observation of embryonic (E) cells in the 10% sucrose layer (67.87 +/- 0.06%, **P < 0.001), resorptive (R) and fibrillar (F) cells in the 20% and 30% sucrose layers (44.71 +/- 0.06%, **P < 0.001, and 43.25 +/- 0.01%, *P < 0.05; respectively), and blister (B) cells in the 40% sucrose layer (63.09 +/- 0.03%, **P < 0.001). The results are a starting point for in vitro studies of heavy metal transport in isolated cells of the mangrove crab U. cordatus, subjected to contamination by metals in the mangrove habitat where they are found.

Glucose metabolism by lymphocytes, macrophages, and tumor cells from Walker 256 tumor-bearing rats supplemented with fish oil for one generation

Here we investigated the effect of lifelong supplementation of the diet with coconut fat (CO, rich in saturated fatty acids) or fish oil (170, rich in n-3 polyunsaturated fatty acids) on tumor growth and lactate production from glucose in Walker 256 tumor cells, peritoneal macrophages, spleen, and gut-associated lymphocytes. Female Wistar rats were supplemented with CO or FO prior to mating and then throughout pregnancy and gestation and then the male offspring were supplemented from weaning until 90 days of age. Then they were inoculated subcutaneously with Walker 256 tumor cells. Tumor weight at 14 days in control rats (those fed standard chow) and CO supplemented was approximately 30 g. Supplementation of the diet with FO significantly reduced tumor growth by 76%. Lactate production (nmol h(-1) mg(-1) protein) from glucose by Walker 256 cells in the group fed regular chow (W) was 381.8 +/- 14.9. Supplementation with coconut fat (WCO) caused a significant reduction in lactate production by 1.6-fold and with fish oil (WFO) by 3.8-fold. Spleen lymphocytes obtained from W and WCO groups had markedly increased lactate production (553 +/- 70 and 635 +/- 150) when compared to non-tumor-bearing rats (similar to 260 +/- 30). FO supplementation reduced significantly the lactate production (297 +/- 50). Gut-associated lymphocytes obtained from W and WCO groups increased lactate production markedly (280 +/- 31 and 276 +/- 25) when compared to non-tumor-bearing rats (similar to 90 +/- 18). FO supplementation reduced significantly the lactate production (168 +/- 14). Lactate production by peritoneal macrophages was increased by tumor burden but there was no difference between the groups fed the various diets. Lifelong consumption of FO protects against tumor growth and modifies glucose metabolism in Walker tumor cells and lymphocytes but not in macrophages. Copyright (C) 2008 John Wiley & Sons, Ltd.

Effects of lipid-lowering drugs on reverse cholesterol transport gene expressions in peripheral blood mononuclear and HepG2 cells

Aims: The ATP-binding cassette transporters, ABCA1 and ABCG1, are LXR-target genes that play an important role in reverse cholesterol transport. We examined the effects of inhibitors of the cholesterol absorption (ezetimibe) and synthesis (statins) on expression of these transporters in HepG2 cells and peripheral blood mononuclear cells (PBMCs) of individuals with primary (and nonfamilial) hypercholesterolemia (HC). Materials & methods: A total of 48 HC individuals were treated with atorvastatin (10 mg/day/4 weeks) and 23 were treated with ezetimibe (10 mg/day/4 weeks), followed by simvastatin (10 mg/day/8 weeks) and simvastatin plus ezetimibe (10 mg of each/day/4 weeks). Gene expression was examined in statin- or ezetimibe-treated and control HepG2 cells as well as PBMCs using real-time PCR. Results: In PBMCs, statins and ezetimibe downregulated ABCA1 and ABCG1 mRNA expression but did not modulate NR1H2 (LxR-beta) and NR1H3 (LXR-alpha) levels. Positive correlations of ABCA1 with ABCG1 and of NR1H2 with NR1H3 expressions were found in all phases of the treatments. In HepG2 cells, ABCA1 mRNA levels remained unaltered while ABCG1 expression was increased by statin (1.0-10.0 mu M) or ezetimibe (5.0 mu M) treatments. Atorvastatin upregulated NR1H2 and NR1H3 only at 10.0 mu M, meanwhile ezetimibe (1.0-5.0 mu M) downregulated NR1H2 but did not change NR1H3 expression. Conclusion: Our findings reveal that lipid-lowering drugs downregulate ABCA1 and ABCG1 mRNA expression in PBMCs of HC individuals and exhibit differential effects on HepG2 cells. Moreover, they indicate that the ABCA1 and ABCG1 transcript levels were not correlated directly to LXR mRNA expression in both cell models treated with lipid-lowering drugs.

Endogenous Hepatocyte Growth Factor Attenuates Inflammatory Response in Glycerol-Induced Acute Kidney Injury

Background: Hepatocyte growth factor (HGF) is overexpressed after acute kidney injury (AKI). The aim of this study was to evaluate the role of endogenous HGF in the progression of the inflammatory response in glycerol-induced AKI (Gly-AKI) in rats. Methods: Renal and systemic HGF expressions were evaluated during the development of Gly-AKI. Subsequently, the blockade of endogenous HGF was analyzed in rats treated with anti-HGF antibody concomitant to glycerol injection. Apoptosis, cell infiltration and chemokine and cytokine profiles were investigated. Results: We detected an early peak of renal and plasma HGF protein expressions 3 h after glycerol injection. The pharmacological blockade of the endogenous HGF exacerbated the renal impairment, the tubular apoptosis, the renal expression of monocyte chemoattractant protein-1 and the macrophage, CD43+, CD4+ and CD8+ T lymphocytes renal infiltration. The analysis of mRNA expressions of Th1 (t-bet, TNF-alpha, IL-1 beta) and Th2 (gata-3, IL-4, IL-10) cytokines showed a Th1-polarized response in Gly-AKI rats that was aggravated with the anti-HGF treatment. Conclusion: Endogenous HGF attenuates the renal inflammatory response, leukocyte infiltration and Th1 polarization after glycerol injection. The control of cellular immune response may partly explain the protective effect of endogenous HGF in the development of Gly-AKI. Copyright (C) 2008 S. Karger AG, Basel