Immune regulatory effect of pHSP65 DNA therapy in pulmonary tuberculosis: Activation of CD8+ cells, interferon-γ recovery and reduction of lung injury

A DNA vaccine based on the heat-shock protein 65 Mycobacterium leprae gene (pHSP65) presented a prophylactic and therapeutic effect in an experimental model of tuberculosis. In this paper, we addressed the question of which protective mechanisms are activated in Mycobacterium tuberculosis-infected mice after immune therapy with pHSP65. We evaluated activation of the cellular immune response in the lungs of infected mice 30 days after infection (initiation of immune therapy) and in those of uninfected mice. After 70 days (end of immune therapy), the immune responses of infected untreated mice, infected pHSP65-treated mice and infected pCDNA3-treated mice were also evaluated. Our results show that the most significant effect of pHSP65 was the stimulation of CD8+ lung cell activation, interferon-γ recovery and reduction of lung injury. There was also partial restoration of the production of tumour necrosis factor-α. Treatment with pcDNA3 vector also induced an immune stimulatory effect. However, only infected pHSP65-treated mice were able to produce significant levels of interferon-γ and to restrict the growth of bacilli.

Stem cell factor estimula células da musculatura lisa da traqueia a produzir TGF-β, FGF-2 E CCL3/MIP-1α

Pós-graduação em Ciências Fisiológicas - FOA

Efeitos do hormônio do crescimento sobre vias de sinalização intracelular na miopatia associada à insuficiência cardíaca de ratos

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Mesenchymal stem cells engrafted in a fibrin scaffold stimulate Schwann cell reactivity and axonal regeneration following sciatic nerve tubulization

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effects of Yersinia enterocolitica O:3 derivatives on B lymphocyte activation in vivo

The potential sequelae of intestinal infection with Yersinia enterocolitica include reactive arthritis, erythema nodosum, Reiter's syndrome and other autoimmune diseases. The role of the immune response in the pathogenesis of these diseases has not been fully defined, but autoimmune manifestations may be a consequence of the increase in autoantibodies as a result of polyclonal B-cell activation induced by Yersinia. We investigated the effects of Y enterocolitica 0:3 derivatives on B lymphocyte activation in vivo. Groups of five specific pathogen free (SPF) Swiss mice were inoculated with bacterial cell extract, Yersinia outermembrane proteins (Yops) or lipopolysaccharide (LPS) obtained from Y enterocolitica 0:3 and their immunoglobulin-secreting spleen cells were detected by isotype-specific protein A plaque assay. The presence of specific anti-Yersinia antibodies and autoantibodies was determined in mouse sera by ELISA. In all experiments a marked increase in the number of secretory cells of different isotypes was observed as early as the third day after inoculation. IgG and IgM anti-Yersinia antibodies were detected in the sera of all inoculated mice, and autoantibodies against myosin in the sera of those inoculated with bacterial cell extract. The sera from animals stimulated with LPS reacted with myelin, actin and laminin, while the sera from mice inoculated with Yops reacted with myelin, thyroglobulin and cardiolipin. These results suggest that SPF Swiss mice inoculated with any one of the Y enterocolitica derivatives tested exhibited polyclonal activation of B lymphocytes as a result of stimulation by various bacterial components and not only LPS stimulation.

Anti-IL-2 Treatment Impairs the Expansion of T-reg Cell Population during Acute Malaria and Enhances the Th1 Cell Response at the Chronic Disease

Plasmodium chabaudi infection induces a rapid and intense splenic CD4(+) T cell response that contributes to both disease pathogenesis and the control of acute parasitemia. The subsequent development of clinical immunity to disease occurs concomitantly with the persistence of low levels of chronic parasitemia. The suppressive activity of regulatory T (T-reg) cells has been implicated in both development of clinical immunity and parasite persistence. To evaluate whether IL-2 is required to induce and to sustain the suppressive activity of T-reg cells in malaria, we examined in detail the effects of anti-IL-2 treatment with JES6-1 monoclonal antibody (mAb) on the splenic CD4(+) T cell response during acute and chronic P. chabaudi AS infection in C57BL/6 mice. JES6-1 treatment on days 0, 2 and 4 of infection partially inhibits the expansion of the CD4(+)CD25(+)Foxp3(+) cell population during acute malaria. Despite the concomitant secretion of IL-2 and expression of high affinity IL-2 receptor by large CD4(+) T cells, JES6-1 treatment does not impair effector CD4+ T cell activation and IFN-gamma production. However, at the chronic phase of the disease, an enhancement of cellular and humoral responses occurs in JES6-1-treated mice, with increased production of TNF-alpha and parasite-specific IgG2a antibodies. Furthermore, JES6-1 mAb completely blocked the in vitro proliferation of CD4(+) T cells from non-treated chronic mice, while it further increased the response of CD4(+) T cells from JES6-1-treated chronic mice. We conclude that JES6-1 treatment impairs the expansion of T-reg cell population during early P. chabaudi malaria and enhances the Th1 cell response in the late phase of the disease.

Liver accumulation of Plasmodium chabaudi-infected red blood cells and modulation of regulatory T Cell and dendritic cell responses

It is postulated that accumulation of malaria-infected Red Blood Cells (iRBCs) in the liver could be a parasitic escape mechanism against full destruction by the host immune system. Therefore, we evaluated the in vivo mechanism of this accumulation and its potential immunological consequences. A massive liver accumulation of P. c. chabaudi AS-iRBCs (PciRBCs) was observed by intravital microscopy along with an over expression of ICAM-1 on day 7 of the infection, as measured by qRT-PCR. Phenotypic changes were also observed in regulatory T cells (Tregs) and dendritic cells (DCs) that were isolated from infected livers, which indicate a functional role for Tregs in the regulation of the liver inflammatory immune response. In fact, the suppressive function of liver-Tregs was in vitro tested, which demonstrated the capacity of these cells to suppress naive T cell activation to the same extent as that observed for spleen-Tregs. On the other hand, it is already known that CD4+ T cells isolated from spleens of protozoan parasite-infected mice are refractory to proliferate in vivo. In our experiments, we observed a similar lack of in vitro proliferative capacity in liver CD4+ T cells that were isolated on day 7 of infection. It is also known that nitric oxide and IL-10 are partially involved in acute phase immunosuppression; we found high expression levels of IL-10 and iNOS mRNA in day 7-infected livers, which indicates a possible role for these molecules in the observed immune suppression. Taken together, these results indicate that malaria parasite accumulation within the liver could be an escape mechanism to avoid sterile immunity sponsored by a tolerogenic environment.

Study of apoptotic deletion mediated by Bifidobacterium longum with construction of recombinant strains for Serpin encoding gene and phenotypes comparison in a pig cell model

The first part of the research project of the Co-Advisorship Ph.D Thesis was aimed to select the best Bifidobacterium longum strains suitable to set the basis of our study. We were looking for strains with the abilities to colonize the intestinal mucosa and with good adhesion capacities, so that we can test these strains to investigate their ability to induce apoptosis in “damaged” intestinal cells. Adhesion and apoptosis are the two process that we want to study to better understand the role of an adhesion protein that we have previously identified and that have top scores homologies with the recent serpin encoding gene identified in B. longum by Nestlè researchers. Bifidobacterium longum is a probiotic, known for its beneficial effects to the human gut and even for its immunomodulatory and antitumor activities. Recently, many studies have stressed out the intimate relation between probiotic bacteria and the GIT mucosa and their influence on human cellular homeostasis. We focused on the apoptotic deletion of cancer cells induced by B. longum. This has been valued in vitro, performing the incubation of three B.longum strains with enterocyte-like Caco- 2 cells, to evidence DNA fragmentation, a cornerstone of apoptosis. The three strains tested were defined for their adhesion properties using adhesion and autoaggregation assays. These features are considered necessary to select a probiotic strain. The three strains named B12, B18 and B2990 resulted respectively: “strong adherent”, “adherent” and “non adherent”. Then, bacteria were incubated with Caco-2 cells to investigate apoptotic deletion. Cocultures of Caco-2 cells with B. longum resulted positive in DNA fragmentation test, only when adherent strains were used (B12 and B18). These results indicate that the interaction with adherent B. longum can induce apoptotic deletion of Caco-2 cells, suggesting a role in cellular homeostasis of the gastrointestinal tract and in restoring the ecology of damaged colon tissues. These results were used to keep on researching and the strains tested were used as recipient of recombinant techniques aimed to originate new B.longum strains with enhanced capacity of apoptotic induction in “damaged” intestinal cells. To achieve this new goal it was decided to clone the serpin encoding gene of B. longum, so that we can understand its role in adhesion and apoptosis induction. Bifidobacterium longum has immunostimulant activity that in vitro can lead to apoptotic response of Caco-2 cell line. It secretes a hypothetical eukaryotic type serpin protein, which could be involved in this kind of deletion of damaged cells. We had previously characterised a protein that has homologies with the hypothetical serpin of B. longum (DD087853). In order to create Bifidobacterium serpin transformants, a B. longum cosmid library was screened with a PCR protocol using specific primers for serpin gene. After fragment extraction, the insert named S1 was sub-cloned into pRM2, an Escherichia coli - Bifidobacterium shuttle vector, to construct pRM3. Several protocols for B. longum transformation were performed and the best efficiency was obtained using MRS medium and raffinose. Finally bacterial cell supernatants were tested in a dotblot assay to detect antigens presence against anti-antitrypsin polyclonal antibody. The best signal was produced by one starin that has been renamed B. longum BLKS 7. Our research study was aimed to generate transformants able to over express serpin encoding gene, so that we can have the tools for a further study on bacterial apoptotic induction of Caco-2 cell line. After that we have originated new trasformants the next step to do was to test transformants abilities when exposed to an intestinal cell model. In fact, this part of the project was achieved in the Department of Biochemistry of the Medical Faculty of the University of Maribor, guest of the abroad supervisor of the Co-Advisorship Doctoral Thesis: Prof. Avrelija Cencic. In this study we examined the probiotic ability of some bacterial strains using intestinal cells from a 6 years old pig. The use of intestinal mammalian cells is essential to study this symbiosis and a functional cell model mimics a polarised epithelium in which enterocytes are separated by tight junctions. In this list of strains we have included the Bifidobacterium longum BKS7 transformant strain that we have previously originated; in order to compare its abilities. B. longum B12 wild type and B. longum BKS7 transformant and eight Lactobacillus strains of different sources were co-cultured with porcine small intestine epithelial cells (PSI C1) and porcine blood monocytes (PoM2) in Transwell filter inserts. The strains, including Lb. gasseri, Lb. fermentum, Lb. reuterii, Lb. plantarum and unidentified Lactobacillus from kenyan maasai milk and tanzanian coffee, were assayed for activation of cell lines, measuring nitric oxide by Griess reaction, H202 by tetramethylbenzidine reaction and O2 - by cytochrome C reduction. Cytotoxic effect by crystal violet staining and induction on metabolic activity by MTT cell proliferation assay were tested too. Transepithelial electrical resistance (TER) of polarised PSI C1 was measured during 48 hours co-culture. TER, used to observe epithelium permeability, decrease during pathogenesis and tissue becomes permeable to ion passive flow lowering epithelial barrier function. Probiotics can prevent or restore increased permeability. Lastly, dot-blot was achieved against Interleukin-6 of treated cells supernatants. The metabolic activity of PoM2 and PSI C1 increased slightly after co-culture not affecting mitochondrial functions. No strain was cytotoxic over PSI C1 and PoM2 and no cell activation was observed, as measured by the release of NO2, H202 and O2 - by PoM2 and PSI C1. During coculture TER of polarised PSI C1 was two-fold higher comparing with constant TER (~3000 ) of untreated cells. TER raise generated by bacteria maintains a low permeability of the epithelium. During treatment Interleukin-6 was detected in cell supernatants at several time points, confirming immunostimulant activity. All results were obtained using Lactobacillus paracasei Shirota e Carnobacterium divergens as controls. In conclusion we can state that both the list of putative probiotic bacteria and our new transformant strain of B. longum are not harmful when exposed to intestinal cells and could be selected as probiotics, because can strengthen epithelial barrier function and stimulate nonspecific immunity of intestinal cells on a pig cell model. Indeed, we have found out that none of the strains tested that have good adhesion abilities presents citotoxicity to the intestinal cells and that non of the strains tested can induce cell lines to produce high level of ROS, neither NO2. Moreover we have assayed even the capacity of producing certain citokynes that are correlated with immune response. The detection of Interleukin-6 was assayed in all our samples, including B.longum transformant BKS 7 strain, this result indicates that these bacteria can induce a non specific immune response in the intestinal cells. In fact, when we assayed the presence of Interferon-gamma in cells supernatant after bacterial exposure, we have no positive signals, that means that there is no activation of a specific immune response, thus confirming that these bacteria are not recognize as pathogen by the intestinal cells and are certainly not harmful for intestinal cells. The most important result is the measure of Trans Epithelial Electric Resistance that have shown how the intestinal barrier function get strengthen when cells are exposed to bacteria, due to a reduction of the epithelium permeability. We have now a new strain of B. longum that will be used for further studies above the mechanism of apoptotic induction to “damaged cells” and above the process of “restoring ecology”. This strain will be the basis to originate new transformant strains for Serpin encoding gene that must have better performance and shall be used one day even in clinical cases as in “gene therapy” for cancer treatment and prevention.

The role of CYLD in dendritic cell function

Deubiquitination of NF-κB members by CYLD is crucial in controlling the magnitude and nature of cell activation. The naturally occurring CYLD splice variant, devoid of exons 7 and 8, lacks TRAF2 and NEMO binding sites. The role of this splice variant in dendritic cell (DC) function was analyzed using CYLDex7/8 mice, which lack the full-length CYLD (FL-CYLD) transcript and over-express the short splice variant (sCYLD). Bone marrow derived DCs (BMDC) from CYLDex7/8 mice display a hyper-reactive phenotype in vitro and in vivo and have a defect in establishing tolerance using DEC205-mediated antigen targeting to resting DCs. This phenotype was accompanied by an increased nuclear translocation of the IκB molecule Bcl-3, and increased degradation of cytoplasmic p105 in CYLDex7/8 BMDCs after stimulation. This suggests that in contrast to FL-CYLD, sCYLD is a positive regulator of NF-κB activity and its over-expression induces a hyper-reactive phenotype in DCs.

Improving adoptive T cell therapies of cancer by ectopic expression of miR181a to repress inhibitory phosphatases

Adoptive T cell therapy using antigen-specific T lymphocytes is a powerful immunotherapeutic approach against cancer. Nevertheless, many T cells against tumor-antigens exhibit only weak anti-tumoral response. To overcome this barrier it is necessary to improve the potency and anti-tumoral efficacy of these T cells. Activation and activity of T cells are tightly controlled to inhibit unwanted T cell responses and to reduce the risk of autoimmunity. Both are regulated by extrinsic signals and intrinsic mechanisms which suppress T cell activation. The intrinsic mechanisms include the expression of phosphatases that counteract the activation-inducing kinases. Modifying the expression of these phosphatases allows the targeted modulation of T cell reactivity. MicroRNAs (miRNAs) are regulatory small noncoding RNA molecules that control gene expression by targeting messenger RNAs in a sequence specific manner. Gene-specific silencing plays a key role in diverse biological processes, such as development, differentiation, and functionality. miR181a has been shown to be highly expressed in immature T cells that recognize low-affinity antigens.rnThe present study successfully shows that ectopic expression of miR181a is able to enhance the sensitivity of both murine and human T cells. In CD4+ T helper cells as well as in CD8+ cytotoxic T cells the overexpression of miR181a leads to downregulation of multiple phosphatases involved in the T cell receptor signaling pathway. Overexpression of miR181a in human T cells achieves a co-stimulatory independent activation and has an anti-apoptotic effect on CD4+ T helper cells. Additionally, increasing the amount of miR181a enhances the cytolytic activity of murine CD8+ TCRtg T cells in an antigen-specific manner.rnTo test miR181a overexpressing T cells in vivo, a mouse tumor model using a B cell lymphoma cell line (A20-HA) expressing the Influenza hemagglutinin (Infl.-HA) antigen was established. The expression of model antigens in tumor cell lines enables targeted elimination of tumors using TCRtg T cells. The transfer of miR181a overexpressing Infl.-HA TCRtg CD8+ T cells alone has no positive effect neither on tumor control nor on survival of A20-HA tumor-bearing mice. In contrast, the co-transfer of miR181a overexpressing Infl.-HA TCRtg CD8+ and CD4+ T cells leads to improved tumor control and prolongs survival of A20-HA tumor-bearing mice. This effect is characterized by higher amounts of effector T cells and the expansion of Infl.-HA TCRtg CD8+ T cells.rnAll effects were achieved by changes in expression of several genes including molecules involved in T cell differentiation, activation, and regulation, cytotoxic effector molecules, and receptors important for the homing process of T cells in miR181a overexpressing T cells. The present study demonstrates that miR181a is able to enhance the anti-tumoral response of antigen-specific T cells and is a promising candidate for improving adoptive cell therapy.

Lysosomal degradation of RhoH protein upon antigen receptor activation in T but not B cells

RhoH is a member of the Rho (ras homologous) GTPase family, yet it lacks GTPase activity and thus remains in its active conformation. Unlike other Rho GTPases, the RhoH gene transcript is restricted to hematopoietic cells and RhoH was shown to be required for adequate T-cell activation through the TCR. Here, we demonstrate that both blood T and B cells, but not neutrophils or monocytes, express RhoH protein under physiological conditions. Upon TCR complex activation, RhoH was degraded in lysosomes of primary and Jurkat T cells. Pharmacologic activation of T cells distal to the TCR complex had no effect on RhoH protein levels suggesting that early events during T-cell activation are required for RhoH protein degradation. In contrast to T cells, activation of the BCR in blood B cells was not associated with changes in RhoH levels. These data suggest that RhoH function might be regulated by lysosomal degradation of RhoH protein following TCR complex but not BCR activation. This newly discovered regulatory pathway of RhoH expression might limit TCR signaling and subsequent T-cell activation upon Ag contact.

Inhibition of direct and indirect TLR-mediated activation of human NK cells by low molecular weight dextran sulfate

NK cells express toll-like receptors (TLR) that recognize conserved pathogen or damage associated molecular patterns and play a fundamental role in innate immunity. Low molecular weight dextran sulfate (DXS), known to inhibit the complement system, has recently been reported by us to inhibit TLR4-induced maturation of human monocyte-derived dendritic cells (MoDC). In this study, we investigated the capability of DXS to interfere with human NK cell activation triggered directly by TLR2 agonists or indirectly by supernatants of TLR4-activated MoDC. Both TLR2 agonists and supernatants of TLR4-activated MoDC activated NK cells phenotypically, as demonstrated by the analysis of NK cell activation markers (CD56, CD25, CD69, NKp30, NKp44, NKp46, DNAM-1 and NKG2D), and functionally as shown by increased NK cell degranulation (CD107a surface expression) and IFN-gamma secretion. DXS prevented the up-regulation of NK cell activation markers triggered by TLR2 ligands or supernatants of TLR4-activated MoDC and dose-dependently abrogated NK cell degranulation and IFN-gamma secretion. In summary our results suggest that DXS may be a useful reagent to inhibit the direct and indirect TLR-mediated activation of NK cells.

An increase in serum tryptase even below 11.4 ng/mL may indicate a mast cell-mediated hypersensitivity reaction: a prospective study in Hymenoptera venom allergic patients

During a systemic hypersensitivity reaction (SR), an increase in serum tryptase compared to the baseline value is an indicator of mast cell activation, most often due to an IgE-mediated mechanism.

Natural killer T cell dysfunction in CD39-null mice protects against concanavalin A-induced hepatitis

Concanavalin A (Con A)-induced injury is an established natural killer T (NKT) cell-mediated model of inflammation that has been used in studies of immune liver disease. Extracellular nucleotides, such as adenosine triphosphate, are released by Con A-stimulated cells and bind to specific purinergic type 2 receptors to modulate immune activation responses. Levels of extracellular nucleotides are in turn closely regulated by ectonucleotidases, such as CD39/NTPDase1. Effects of extracellular nucleotides and CD39 on NKT cell activation and upon hepatic inflammation have been largely unexplored to date. Here, we show that NKT cells express both CD39 and CD73/ecto-5'-nucleotidase and can therefore generate adenosine from extracellular nucleotides, whereas natural killer cells do not express CD73. In vivo, mice null for CD39 are protected from Con A-induced liver injury and show substantively lower serum levels of interleukin-4 and interferon-gamma when compared with matched wild-type mice. Numbers of hepatic NKT cells are significantly decreased in CD39 null mice after Con A administration. Hepatic NKT cells express most P2X and P2Y receptors; exceptions include P2X3 and P2Y11. Heightened levels of apoptosis of CD39 null NKT cells in vivo and in vitro appear to be driven by unimpeded activation of the P2X7 receptor. CONCLUSION: CD39 and CD73 are novel phenotypic markers of NKT cells. Deletion of CD39 modulates nucleotide-mediated cytokine production by, and limits apoptosis of, hepatic NKT cells providing protection against Con A-induced hepatitis. This study illustrates a further role for purinergic signaling in NKT-mediated mechanisms that result in liver immune injury.

Mesenchymal progenitor cell markers in human articular cartilage: normal distribution and changes in osteoarthritis

INTRODUCTION: Recent findings suggest that articular cartilage contains mesenchymal progenitor cells. The aim of this study was to examine the distribution of stem cell markers (Notch-1, Stro-1 and VCAM-1) and of molecules that modulate progenitor differentiation (Notch-1 and Sox9) in normal adult human articular cartilage and in osteoarthritis (OA) cartilage. METHODS: Expression of the markers was analyzed by immunohistochemistry (IHC) and flow cytometry. Hoechst 33342 dye was used to identify and sort the cartilage side population (SP). Multilineage differentiation assays including chondrogenesis, osteogenesis and adipogenesis were performed on SP and non-SP (NSP) cells. RESULTS: A surprisingly high number (>45%) of cells were positive for Notch-1, Stro-1 and VCAM-1 throughout normal cartilage. Expression of these markers was higher in the superficial zone (SZ) of normal cartilage as compared to the middle zone (MZ) and deep zone (DZ). Non-fibrillated OA cartilage SZ showed reduced Notch-1 and Sox9 staining frequency, while Notch-1, Stro-1 and VCAM-1 positive cells were increased in the MZ. Most cells in OA clusters were positive for each molecule tested. The frequency of SP cells in cartilage was 0.14 +/- 0.05% and no difference was found between normal and OA. SP cells displayed chondrogenic and osteogenic but not adipogenic differentiation potential. CONCLUSIONS: These results show a surprisingly high number of cells that express putative progenitor cell markers in human cartilage. In contrast, the percentage of SP cells is much lower and within the range of expected stem cell frequency. Thus, markers such as Notch-1, Stro-1 or VCAM-1 may not be useful to identify progenitors in cartilage. Instead, their increased expression in OA cartilage implicates involvement in the abnormal cell activation and differentiation process characteristic of OA.