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BACKGROUND: We analyzed the gammadelta T cell composition and responses in the peripheral blood and cerebrospinal fluid (CSF) of children affected by tuberculous meningitis (TBM) and in control children. MATERIALS AND METHODS: Peripheral blood and CSF samples were stimulated with different phosphoantigens and IL-2, and expansion of Vgamma9/Vdelta2 T cells assessed by FACS analysis. Vgamma9/Vdelta2 lines were obtained by culturing CSF or peripheral blood mononuclear cells (PBMC) in vitro with phosphoantigens and IL-2 for 2 months, and tested for proliferation and cytokine production in response to phosphoantigens. Vdelta2(D)Jdelta junctional sequence length was assessed by PCR. RESULTS: The repertoire of gammadelta T cells from the CSF of TBM patients was characterized by the predominance of Vgamma9/Vdelta2 T lymphocytes, which accounted for >80% of gammadelta T cells. Vgamma9/Vdelta2 cells from the CSF of TBM children responded to different synthetic and natural (mycobacterial) phosphoantigens and produced discrete amounts of IFN-gamma and TNF-alpha. The in vitro expansion of Vgamma9/Vdelta2 T cells from CSF and peripheral blood of TBM patients prominently decreased following chemotherapy, and similarly, the proportion of ex vivo unstimulated Vgamma9/Vdelta2 T cells in CSF of TBM patients decreased to levels detected in the CSF of control subjects. Vdelta2 CDR3 TCR analysis showed that the remaining Vdelta2 cells in the CSF of TBM patients were still polyclonal. CONCLUSIONS: These findings are consistent with an involvement of Vgamma9/Vdelta2 T cells in TBM. http://link. springer-ny.com/link/service/journals/00020/bibs/5n5p301. html

The beneficial health properties of the Mediterranean diet are well recognized. The principle source of fat in Mediterranean diet is extra-virgin olive oil (EVOO). Oleocanthal (OC) is a naturally occurring minor phenolic compound isolated from EVOO, which has shown a potent anti-inflammatory activity, by means of its ability to inhibit the cyclooxygenase (COX) enzymes COX-1 and COX-2. A large body of evidence indicates that phenols exhibit anticancer activities. The aim of the present study was to evaluate the potential anticancer effects of OC in hepatocellular carcinoma (HCC) and colorectal carcinoma (CRC) models. A panel of human HCC (HepG2, Huh7, Hep3B and PLC/PRF/5) and CRC (HT29, SW480) cell lines was used. Cells were treated with OC, and cell viability and apoptosis were evaluated. Compared with classical commercially available COX inhibitors (ibuprofen, indomethacin, nimesulide), OC was more effective in inducing cell growth inhibition in HCC and CRC cells. Moreover, OC inhibited colony for mation and i nduced ap optosis, as confirmed by PARP cleavage, activation of caspases 3/7 and chromatin condensation. OC treatment in a dose dependent-manner induced expression of γH2AX, a marker of DNA damage, increased intracellular ROS production and caused mitochondrial depolarization. Moreover, the effects of OC were suppressed by the ROS scavenger N-acetyl-L-cysteine. Finally, OC was not toxic in primary normal human hepatocytes. In conclusion, OC treatment was found to exert a potent anticancer activity against HCC and CRC cells. Taken together, our findings provide preclinical support of the chemotherapeutic potential of EVOO against cancer.

Background Reduced innate immunity responses as well as reduced T regulatory activities characterise bronchial asthma. Objectives In this study the effect of budesonide on the expression of TLR4 and TLR2 in T regulatory lymphocyte sub-population was assessed. Methods TLR4 and TLR2 expression in total peripheral blood mononuclear cells (PBMC), in CD4+/CD25+ and in CD4+/CD25− was evaluated, by flow cytometric analysis, in mild intermittent asthmatics (n = 14) and in controls (n = 11). The in vitro effects of budesonide in modulating: TLR4 and TLR2 expression in controls and in asthmatics; IL-10 expression and cytokine release (IL-6 and TNF-α selected by a multiplex assay) in asthmatics were also explored. Results TLR4 and TLR2 were reduced in total PBMC from asthmatics in comparison to PBMC from controls. CD4+CD25+ cells expressed at higher extent TLR2 and TLR4 in comparison to CD4+CD25− cells. Budesonide was able to increase the expression of TLR4, TLR2 and IL-10 in CD4+/CD25highly+ cells from asthmatics. TLR4 ligand, LPS induced Foxp3 expression. Budesonide was also able to reduce the release of IL-6 and TNF-α by PBMC of asthmatics. Conclusions Budesonide potentiates the activity of Treg by increasing TLR4, TLR2 and IL-10 expression. This event is associated to the decreased release of IL-6 and TNF-α in PBMC treated with budesonide. These findings shed light on new mechanisms by which corticosteroids, drugs widely used for the clinical management of bronchial asthma, control T lymphocyte activation.

Hepatocellular carcinoma (HCC) is one of the common malignancies and is an increasingly important cause of cancer death worldwide. Surgery, chemotherapy, and radiation therapy extend the 5-year survival limit in HCC patients by only 6%. Therefore, there is a need to develop new therapeutic approaches for the treatment of this disease. The orally bioavailable proteasome inhibitor MLN2238 (ixazomib) has been demonstrated to have anticancer activity. In the present study, we investigated the preclinical therapeutic efficacy of MLN2238 in HCC cells through in vitro and in vivo models, and examined its molecular mechanisms of action. MLN2238 inhibited cell viability in human HCC cells HepG2, Hep3B, and SNU475 in a time- and dose-dependent manner. Flow cytometry analysis demonstrated that MLN2238 induced G2/M cell cycle arrest and cellular apoptosis in HCC cells. Cell cycle arrest was associated with increased expression levels of p21 and p27. MLN2238-induced apoptosis was confirmed by caspase-3/7 activation, PARP cleavage and caspase-dependent β-catenin degradation. In addition, MLN2238 activated ER stress genes in HCC cells and increased the expression of the stress-inducible gene nuclear protein-1. Furthermore, MLN2238 treatment induced upregulation of myeloid cell leukemia-1 (Mcl-1) protein, and Mcl-1 knockdown sensitized HCC cells to MLN2238 treatment, suggesting the contribution of Mcl-1 expression to MLN2238 resistance. This result was also confirmed using the novel Mcl-1 small molecule inhibitor A1210477. Association of A1210477 and MLN2238 determined synergistic antitumor effects in HCC cells. Finally, in vivo orally administered MLN2238 suppressed tumor growth of Hep3B cells in xenograft models in nude mice. In conclusion, our results offer hope for a new therapeutic opportunity in the treatment of HCC patients.

Airway epithelium is a regulator of innate immune responses to a variety of insults including cigarette smoke. Cigarette smoke alters the expression and the activation of Toll Like Receptor 4 (TLR4), an innate immunity receptor. IL-33, an alarmin, increases innate immunity Th2 responses. The aims of this study were to explore whether mini-bronchoalveolar lavage (mini-BAL) or sera from smokers have altered concentrations of IL-33 and whether cigarette smoke extracts (CSE) alter both intracellular expression (mRNA and protein) and release of IL-33 in bronchial epithelial cells. The role of TLR4 in the expression of IL-33 was also explored.Mini-BALs, but not sera, from smokers show reduced concentrations of IL-33. The expression of IL-33 was increased also in bronchial epithelium from smokers. 20% CSE reduced IL-33 release but increased the mRNA for IL-33 by real time PCR and the intracellular expression of IL-33 in bronchial epithelial cells as confirmed by flow cytometry, immunocytochemistry and western blot analysis. The effect of CSE on IL-33 expression was also observed in primary bronchial epithelial cells. IL-33 expression was mainly concentrated within the cytoplasm of the cells. LPS, an agonist of TLR4, reduced IL-33 expression, and an inhibitor of TLR4 increased the intracellular expression of IL-33. In conclusion, the release of IL-33 is tightly controlled and, in smokers, an altered activation of TLR4 may lead to an increased intracellular expression of IL-33 with a limited IL-33 release.

TGF-beta-targeting structural and inflammatory cells has been implicated in the mechanisms leading to the inflammatory and restructuring processes in asthma, suggesting an impact of TGF-beta1 signaling on the development and persistency of this disease. We investigated the potential early involvement of TGF-beta1 activity in the immunological and molecular mechanisms underlying progression of inflammation in childhood asthma. We evaluated the levels of TGF-beta1 in induced sputum supernatants (ISSs) and the expression of small mother cell against decapentaplegic (Smad) 2 and Smad7 proteins in induced sputum cells (ISCs) from children with intermittent asthma (IA), moderate asthma (MA) and control subjects (C). Furthermore, we investigated the regulatory role of TGF-beta1 activity on eosinophil and neutrophil adhesion to epithelial cells using adhesion assay, and on the granulocyte expression of adhesion molecule CD11b/CD18 Macrophage-1 antigen (MAC-1), by flow cytometry. We found that the levels of TGF-beta1 are increased in ISSs of IA and MA in comparison to C, concomitantly to the activation of intracellular signaling TGFbeta/Smads pathway in ISCs. In MA, TGF-beta1 levels correlated with the number of sputum eosinophils and neutrophils. Furthermore, we showed the ability of sputum TGF-beta1 to promote eosinophil and neutrophil adhesion to epithelial cells, and to increase the expression of MAC-1 on the granulocyte surface. This study shows the activation of TGFbeta/Smad signaling pathway in the airways of children with IA and, despite the regular ICS treatment, in children with MA, and provides evidence for the contribution of TGF-beta1 in the regulation of granulocyte activation and trafficking.

Two human leukocyte antigen (HLA) class I loci (HLA-A and HLA-B) and one class II locus (HLA-DR) were typed at the DNA level in the Sicilian population. Study participants were of Sicilian origin (183 for class I loci and 260 for class II loci) and live in three towns, chosen on the basis of geographic position and different historical events. These towns are Sciacca (southwest Sicily, located at sea level, conquered by Arabs in a.d. 814), Piana degli Albanesi (northwest Sicily, 720 m above sea level, has maintained religious, cultural, and linguistic peculiarities traced to Albanian settlement in 1488), and Troina (northeast Sicily, 1,120 m above sea level, known as the first settlement of Normans). The assumptions underlying the study of genetic structure, based on HLA allele polymorphism, are that these three towns are located in areas that can be distinguished according to historical criteria and that they are likely to have contributed to cultural and probably genetic differences. As such, the high frequency of some alleles in Sciacca and Troina seems to be correlated with Greek, Phoenician, North African, and Arab influence. In accordance with different human settlements in Sicily, we found that the HLA allele frequencies support the existence of genetic differentiation between the western and eastern sides of Sicily. This separation is attributed to Greek colonization in the east and to Phoenician-Carthaginian-Arab influence in the west. Moreover, the comparisons of all allele frequencies between Mediterranean and African populations show the same trend, highlighting in some cases European origin and in other cases non-European origin.

The alphabeta and gammadelta T cell responses were analyzed in the peripheral blood of children affected by active tuberculosis (TB) and in healthy children who tested positive (PPD+) or negative (PPD-) for purified protein derivative. PPD+ healthy and diseased children responded equally well to PPD in vitro. In contrast, only 18% of PPD+ TB patients responded to peptide p38G derived from the 38-kDa protein of Mycobacterium tuberculosis. Analysis of the whole gammadelta T cell population and of its Vgamma9/Vdelta2 subset showed similar frequencies in PPD+ children with TB and in healthy PPD+ and PPD- children. Vgamma9/Vdelta2 cells from children with TB responded to 5 different phosphoantigens similarly to those from healthy PPD+ children, but healthy PPD- children responded very poorly. Chemotherapy had contrasting effects on the tested lymphocyte population, represented by increase of alphabeta and decline of Vgamma9/Vdelta2 T cell responses. T cell responses in childhood TB may be similar to those in adult TB.

BACKGROUND: Increased activation and increased survival of T lymphocytes characterise bronchial asthma. OBJECTIVES: In this study the effect of budesonide on T cell survival, on inducible co-stimulator T cells (ICOS), on Foxp3 and on IL-10 molecules in T lymphocyte sub-populations was assessed. METHODS: Cell survival (by annexin V binding) and ICOS in total lymphocytes, in CD4+/CD25+ and in CD4+/CD25- and Foxp3 and IL-10 in CD4+/CD25+ and in CD4+/CD25-cells was evaluated, by cytofluorimetric analysis, in mild intermittent asthmatics (n = 19) and in controls (n = 15). Allergen induced T lymphocyte proliferation and the in vivo effects of budesonide in mild persistent asthmatics (n = 6) were also explored. RESULTS: Foxp3 was reduced in CD4+/CD25- and in CD4+/CD25+ cells and ICOS was reduced in CD4+/CD25+ cells but it was increased in CD4+CD25-in asthmatics when compared to controls. In asthmatics, in vitro, budesonide was able to: 1) increase annexin V binding and to reduce ICOS in total lymphocytes; 2) increase annexin V binding and Foxp3 and to reduce ICOS in CD4+/CD25- cells; 3) reduce annexin V binding and to increase IL-10 and ICOS in CD4+/CD25+ cells; 4) reduce cell allergen induced proliferation. In vivo, budesonide increased ICOS in CD4+/CD25+ while it increased Foxp3 and IL-10 in CD4+/CD25+ and in CD4+/CD25- cells. CONCLUSIONS: Budesonide modulates T cell survival, ICOS, Foxp3 and IL-10 molecules differently in T lymphocyte sub-populations. The findings provided shed light on new mechanisms by which corticosteroids, drugs widely used for the clinical management of bronchial asthma, control T lymphocyte activation.

CD94/NKG2A is an inhibitory receptor expressed by NK cells and cytotoxic lymphocytes and, upon activation by HLA-E, downregulates the cytolytic activities of these cells thus representing a tumour immune escape mechanism. This study was aimed at assessing whether cytotoxic lymphocytes (CD8+) and NK cells from malignant pleural effusions have a deregulated expression of CD94/NKG2A. The expression of membrane CD94/NKG2A and perforin was evaluated by flow-cytometry in CD8+ and NK cells from pleural effusions and autologous peripheral blood of cancer (n=19) and congestive heart failure (CHF) (n=11) patients. Intracellular CD94/NKG2A expression was evaluated by flow-cytometry in pleural effusion CD8+ and NK cells from cancer patients (n=10). Cytotoxic activity against cancer cells exerted by pleural and autologous peripheral blood T lymphocytes from cancer patients was assessed by flow-cytometry assay. Pleural CD8+ from cancer patients showed a reduced expression of membrane CD94/NKG2A and perforin when compared to autologous peripheral blood and CHF pleural effusions. Reduced numbers of NK cells were present in pleural effusions from both cancer and CHF patients. Pleural NK from cancer patients showed a reduced expression of membrane CD94/NKG2A and perforin when compared to autologous peripheral blood. Pleural T lymphocytes from cancer patients exhibited a reduced cytotoxic activity against cancer cells when compared to autologous peripheral blood T lymphocytes. The intracellular expression of CD94/NKG2A in CD8+ and NK cells from cancer patients was higher than membrane expression. In conclusion, this study provides compelling evidences of new mechanisms underlying the reduced host defence against cancer within the pleural space.

We previously reported that an inhibition of antigen-specific Interferon-γ release and cytotoxicity occurs after a continuous infusion of an HY immunodominant peptide although this treatment is not able to cause a significant delay of male skin grafts rejection. In vivo administration of high doses of an HY peptide, through mini-osmotic pumps, in naive female mice was used to study the effects on the male skin grafts rejection. A continuous infusion of 1 mg of an HY peptide induces a significant delay of male skin graft rejection. In vitro HY-specific Interferon-γ release was inhibited adding peptide-specific suppressor cells: the ability to inhibit Interferon-γ release was evident when two HY peptides were present on the same dendritic cells indicating that the suppressor cells exert “linked-suppression”. The phenotype of the suppressor cells is CD8 + CD28 − and these cells express more CD62 ligand and FOXP3 than controls. Suppressor cells were able to cause a significant delay of rejection of male skin grafts when injected in naive female mice. The inhibitory effects of these suppressor cells seem to be due to the impairment of antigen presentation; down-regulation of B7 molecules on dendritic cells occurred. Taken all together, our data demonstrate that a continuous infusion of an immunodominant HY peptide induces a T CD8 suppressor subset able to inhibit immune responses to male tissues and cells.

Transgenic mice expressing human major histocompatibility complex (MHC) class II molecules would provide a valuable model system for studying murine anti-human MHC immune response. We have previously shown that skin from HLA-DR1 transgenic mice was rejected by control littermates and spleen cells from rejecting mice were able to proliferate to donor cells. The aim of this paper is to analyze the mechanism of recognition of this xenoantigen and the possible involvement of antibody response in anti-HLA-DR1 immune response. Control littermates were immunized with spleen cells from HLA-DR1 transgenic (TG) mice; at indicated times, xenoantigen-specific proliferation and IFNgamma production was assessed using APC obtained from HLA-DR1 TG mice. Mixed direct-indirect pathway of xenoantigen recognition was suggested by the following findings: i)T cell response to HLA-DR1 was inhibited adding in culture monoclonal antibodies directed either to donor (HLA-DR) or to recipient MHC (I-A); ii) APC from control mice pulsed with purified DR1 molecules were able to induce proliferation by FVB/N mice immunized with transgenic spleen cells. HLA-DR1 recognition permits DR peptide-specific T cell response by lymphocytes of control littermates immunized with the xenoantigen. In addition, we detected xenoreactive IgM and IgG2 antibodies. Our data suggest that HLA-DR1 xenoantigen may be recognized through direct or indirect pathway and provide additional information on mouse anti-human HLA immune response.