HDAC inhibitors tune miRNAs in extracellular vesicles of dystrophic muscle-resident mesenchymal cells.

We show that extracellular vesicles (EVs) released by mesenchymal cells (i.e., fibro-adipogenic progenitors-FAPs) mediate microRNA (miR) transfer to muscle stem cells (MuSCs) and that exposure of dystrophic FAPs to HDAC inhibitors (HDACis) increases the intra-EV levels of a subset of miRs, which cooperatively target biological processes of therapeutic interest, including regeneration, fibrosis, and inflammation. Increased levels of miR-206 in EVs released by FAPs of muscles from Duchenne muscular dystrophy (DMD) patients or mdx mice exposed to HDACi are associated with enhanced regeneration and decreased fibrosis. Consistently, EVs from HDACi-treated dystrophic FAPs can stimulate MuSC activation and expansion ex vivo, and promote regeneration, while inhibiting fibrosis and inflammation of dystrophic muscles, upon intramuscular transplantation in mdx mice, in vivo. AntagomiR-mediated blockade of individual miRs reveals a specific requirement of miR-206 for EV-induced expansion of MuSCs and regeneration of dystrophic muscles, and indicates that cooperative activity of HDACi-induced miRs accounts for the net biological effect of these EVs. These data point to pharmacological modulation of EV content as novel strategy for therapeutic interventions in muscular dystrophies.

Nanovesicles released by OKT3 hybridoma express fully active antibodies.

Recent findings have shown that nanovesicles preparations from either primary immune cells culture supernatants or plasma contain immunoglobulins, suggesting that a natural way of antibody production may be through exosome release. To verify this hypothesis, we used the OKT3 hybridoma clone, which produces a murine IgG2a monoclonal antibody used to reduce rejection in patients undergoing organ transplantation. We showed exosome-associated immunoglobulins in hybridoma supernatants, by Western blot, nanoscale flow cytometry and immunocapture-based ELISA. The OKT3-exo was also being able to trigger cytokines production in both CD4 and CD8 T cells. These results show that nanovesicles contain immunoglobulin and could be used for immunotherapy. These data could lead to a new approach to improve the effectiveness of therapeutic antibodies by exploiting their natural property to be expressed on nanovesicle membrane, that probably render them more stable and as a consequence more capable to interact with their specific ligand in the best way.

Sexually Dimorphic Immune and Neuroimmune Changes Following Peripheral Nerve Injury in Mice: Novel Insights for Gender Medicine.

Neuropathic pain (NeP) in humans is often a life-long condition with no effective therapy available. The higher incidence of female gender in NeP onset is worldwide reported, and although the cause is generally attributed to sex hormones, the actual mechanisms and the players involved are still unclear. Glial and immune cells take part in NeP development, and orchestrate the neuroimmune and inflammatory response, releasing pro-inflammatory factors with chemoattractant properties that activate resident immune cells and recruit immune cells from circulation. The neuro-immune crosstalk is a key contributor to pain hypersensitivity following peripheral nervous system injury. Our previous works showed that in spite of the fact that female mice had an earlier analgesic response than males following nerve lesion, the recovery from NeP was never complete, suggesting that this difference could occur in the very early stages after injury. To further investigate gender differences in immune and neuroimmune responses to NeP, we studied the main immune cells and mediators elicited both in plasma and sciatic nerves by peripheral nerve lesion. After injury, we found a different pattern of distribution of immune cell populations showing either a higher infiltration of T cells in nerves from females or a higher infiltration of macrophages in nerves from males. Moreover, in comparison to male mice, the levels of cytokines and chemokines were differently up- and down-regulated in blood and nerve lysates from female mice. Our study provides some novel insights for the understanding of gender-associated differences in the generation and perseveration of NeP as well as for the isolation of specific neurodegenerative mechanisms underlying NeP. The identification of gender-associated inflammatory profiles in neuropathy is of key importance for the development of differential biomarkers and gender-specific personalized medicine.

Plasmatic exosomes from prostate cancer patients show increased carbonic anhydrase IX expression and activity and low pH.

Acidity, hypoxia and increased release of exosomes are severe phenotypes of tumours. The regulation of pH in tumours involves the interaction of several proteins, including the carbonic anhydrases which catalyze the formation of bicarbonate and protons from carbon dioxide and water. Among CA isoforms, CA IX is over-expressed in a large number of solid tumours, conferring to cancer cells a survival advantage in hypoxic and acidic microenvironment, but there isn't evidence that CA IX expression could have a real clinical impact. Therefore, in this study for the first time the expression and activity of CA IX have been investigated in the plasmatic exosomes obtained from patients with prostate carcinoma (PCa). For this purpose, the study was performed through different methodological approaches, such as NTA, western blot analysis, enzyme activity assay, Nanoscale flow cytometry, ELISA, confocal microscopy. The results showed that PCa exosomes significantly overexpressed CA IX levels and related activity as compared to healthy donors. Furthermore, CA IX expression and activity were correlated to the exosome intraluminal pH, demonstrating for the first time that PCa exosomes are acidic. Our data suggest the possible use of the exosomal CA IX expression and activity as a biomarker of cancer progression in PCa.

Altered intestinal permeability in patients with relapsing-remitting multiple sclerosis: A pilot study.

BACKGROUND: Alterations of intestinal permeability (IP) may contribute to the pathophysiology of immune-mediated diseases. OBJECTIVE: We investigated the possible association between IP changes and multiple sclerosis (MS). METHODS: We studied 22 patients with relapsing-remitting multiple sclerosis (RRMS) and 18 age- and sex-matched healthy donors (HDs), including five twin pairs (one concordant, and four discordant for disease). Measurement of lactulose (L) and mannitol (M; two non-metabolized sugars) levels in urine samples, after an oral load, allowed to quantify gut dysfunction. RESULTS: The proportion of participants with increased IP was significantly higher in patients than in HDs (16/22 (73%) versus 5/18 (28%); p = 0.001). Accordingly, the L/M urinary ratio showed significantly higher values in patients than in controls ( p = 0.0284). Urinary mannitol concentration was significantly lower in patients than in controls ( p = 0.022), suggesting a deficit of absorption from intestinal lumen. Such changes did not appear related to patients' clinical-radiological features. CONCLUSION: The relatively high proportion of IP changes in RR-MS patients seems to confirm our work hypothesis and warrants more work to confirm the result on a larger sample, and to understand the implications for related immunological disturbances and intestinal microbiota alterations. Our finding may also have relevance for oral treatments, recently introduced in clinical practice.

Evaluation of the Impact of a New Synthetic Vitamin E-Bonded Membrane on the Hypo-Responsiveness to the Erythropoietin Therapy in Hemodialysis Patients: A Multicenter Study.

BACKGROUND: Oxidative stress has been related to hypo-response to erythropoiesis-stimulating agents (ESAs) in hemodialysis (HD) patients. The aim of this study was to verify whether vitamin E (ViE) on a synthetic polysulfone dialyzer can improve ESA responsiveness. METHODS: This controlled, multicenter study involved 93 HD patients on stable ESA therapy, who were randomized to either ViE-coated polysulfone dialyzer or to a low-flux synthetic dialyzer. The primary outcome measure was the change in ESA resistance index (ERI) from baseline. RESULTS: Mean ERI decreased in the ViE group by 1.45 IU/kg*g/dl and increased in the control group by 0.53 IU/kg*g/dl, with a mean difference of 1.98 IU/kg*g/dl (p = 0.001 after adjusting for baseline ERI, as foreseen by the study protocol). Baseline ERI was inversely related to its changes during follow-up only in the control group (R2 = 0.29). CONCLUSIONS: The ViE dialyzer can improve ESA response in HD patients. Changes in ERI during follow-up are independent from baseline ERI only in the ViE group. Video Journal Club 'Cappuccino with Claudio Ronco' at http://www.karger.com/?doi=453442.

Body mass index, fat free mass, uric acid, and renal function as blood pressure levels determinants in young adults.

AIM: We assessed some major determinants of blood pressure (BP) in young adulthood to plan a lifestyle changes policy METHODS: A cross sectional survey was held, involving 2373 high school people (age 18-21), measuring BP, body mass index (BMI), waist circumference (WCirc), fat free mass (FFM); alcohol and smoking habits were evaluated by a questionnaire. In a subset of this population (n = 60) uric acid (UA), estimated glomerular filtration rate (eGFR) were also evaluated. RESULTS: Smoking and not alcohol was correlated to systolic blood pressure (SBP) through quartiles (31.7%, 39.1%, 46.5%, 45.5%). Systolic BP was significantly correlated with FFM in the whole population (r = 0.51) as well as in SBP quartiles (r = 0.243, 0.138, 0.118, 0.204). FFM-SBP cluster analysis gave two centroids corresponding to sexes; females n = 998; coordinates (116.4 mmHg, 38.9 kg) and males n = 1068; coordinates (131.3 mmHg, 56.7 kg). In the n = 60 substudy a multiple linear regression model (multiple R = 0.741) with SBP as dependent variable and UA, FFM, BMI, eGFR as explicative ones, only UA (ß coefficent = 0.363, partial r = 0.240, P < 0.01) was the determinant of BP particularly in men. Moreover in the same group we found an inverse relationship between eGFR (albeit always in the normal range) and UA, as well as for women (r = -0.54, P < 0.01) and men (r = -0.43, P < 0.01) analyzed separately. CONCLUSIONS: A significant correlation exists between BP and FFM; UA has proven to be the most important SBP determinant. At variance with paediatric age UA was negatively correlated with renal function. Dietary intervention on UA and alcohol habits in young adults seems advisable to prevent hypertension.

Acridine Orange/exosomes increase the delivery and the effectiveness of Acridine Orange in human melanoma cells: A new prototype for theranostics of tumors.

Specifically targeted drug delivery systems with low immunogenicity and toxicity are deemed to increase efficacy of cancer chemotherapy. Acridine Orange (AO) is an acidophilic dye with a strong tumoricidal action following excitation with a light source at 466 nm. However, to date the clinical use of AO is limited by the potential side effects elicited by systemic administration. The endogenous nanocarrier exosomes have been recently introduced as a natural delivery system for therapeutic molecules. In this article, we show the outcome of the administration to human melanoma cells of AO charged Exosomes (Exo-AO), in both monolayer and spheroid models. The results showed an extended drug delivery time of Exo-AO to melanoma cells as compared to the free AO, improving the cytotoxicity of AO. This study shows that Exo-AO have a great potential for a real exploitation as a new theranostic approach against tumors based on AO delivered through the exosomes.

BRCA1, PARP1 and γH2AX in acute myeloid leukemia: Role as biomarkers of response to the PARP inhibitor olaparib.

Olaparib (AZD-2281, Ku-0059436) is an orally bioavailable and well-tolerated poly(ADP-ribose) polymerase (PARP) inhibitor currently under investigation in patients with solid tumors. To study the clinical potential of olaparib as a single-agent for the treatment of acute myeloid leukemia (AML) patients, we analyzed the in vitro sensitivity of AML cell lines and primary blasts. Clinically achievable concentrations of olaparib were able to induce cell death in the majority of primary AML case samples (88%) and tested cell lines. At these concentrations, olaparib preferentially killed leukemic blasts sparing normal lymphocytes derived from the same patient and did not substantially affect the viability of normal bone marrow and CD34-enriched peripheral blood cells obtained from healthy donors. Most primary AML analyzed were characterized by low BRCA1 mRNA level and undetectable protein expression that likely contributed to explain their sensitivity to olaparib. Noteworthy, while PARP1 over-expression was detected in blasts not responsive to olaparib, phosphorylation of the histone H2AFX (γH2AX) was associated with drug sensitivity. As to genetic features of tested cases the highest sensitivity was shown by a patient carrying a 11q23 deletion. The high sensitivity of AML blasts and the identification of biomarkers potentially able to predict response and/or resistance may foster further investigation of olaparib monotherapy for AML patients unfit to conventional chemotherapy.

Increased CD8+ T cell response to Epstein-Barr virus lytic antigens in the active phase of multiple sclerosis.

It has long been known that multiple sclerosis (MS) is associated with an increased Epstein-Barr virus (EBV) seroprevalence and high immune reactivity to EBV and that infectious mononucleosis increases MS risk. This evidence led to postulate that EBV infection plays a role in MS etiopathogenesis, although the mechanisms are debated. This study was designed to assess the prevalence and magnitude of CD8+ T-cell responses to EBV latent (EBNA-3A, LMP-2A) and lytic (BZLF-1, BMLF-1) antigens in relapsing-remitting MS patients (n = 113) and healthy donors (HD) (n = 43) and to investigate whether the EBV-specific CD8+ T cell response correlates with disease activity, as defined by clinical evaluation and gadolinium-enhanced magnetic resonance imaging. Using HLA class I pentamers, lytic antigen-specific CD8+ T cell responses were detected in fewer untreated inactive MS patients than in active MS patients and HD while the frequency of CD8+ T cells specific for EBV lytic and latent antigens was higher in active and inactive MS patients, respectively. In contrast, the CD8+ T cell response to cytomegalovirus did not differ between HD and MS patients, irrespective of the disease phase. Marked differences in the prevalence of EBV-specific CD8+ T cell responses were observed in patients treated with interferon-ß and natalizumab, two licensed drugs for relapsing-remitting MS. Longitudinal studies revealed expansion of CD8+ T cells specific for EBV lytic antigens during active disease in untreated MS patients but not in relapse-free, natalizumab-treated patients. Analysis of post-mortem MS brain samples showed expression of the EBV lytic protein BZLF-1 and interactions between cytotoxic CD8+ T cells and EBV lytically infected plasma cells in inflammatory white matter lesions and meninges. We therefore propose that inability to control EBV infection during inactive MS could set the stage for intracerebral viral reactivation and disease relapse.

Dysfunctional Vγ9Vδ2 T cells are negative prognosticators and markers of dysregulated mevalonate pathway activity in chronic lymphocytic leukemia cells.

The role of Vγ9Vδ2 T cells in chronic lymphocytic leukemia (CLL) is unexplored, although these cells have a natural inclination to react against B-cell malignancies. Proliferation induced by zoledronic acid was used as a surrogate of γδ TCR-dependent stimulation to functionally interrogate Vγ9Vδ2 T cells in 106 untreated CLL patients. This assay permitted the identification of responder and low-responder (LR) patients. The LR status was associated with greater baseline counts of Vγ9Vδ2 T cells and to the expansion of the effector memory and terminally differentiated effector memory subsets. The tumor immunoglobulin heavy chain variable region was more frequently unmutated in CLL cells of LR patients, and the mevalonate pathway, which generates Vγ9Vδ2 TCR ligands, was more active in unmutated CLL cells. In addition, greater numbers of circulating regulatory T cells were detected in LR patients. In multivariate analysis, the LR condition was an independent predictor of shorter time-to-first treatment. Accordingly, the time-to-first treatment was significantly shorter in patients with greater baseline numbers of total Vγ9Vδ2 T cells and effector memory and terminally differentiated effector memory subpopulations. These results unveil a clinically relevant in vivo relationship between the mevalonate pathway activity of CLL cells and dys-functional Vγ9Vδ2 T cells.

Non-myeloablative autologous haematopoietic stem cell transplantation expands regulatory cells and depletes IL-17 producing mucosal-associated invariant T cells in multiple sclerosis.

Autologous haematopoietic stem cell transplantation has been tried as one experimental strategy for the treatment of patients with aggressive multiple sclerosis refractory to other immunotherapies. The procedure is aimed at ablating and repopulating the immune repertoire by sequentially mobilizing and harvesting haematopoietic stem cells, administering an immunosuppressive conditioning regimen, and re-infusing the autologous haematopoietic cell product. 'Non-myeloablative' conditioning regimens to achieve lymphocytic ablation without marrow suppression have been proposed to improve safety and tolerability. One trial with non-myeloablative autologous haematopoietic stem cell transplantation reported clinical improvement and inflammatory stabilization in treated patients with highly active multiple sclerosis. The aim of the present study was to understand the changes in the reconstituted immune repertoire bearing potential relevance to its mode of action. Peripheral blood was obtained from 12 patients with multiple sclerosis participating in the aforementioned trial and longitudinally followed for 2 years. We examined the phenotype and function of peripheral blood lymphocytes by cell surface or intracellular staining and multi-colour fluorescence activated cell sorting alone or in combination with proliferation assays. During immune reconstitution post-transplantation we observed significant though transient increases in the proportion of CD4+ FoxP3+ T cells and CD56(high) natural killer cell subsets, which are cell subsets associated with immunoregulatory function. CD8+ CD57+ cytotoxic T cells were persistently increased after therapy and were able to suppress CD4+ T cell proliferation with variable potency. In contrast, a CD161(high) proinflammatory CD8+ T cell subset was depleted at all time-points post-transplantation. Phenotypic characterization revealed that the CD161(high)CD8+ T cells were mucosal-associated invariant T cells, a novel cell population originating in the gut mucosa but expressing the central nervous system-homing receptor CCR6. Detection of mucosal-associated invariant T cells in post-mortem multiple sclerosis brain white matter active lesions confirmed their involvement in the disease pathology. Intracellular cytokine staining demonstrated interferon γ and interleukin 17 production and lack of interleukin 10 production, a pro-inflammatory profile. Mucosal-associated invariant T cell frequency did not change in patients treated with interferon ß; and was more depleted after autologous haematopoietic stem cell transplantation than in patients who had received high-dose cyclophosphamide (n = 7) or alemtuzumab (n = 21) treatment alone, suggesting an additive or synergistic effect of the conditioning regime components. We propose that a favourably modified balance of regulatory and pro-inflammatory lymphocytes underlies the suppression of central nervous system inflammation in patients with multiple sclerosis following non-myeloablative autologous haematopoietic stem cell transplantation with a conditioning regimen consisting of cyclophosphamide and alemtuzumab.

Pan-neuronal expression of human mutant SOD1 in Drosophila impairs survival and motor performance, induces early neuroinflammation and chromosome aberrations.

Several mutations in the SOD1 gene encoding for the antioxidant enzyme Superoxide Dismutase 1, are associated with amyotrophic lateral sclerosis, a rare and devastating disease characterized by motor neuron degeneration and patients' death within 2-5 years from diagnosis. Motor neuron loss and related symptomatology manifest mostly in adult life and, to date, there is still a gap of knowledge on the precise cellular and molecular events preceding neurodegeneration. To deepen our awareness of the early phases of the disease, we leveraged two Drosophila melanogaster models pan-neuronally expressing either the mutation A4V or G85R of the human gene SOD1 (hSOD1A4V or hSOD1G85R). We demonstrate that pan-neuronal expression of the hSOD1A4V or hSOD1G85R pathogenic construct impairs survival and motor performance in transgenic flies. Moreover, protein and transcript analysis on fly heads indicates that mutant hSOD1 induction stimulates the glial marker Repo, up-regulates the IMD/Toll immune pathways through antimicrobial peptides and interferes with oxidative metabolism. Finally, cytological analysis of larval brains demonstrates hSOD1-induced chromosome aberrations. Of note, these parameters are found modulated in a timeframe when neurodegeneration is not detected. The novelty of our work is twofold: we have expressed for the first time hSOD1 mutations in all neurons of Drosophila and confirmed some ALS-related pathological phenotypes in these flies, confirming the power of SOD1 mutations in generating ALS-like phenotypes. Moreover, we have related SOD1 pathogenesis to chromosome aberrations and antimicrobial peptides up-regulation. These findings were unexplored in the SOD1-ALS field.

Macrophages treated with interferons induce different responses in lymphocytes via extracellular vesicles.

Limited information exists regarding the impact of interferons (IFNs) on the information carried by extracellular vesicles (EVs). This study aimed at investigating whether IFN-α2b, IFN-ß, IFN-γ, and IFN-λ1/2 modulate the content of EVs released by primary monocyte-derived macrophages (MDM). Small-EVs (sEVs) were purified by size exclusion chromatography from supernatants of MDM treated with IFNs. To characterize the concentration and dimensions of vesicles, nanoparticle tracking analysis was used. SEVs surface markers were examined by flow cytometry. IFN treatments induced a significant down-regulation of the exosomal markers CD9, CD63, and CD81 on sEVs, and a significant modulation of some adhesion molecules, major histocompatibility complexes and pro-coagulant proteins, suggesting IFNs influence biogenesis and shape the immunological asset of sEVs. SEVs released by IFN-stimulated MDM also impact lymphocyte function, showing significant modulation of lymphocyte activation and IL-17 release. Altogether, our results show that sEVs composition and activity are affected by IFN treatment of MDM.

A minimal promoter region of Kit gene recapitulates mast cell differentiation in development, aging and inflammation.

To follow mast cells (MCs) distribution during aging and inflammation, we characterized two transgenic mouse models in which the EGFP expression is controlled by 9 kb or 12 kb of Kit gene promoter, defined as p18 and p70, respectively. We detected EGFP-positive cells in the serosal surfaces of the peritoneum, pleuras and pericardium, mucosal cavities, and connective tissue of almost all organs including gonads of p70, but not of p18 mice. By FACS and immunofluorescence for FcεR1, Kit and ß7-integrin, we found that these EGFP positive cells were MCs. In non-inflammatory conditions, a higher percentage of EGFP positive cells was found in juvenile with respect to adult serosal surfaces, but no differences between males and females at both developmental ages. We found, however, a striking difference in developing gonads, with low numbers of EGFP positive cells in fetal ovaries compared to age matched testes. Under inflammatory conditions caused by high fat diet (HFD), mice showed an increase in serosal EGFP positve cells. Altogether our results identify a regulatory region of the Kit gene, activated in MCs and that directing EGFP expression, can be employed to trace this immune cell type throughout the organism and in different animal conditions.

YY1 Knockdown Relieves the Differentiation Block and Restores Apoptosis in AML Cells.

In this study we analyzed the expression of Yin and Yang 1 protein (YY1), a member of the noncanonical PcG complexes, in AML patient samples and AML cell lines and the effect of YY1 downregulation on the AML differentiation block. Our results show that YY1 is significantly overexpressed in AML patient samples and AML cell lines and that YY1 knockdown relieves the differentiation block. YY1 downregulation in two AML cell lines (HL-60 and OCI-AML3) and one AML patient sample restored the expression of members of the CEBP protein family, increased the expression of extrinsic growth factors/receptors and surface antigenic markers, induced morphological cell characteristics typical of myeloid differentiation, and sensitized cells to retinoic acid treatment and to apoptosis. Overall, our data show that YY1 is not a secondary regulator of myeloid differentiation but that, if overexpressed, it can play a predominant role in myeloid differentiation block.

Cytogenetic and molecular diagnostic characterization combined to postconsolidation minimal residual disease assessment by flow cytometry improves risk stratification in adult acute myeloid leukemia.

A total of 143 adult acute myeloid leukemia (AML) patients with available karyotype (K) and FLT3 gene mutational status were assessed for minimal residual disease (MRD) by flow cytometry. Twenty-two (16%) patients had favorable, 115 (80%) intermediate, and 6 (4%) poor risk K; 19 of 129 (15%) carried FLT3-ITD mutation. Considering postconsolidation MRD status, patients with good/intermediate-risk K who were MRD(-) had 4-year relapse-free survival (RFS) of 70% and 63%, and overall survival (OS) of 84% and 67%, respectively. Patients with good- and intermediate-risk K who were MRD(+) had 4-year RFS of 15% and 17%, and OS of 38% and 23%, respectively (P < .001 for all comparisons). FLT3 wild-type patients achieving an MRD(-) status, had a better outcome than those who remained MRD(+) (4-year RFS, 54% vs 17% P < .001; OS, 60% vs 23%, P = .002). Such an approach redefined cytogenetic/genetic categories in 2 groups: (1) low-risk, including good/intermediate K-MRD(-) with 4-year RFS and OS of 58% and 73%, respectively; and (2) high risk, including poor-risk K, FLT3-ITD mutated cases, good/intermediate K-MRD(+) categories, with RFS and OS of 22% and 17%, respectively (P < .001 for all comparisons). In AML, the integrated evaluation of baseline prognosticators and MRD improves risk-assessment and optimizes postremission therapy.

TLR stimulation of prostate tumor cells induces chemokine-mediated recruitment of specific immune cell types.

TLRs boost antimicrobial response mechanisms by epithelial cells and represent the first line of defense at mucosal sites. In view of these immunomodulatory properties, TLR stimulation may represent a novel means to activate anticancer immune responses. In the present study, the ability of TLR ligands to affect the recruitment of different immune cell populations by human prostate cancer cell lines and the underlying mechanisms were investigated. We showed that LNCaP and DU-145 cells express functionally active TLR3 and TLR5. Treatment with their respective agonists, polyinosinic:polycytidylic acid and flagellin, rapidly triggered NF-kappaB-dependent upregulation of different inflammatory molecules, as assayed by microarray and ELISA. Furthermore, we demonstrated that conditioned media from polyinosinic:polycytidylic acid- and flagellin-treated LNCaP and DU-145 cells induced the recruitment of different leukocyte subpopulations, suggesting that TLR stimulation is able to activate the earliest step of immune response mediated by soluble factors. Interestingly, the more aggressive cancer cell line PC3 expressed TLR3 and TLR5 but failed to respond to TLR agonists in terms of NF-kappaB activation and the ability to attract immune effectors. Overall, these data show for the first time that TLR3 and TLR5 stimulation of human prostate cancer cells triggers the production of chemokines, which, in turn, favor the attraction of immune effectors, thereby representing a tool to enhance the efficacy of conventional therapies by stimulating anticancer immune responses.

CD161(high)CD8+T cells bear pathogenetic potential in multiple sclerosis.

To identify differentially expressed genes in multiple sclerosis, microarrays were used in a stringent experimental setting-leukapheresis from disease-discordant monozygotic twins and gene expression profiling in CD4(+) and CD8(+) T-cell subsets. Disease-related differences emerged only in the CD8(+) T-cell subset. The five differentially expressed genes identified included killer cell lectin-like receptor subfamily B, member 1, also known as natural killer receptor protein 1a/CD161, presented by the International Multiple Sclerosis Genetics Consortium as one of the non-MHC candidate loci. Flow cytometric analysis on peripheral blood of healthy donors and patients with multiple sclerosis and rheumatoid arthritis confirmed an upregulation of CD161 at the protein level, showing also a significant excess of CD161(high)CD8(+) T cells in multiple sclerosis. This subset prevalently included chemokine (C-C motif) receptor 6(+), cytokine-producing, effector-memory T cells with proinflammatory profiles. It also included all circulating interleukin-17(+)CD8(+) T cells. In the CD161(high)CD8(+) subset, interleukin-12 facilitated proliferation and interferon-γ production, with CD161 acting as a co-stimulatory receptor. CD161(+)CD8(+)CD3(+) T cells producing interferon-γ were part of intralesional immune infiltrates and ectopic B cell follicles in autopsy multiple sclerosis brains. Variations of CD161 expression on CD8(+) T cells identify a subset of lymphocytes with proinflammatory characteristics that have not been previously reported in multiple sclerosis and are likely to contribute to disease immunopathology.