Donor natural killer cells trigger production of ß-2-microglobulin to enhance post-bone marrow transplant immunity.

Allogeneic hematopoietic transplantation is a powerful treatment for hematologic malignancies. Posttransplant immune incompetence exposes patients to disease relapse and infections. We previously demonstrated that donor alloreactive natural killer (NK) cells ablate recipient hematopoietic targets, including leukemia. Here, in murine models, we show that infusion of donor alloreactive NK cells triggers recipient dendritic cells (DCs) to synthesize ß-2-microglobulin (B2M) that elicits the release of c-KIT ligand and interleukin-7 that greatly accelerate posttransplant immune reconstitution. An identical chain of events was reproduced by infusing supernatants of alloreactive NK/DC cocultures. Similarly, human alloreactive NK cells triggered human DCs to synthesize B2M that induced interleukin-7 production by thymic epithelial cells and thereby supported thymocyte cellularity in vitro. Chromatography fractionation of murine and human alloreactive NK/DC coculture supernatants identified a protein with molecular weight and isoelectric point of B2M, and mass spectrometry identified amino acid sequences specific of B2M. Anti-B2M antibody depletion of NK/DC coculture supernatants abrogated their immune-rebuilding effect. B2M knock-out mice were unable to undergo accelerated immune reconstitution, but infusion of (wild-type) NK/DC coculture supernatants restored their ability to undergo accelerated immune reconstitution. Similarly, silencing the B2M gene in human DCs, before coculture with alloreactive NK cells, prevented the increase in thymocyte cellularity in vitro. Finally, human recombinant B2M increased thymocyte cellularity in a thymic epithelial cells/thymocyte culture system. Our studies uncover a novel therapeutic principle for treating posttransplant immune incompetence and suggest that, upon its translation to the clinic, patients may benefit from adoptive transfer of large numbers of cytokine-activated, ex vivo-expanded donor alloreactive NK cells.

The Cross-Talk between Myeloid and Mesenchymal Stem Cells of Human Bone Marrow Represents a Biomarker of Aging That Regulates Immune Response and Bone Reabsorption.

One of the mechanisms that characterizes the aging process of different organs is the accumulation of fat. Different authors have demonstrated that adipose tissue replaces the loss of other cell types, deriving from mesenchymal cells. During aging, there is substitution or trans-differentiation of mesenchymal cells with other cells having the same embryological origin. Newly formed adipocytes were also observed in the trabecular matrix of elderly people's bones, associated with myeloid cells. In this study, we have investigated the relationship between immature myeloid-derived suppressor cells (I-MDSCs) and mesenchymal stem cells (MSCs) in bone marrow (BM) samples harvested from 57 patients subjected to different orthopedic surgeries. Patients aged from 18 to 92 years were considered in order to compare the cellular composition of bone marrow of young and elderly people, considered a biomarker of immunity, inflammation, and bone preservation. The I-MDSC percentage was stable during aging, but in elderly people, it was possible to observe a strong basal immunosuppression of autologous and heterologous T cells' proliferation. We hypothesized that this pattern observed in elders depends on the progressive accumulation in the BM of activating stimuli, including cell-cell contact, or the production of different cytokines and proteins that induce the differentiation of bone marrow mesenchymal stem cells in adipocytes. The collected data provided underline the importance of specific biomarkers of aging that promote a reduction in immune response and incremented inflammatory pathways, leading to bone reabsorption in elderly people.

Prostate-specific membrane antigen (PSMA) assembles a macromolecular complex regulating growth and survival of prostate cancer cells "in vitro" and correlating with progression "in vivo".

The expression of Prostate Specific-Membrane Antigen (PSMA) increases in high-grade prostate carcinoma envisaging a role in growth and progression. We show here that clustering PSMA at LNCaP or PC3-PSMA cell membrane activates AKT and MAPK pathways thus promoting proliferation and survival. PSMA activity was dependent on the assembly of a macromolecular complex including filamin A, beta1 integrin, p130CAS, c-Src and EGFR. Within this complex beta1 integrin became activated thereby inducing a c-Src-dependent EGFR phosphorylation at Y1086 and Y1173 EGF-independent residues. Silencing or blocking experiments with drugs demonstrated that all the complex components were required for full PSMA-dependent promotion of cell growth and/or survival in 3D culture, but that p130CAS and EGFR exerted a major role. All PSMA complex components were found assembled in multiple samples of two high-grade prostate carcinomas and associated with EGFR phosphorylation at Y1086. The expression of p130CAS and pEGFRY1086 was thus analysed by tissue micro array in 16 castration-resistant prostate carcinomas selected from 309 carcinomas and stratified from GS 3+4 to GS 5+5. Patients with Gleason Score ≤5 resulted negative whereas those with GS≥5 expressed p130CAS and pEGFRY1086 in 75% and 60% of the cases, respectively.Collectively, our results demonstrate for the first time that PSMA recruits a functionally active complex which is present in high-grade patients. In addition, two components of this complex, p130CAS and the novel pEGFRY1086, correlate with progression in castration-resistant patients and could be therefore useful in therapeutic or surveillance strategies of these patients.

An electrochemiluminescence-supramolecular approach to sarcosine detection for early diagnosis of prostate cancer.

Monitoring Prostate Cancer (PCa) biomarkers is an efficient way to diagnosis this disease early, since it improves the therapeutic success rate and suppresses PCa patient mortality: for this reason a powerful analytical technique such as electrochemiluminescence (ECL) is already used for this application, but its widespread usability is still hampered by the high cost of commercial ECL equipment. We describe an innovative approach for the selective and sensitive detection of the PCa biomarker sarcosine, obtained by a synergistic ECL-supramolecular approach, in which the free base form of sarcosine acts as co-reagent in a Ru(bpy)3(2+)-ECL process. We used magnetic micro-beads decorated with a supramolecular tetraphosphonate cavitand (Tiiii) for the selective capture of sarcosine hydrochloride in a complex matrix like urine. Sarcosine determination was then obtained with ECL measurements thanks to the complexation properties of Tiiii, with a protocol involving simple pH changes - to drive the capture-release process of sarcosine from the receptor - and magnetic micro-bead technology. With this approach we were able to measure sarcosine in the µM to mM window, a concentration range that encompasses the diagnostic urinary value of sarcosine in healthy subjects and PCa patients, respectively. These results indicate how this ECL-supramolecular approach is extremely promising for the detection of sarcosine and for PCa diagnosis and monitoring, and for the development of portable and more affordable devices.

Undiagnosed HIV infection in a population of MSM from six European cities: results from the Sialon project.

OBJECTIVES: The purpose of this article is to assess the distribution of undiagnosed HIV infection in men who have sex with men (MSM) in Southern and Eastern European countries, to describe the differences in epidemiology and behaviour between undiagnosed, diagnosed HIV-positive and HIV-negative MSM and to identify factors associated with undiagnosed HIV infection in the study population. METHODS: A multi-centre biological and behavioural cross-sectional study was conducted in 2008. Time-location sampling was used to recruit men attending different venues. A self-administered questionnaire was completed and oral fluid samples were collected to estimate HIV prevalence. RESULTS: HIV prevalence was 17% in Barcelona, 12% in Verona, 6% in Bratislava, 5% in Ljubljana, 5% in Bucharest and 3% in Prague while undiagnosed HIV infection was 47, 62, 67, 83, 85 and 57%, respectively. Diagnosed HIV-positive men reported more casual partners than HIV-negative MSM (mean: 19 and 9, respectively) (P < 0.001), and they were more likely to self-reported condyloma in the last year than undiagnosed HIV-positive and HIV-negative men (15, 1 and 3%, respectively) (P < 0.001). Factors associated with undiagnosed HIV infection included attending sex-focused venues (OR = 2.49), reporting syphilis in the previous 12 months (OR = 2.56), using poppers at last sexual intercourse (OR = 3.36) and having had an HIV test in the previous year (OR = 2.00). CONCLUSIONS: Many HIV infections remain undiagnosed, and there is evidence of the persistence of frequent risk behaviours and sexually transmitted infections (STI) despite knowledge of HIV-positive status, emphasising the need for a multidimensional approach to HIV/STI prevention. Access to HIV testing should be considered a priority in prevention programs targeted at MSM, especially in Eastern Europe.

Discriminatory Role of Detergent-Resistant Membranes in the Dimerization and Endocytosis of Prostate-Specific Membrane Antigen.

Prostate-specific membrane antigen (PSMA) is a type-II membrane glycoprotein that was initially identified in LNCaP cells. It is expressed at elevated levels in prostate cancer. In view of the correlation between the expression levels of PSMA and disease grade and stage, PSMA is considered to be one of the most promising biomarkers in the diagnosis and treatment of prostate cancer. In LNCaP cells PSMA undergoes internalization via clathrin-coated pits followed by accumulation in the endosomes. PSMA associates with different types of detergent-resistant membranes (DRMs) along the secretory pathway. Its mature form is mainly insoluble in Lubrol WX, but does not associate with Triton X-100-DRMs. To understand the mechanism of PSMA internalization we investigated its association during internalization with DRMs. For this purpose, internalization was induced by antibody cross-linking. We demonstrate at the biochemical and cell biological levels that: [i] exclusively homodimers of PSMA are associated with Lubrol WX-DRMs, [ii] antibody-induced cross-linking of PSMA molecules results in a time-dependent partitioning into another DRMs type, namely Triton X-100-DRMs, and [iii] concomitant with its association with Triton-X-100-DRMs internalization of PSMA occurs along tubulin filaments. In a previous work (Colombatti et al. (2009) PLoS One 4: e4608) we demonstrated that the small GTPases RAS and RAC1 and the MAPKs p38 and ERK1/2 are activated during antibody cross-linking. As downstream effects of this activation we observed a strong induction of NF-kB associated with an increased expression of IL-6 and CCL5 genes and that IL-6 and CCL5 enhanced the proliferative potential of LNCaP cells synergistically. These observations together with findings reported here hypothesize a fundamental role of DRMs during activation of PSMA as platforms for trafficking, endocytosis and signalling. Understanding these mechanisms constitutes an essential prerequisite for utilization of PSMA as a therapeutically suitable target in prostate cancer.

The proinflammatory cytokine, IL-6, and its interference with bFGF signaling and PSMA in prostate cancer cells.

The aim of the present work was to study the expression of the proinflammatory cytokine, interleukin-6 (IL-6), mediated by bFGF signaling and its possible crosstalk with prostate-specific membrane antigen (PSMA) in LNCaP and PC3-PSMA prostate cancer cell lines. PC3 cells stably transfected with PSMA gene were used for restoring PSMA expression. LNCaP and PC3-PSMA cells were exposed to 10 ng/mL of basic fibroblast growth factor (bFGF). IL-6 production was measured by ELISA assay, and levels of PSMA expression were assessed by flow cytometry. AKT, ERK1/2, and p38 phosphorylation were detected by Western blot. bFGF enhances IL-6 production in LNCaP and PC3-PSMA prostate cancer cells. The effect of bFGF on stimulating IL-6 secretion was greater in LNCaP than in PC3-PSMA cells. In the presence of bFGF, PSMA expression was activated after 4 days of treatment in LNCaP and PC3-PSMA cells. This activation was not maintained after long term of treatment in both metastatic cell lines. Solely MAPKs pathways (ERK1/2 and p38) were activated after bFGF stimulation in both metastatic cell lines, whereas AKT did not show any activation. The interference of the proinflammatory cytokine, IL-6, with bFGF signaling and PSMA, should be of high clinical relevance in the treatment of metastatic prostate cancer. In developing novel therapeutic modalities targeting IL-6, significant attention should be given to PSMA and its inactivation to fight against prostate cancer.

The puzzling uniqueness of the heterotrimeric G15 protein and its potential beyond hematopoiesis.

Heterotrimeric G proteins transduce the signals of the largest family of membrane receptors (G protein-coupled receptors, GPCRs) hence triggering the activation of a wide variety of physiological responses. G15 is a G protein characterized by a number of functional peculiarities that make its signaling exceptional: 1) it can couple a variety of Gs-, Gi/o-, and Gq-linked receptors to phospholipase C activation; 2) relatively to other G proteins, it is poorly affected by beta-arrestin-dependent desensitization, the general mechanism that regulates GPCR function and 3) at the protein level, its expression is only detected in highly specific cell types (hematopoietic and epithelial cells). G15 alpha-subunit displays unique structural and biochemical properties, and is phylogenetically the most recent and divergent component of the Galphaq/11 subfamily. All these aspects shed a mysterious light on G15 biological role, which remains substantially elusive. Thus, far, G15 signaling has been analyzed in the context of hematopoiesis. Here, we highlight observations supporting the view that G15 functions may extend further beyond the immune system. In addition, we describe puzzling aspects of G15 signaling that offer a novel perspective in the understanding of its physiological role.

The prostate specific membrane antigen regulates the expression of IL-6 and CCL5 in prostate tumour cells by activating the MAPK pathways.

The interleukin-6 (IL-6) and the chemokine CCL5 are implicated in the development and progression of several forms of tumours including that of the prostate. The expression of the prostate specific membrane antigen (PSMA) is augmented in high-grade and metastatic tumors. Observations of the clinical behaviour of prostate tumors suggest that the increased secretion of IL-6 and CCL5 and the higher expression of PSMA may be correlated. We hypothesized that PSMA could be endowed with signalling properties and that its stimulation might impact on the regulation of the gene expression of IL-6 and CCL5. We herein demonstrate that the cross-linking of cell surface PSMA with specific antibodies activates the small GTPases RAS and RAC1 and the MAPKs p38 and ERK1/2 in prostate carcinoma LNCaP cells. As downstream effects of the PSMA-fostered RAS-RAC1-MAPK pathway activation we observed a strong induction of NF-kappaB activation associated with an increased expression of IL-6 and CCL5 genes. Pharmacological blockade with specific inhibitors revealed that both p38 and ERK1/2 participate in the phenomenon, although a major role exerted by p38 was evident. Finally we demonstrate that IL-6 and CCL5 enhanced the proliferative potential of LNCaP cells synergistically and in a dose-dependent manner and that CCL5 functioned by receptor-mediated activation of the STAT5-Cyclin D1 pro-proliferative pathway. The novel functions attributable to PSMA which are described in the present report may have profound influence on the survival and proliferation of prostate tumor cells, accounting for the observation that PSMA overexpression in prostate cancer patients is related to a worse prognosis.

Constitutive activation of p38 and ERK1/2 MAPKs in epithelial cells of myasthenic thymus leads to IL-6 and RANTES overexpression: effects on survival and migration of peripheral T and B cells.

Myasthenia gravis (MG) is an autoimmune disease of neuromuscular junctions where thymus plays a pathogenetic role. Thymectomy benefits patients, and thymic hyperplasia, a lymphoid infiltration of perivascular spaces becoming site of autoantibody production, is recurrently observed. Cytokines and chemokines, produced by thymic epithelium and supporting survival and migration of T and B cells, are likely to be of great relevance in pathogenesis of thymic hyperplasia. In thymic epithelial cell (TEC) cultures derived "in vitro" from normal or hyperplastic age-matched MG thymuses, we demonstrate by gene profiling analysis that MG-TEC basally overexpress genes coding for p38 and ERK1/2 MAPKs and for components of their signaling pathways. Immunoblotting experiments confirmed that p38 and ERK1/2 proteins were overexpressed in MG-TEC and, in addition, constitutively activated. Pharmacological blockage with specific inhibitors confirmed their role in the control of IL-6 and RANTES gene expression. According to our results, IL-6 and RANTES levels were abnormally augmented in MG-TEC, either basally or upon induction by adhesion-related stimuli. The finding that IL-6 and RANTES modulate, respectively, survival and migration of peripheral lymphocytes of myasthenic patients point to MAPK transcriptional and posttranscriptional abnormalities of MG-TEC as a key step in the pathological remodelling of myasthenic thymus.

p38 MAPK is a critical regulator of the constitutive and the beta4 integrin-regulated expression of IL-6 in human normal thymic epithelial cells.

Cytokines and adhesion receptors are key mediators in the dialog occurring between thymic epithelial cells (TEC) and thymocytes and regulating T cell maturation and epithelial embryonic differentiation. Among cytokines, IL-6 can be critical in the thymus, fostering proliferation, differentiation and/or survival of both TEC and thymocytes. We have previously reported in human normal TEC that clustering of the laminin receptor alpha6beta4 integrin induced by thymocyte contact or monoclonal antibody-mediated cross-linking regulates IL-6 gene expression via activation of NF-kappaB and NF-IL6 transactivators. Here we show that alpha6beta4 integrin activates p38 mitogen-activated protein kinase (MAPK) and that p38 is essential for IL-6 gene expression. In fact, beta4 cross-linking activated p38 and extracellular signal-regulated kinase (ERK) MAPK, Rac1, p21-activated protein kinase 1 (PAK1) and MAPK kinases (MKK) 3/MKK6. However, pharmacological blockade of p38 or ERK demonstrated that p38 inhibition abrogated both basal and beta4 integrin-induced production of IL-6 preventing NF-kappaB and NF-IL6 activation, whereas ERK inhibition reduced IL-6 production, hampering only NF-kappaB activation. Overall, our results indicate that p38 MAPK and alpha6beta4 integrin, expressed by TEC throughout their life, are critical regulators of the intrathymic availability of a cytokine controlling fate and functions of cells governing development and maintenance of thymic architecture and immune responses.

Death of T cell precursors in the human thymus: a role for CD38.

Thymic T cell maturation depends on interactions between thymocytes and cells of epithelial and hematopoietic lineages that control a selective process whereby developing T cells with inappropriate or self-reactive receptors die. Molecules involved in this process are the TCR expressed on thymocytes together with the CD3 complex and MHC-peptide on accessory cells. However, other molecules may favor or prevent death of thymocytes, thus playing a role in selection. CD38 is expressed by the majority of human thymocytes, mainly at the double-positive (DP) stage. In contrast, CD38 is not found on subcapsular double-negative (DN) thymocytes and on a proportion of medullary single-positive (SP) thymocytes. CD38 enhances death of thymocytes when it is cross-linked by goat anti-mouse (GAM) antiserum or by one of its ligands, CD31, expressed by thymic epithelial cells or transfected into murine fibroblasts (L cells). As most thymocytes are at an intermediate (DP) stage of development, it is likely that these cells are most vulnerable to death mediated via MHC-peptide-TCR interactions that is increased by CD38 cross-linking. DN and SP thymocytes are refractory to CD38-induced apoptosis. Accessory molecules, e.g. CD38, are expressed during thymic cell maturation and their presence is relevant for the survival or death of DP T cells in the course of selection. Based on our data, CD38 enhances thymocyte death by interacting with CD31 expressed by accessory cells. In addition, CD28 expression on developing thymocytes also appears to play a role for their selection and it synergizes with CD38 to induce apoptosis of DP thymocytes.