Optimized protocol for the detection of multifunctional epitope-specific CD4+ T cells combining MHC-II tetramer and intracellular cytokine staining technologies

Analysis of multifunctional CD4+ T cells is fundamental for characterizing the immune responses to vaccination or infection. Major histocompatibility complex (MHC)/peptide tetramers represent a powerful technology for the detection of antigen-specific T cells by specific binding to their T-cell receptor, and their combination with functional assays is fundamental for characterizing the antigen-specific immune response. Here we optimized a protocol for the detection of multiple intracellular cytokines within epitope-specific CD4+ T cells identified by the MHC class II tetramer technology. The optimal procedure for assessing the functional activity of tetramer-binding CD4+ T cells was based on the simultaneous intracellular staining with both MHC tetramers and cytokine-specific antibodies upon in vitro restimulation of cells with the vaccine antigen. The protocol was selected among procedures that differently combine the steps of cellular restimulation and tetramer staining with intracellular cytokine labeling. This method can be applied to better understand the complex functional profile of CD4+ T-cell responses upon vaccination or infection

In Chronic Lymphocytic Leukemia the JAK2/STAT3 Pathway Is Constitutively Activated and Its Inhibition Leads to CLL Cell Death Unaffected by the Protective Bone Marrow Microenvironment

The bone marrow microenvironment promotes proliferation and drug resistance in chronic lymphocytic leukemia (CLL). Although ibrutinib is active in CLL, it is rarely able to clear leukemic cells protected by bone marrow mesenchymal stromal cells (BMSCs) within the marrow niche. We investigated the modulation of JAK2/STAT3 pathway in CLL by BMSCs and its targeting with AG490 (JAK2 inhibitor) or Stattic (STAT3 inhibitor). B cells collected from controls and CLL patients, were treated with medium alone, ibrutinib, JAK/Signal Transducer and Activator of Transcription (STAT) inhibitors, or both drugs, in the presence of absence of BMSCs. JAK2/STAT3 axis was evaluated by western blotting, flow cytometry, and confocal microscopy. We demonstrated that STAT3 was phosphorylated in Tyr705 in the majority of CLL patients at basal condition, and increased following co-cultures with BMSCs or IL-6. Treatment with AG490, but not Stattic, caused STAT3 and Lyn dephosphorylation, through re-activation of SHP-1, and triggered CLL apoptosis even when leukemic cells were cultured on BMSC layers. Moreover, while BMSCs hamper ibrutinib activity, the combination of ibrutinib+JAK/STAT inhibitors increase ibrutinib-mediated leukemic cell death, bypassing the pro-survival stimuli derived from BMSCs. We herein provide evidence that JAK2/STAT3 signaling might play a key role in the regulation of CLL-BMSC interactions and its inhibition enhances ibrutinib, counteracting the bone marrow niche

Old open clusters and the Galactic metallicity gradient: Berkeley 20, Berkeley 66, and Tombaugh 2

To study the crucial range of Galactocentric distances between 12 and 16 kpc, where little information is available, we have obtained VI CCD imaging of Berkeley 20 and BVI CCD imaging of Berkeley 66 and Tombaugh 2, three distant, old open clusters. Using the synthetic colour magnitude diagram (CMD) technique with three types of evolutionary tracks of different metallicities, we have determined age, distance, reddening and indicative metallicity of these systems. The CMD of Be 20 is best reproduced by stellar models with a metallicity about half of solar (Z=0.008 or 0.01), in perfect agreement with high resolution spectroscopic estimates. Its age is between 5 and 6 Gyr from stellar models with overshooting and between 4.3 and 4.5 Gyr from models without it. The distance modulus from the best fitting models is always (m-M)0=14.7 (corresponding to a Galactocentric radius of about 16 kpc), and the reddening E(B-V) ranges between 0.13 and 0.16. A slightly lower metallicity (Z ~ 0.006) appears to be more appropriate for Be 66. This cluster is younger, (age of 3 Gyr), and closer, (m-M)0=13.3 (i.e., at 12 kpc from the Galactic centre), than Be 20, and suffers from high extinction, 1.2 < E(B-V) < 1.3, variable at the 2-3 per cent level. Finally, the results for To 2 indicate that it is an intermediate age cluster, with an age of about 1.4 Gyr or 1.6-1.8 Gyr for models without and with overshooting, respectively. The metallicity is about half of solar (Z=0.006 to 0.01), in agreement with spectroscopic determinations. The distance modulus is (m-M)0=14.5, implying a distance of about 14 kpc from the Galactic centre; the reddening E(B-V) is 0.31-0.4, depending on the model and metallicity, with a preferred value around 0.34.Comment: 19 pages, 19 figures, 8 tables Accepted 2010 November 5. Received 2010 Novembe

Age Determination of Six Intermediate-age SMC Star Clusters with HST/ACS

We present a photometric analysis of the star clusters Lindsay 1, Kron 3, NGC339, NGC416, Lindsay 38, and NGC419 in the Small Magellanic Cloud (SMC), observed with the Hubble Space Telescope Advanced Camera for Surveys (ACS) in the F555W and F814W filters. Our color magnitude diagrams (CMDs) extend ~3.5 mag deeper than the main-sequence turnoff points, deeper than any previous data. Cluster ages were derived using three different isochrone models: Padova, Teramo, and Dartmouth, which are all available in the ACS photometric system. Fitting observed ridgelines for each cluster, we provide a homogeneous and unique set of low-metallicity, single-age fiducial isochrones. The cluster CMDs are best approximated by the Dartmouth isochrones for all clusters, except for NGC419 where the Padova isochrones provided the best fit. The CMD of NGC419 shows several main-sequence turn-offs, which belong to the cluster and to the SMC field. We thus derive an age range of 1.2-1.6 Gyr for NGC419. Interestingly, our intermediate-age star clusters have a metallicity spread of ~0.6 dex, which demonstrates that the SMC does not have a smooth, monotonic age-metallicity relation. We find an indication for centrally concentrated blue straggler star candidates in NGC416, while for the other clusters these are not present. Using the red clump magnitudes, we find that the closest cluster, NGC419 (~50kpc), and the farthest cluster, Lindsay 38 (~67kpc), have a relative distance of ~17kpc, which confirms the large depth of the SMC.Comment: 25 pages, 45 Figure

The chemical evolution of Omega Centauri's progenitor system

Chemical evolution models are presented for the anomalous globular cluster Omega Centauri. After demonstrating that the chemical features of Omega Cen can not be reproduced in the framework of the closed-box self-enrichment scenario, we discuss a model in which this cluster is the remnant of a dwarf spheroidal galaxy evolved in isolation and then swallowed by the Milky Way. Both infall of primordial matter and metal-enriched gas outflows have to be considered in order to reproduce the stellar metallicity distribution function, the age-metallicity relation and several abundance ratios. Yet, as long as an ordinary stellar mass function and standard stellar yields are assumed, we fail by far to get the enormous helium enhancement required to explain the blue main sequence (and, perhaps, the extreme horizontal branch) stellar data. Rotating models of massive stars producing stellar winds with large helium excesses at low metallicities have been put forward as promising candidates to solve the `helium enigma' of Omega Cen (Maeder & Meynet, 2006, A&A, 448, L37). However, we show that for any reasonable choice of the initial mass function the helium-to-metal enrichment of the integrated stellar population is unavoidably much lower than 70 and conclude that the issue of the helium enhancement in Omega Cen still waits for a satisfactory explanation. We briefly speculate upon possible solutions.Comment: 11 pages, 9 figures; accepted for publication in MNRA

Pro-inflammatory cells sustain leukemic clonal expansion in T-cell large granular lymphocyte leukemia

T-cell large granular lymphocyte leukemia (T-LGLL) is a chronic lymphoproliferative disorder characterized by the clonal expansion of T-cell large granular lymphocytes (T-LGL). Immunophenotypic and genotypic features contribute to discriminate symptomatic (CD8+ STAT3-mutated T-LGLL) from clinically indolent patients, this latter group including CD8+ wildtype (wt), CD4+ STAT5B-mutated and wt cases. T-LGL lymphoproliferation is sustained both by somatic gain-offunction mutations (i.e., STAT3 and STAT5B) and by pro-inflammatory cytokines, but little information is available on the activity of T-LGLL non-leukemic cells. In this study, we characterized pro-inflammatory cells in the peripheral blood of T-LGLL patients and analyzed their role in supporting the leukemic growth. In symptomatic patients we found that cell populations not belonging to the leukemic component showed a discrete pro-inflammatory pattern. In particular, CD8+ STAT3-mutated cases showed a skewed Th17/Treg ratio and an abnormal distribution of monocyte populations characterized by increased intermediate and non-classical monocytes. We also demonstrated that monocytes released high levels of interleukin-6 after CCL5 stimulation, a chemokine specifically expressed only by leukemic LGL. Conversely, in asymptomatic cases an altered distribution of monocyte populations was not detected. Moreover, T-LGLL patients’ monocytes showed abnormal activation of signaling pathways, further supporting the different pathogenic role of monocytes in patients in discrete clinical settings. Altogether, our data contribute to deepening the knowledge on the different cell subtypes in T-LGLL, focusing particularly on non-leukemic cell populations and thus offering the rationale for new therapeutic strategies

Psychological treatments and psychotherapies in the neurorehabilitation of pain. Evidences and recommendations from the italian consensus conference on pain in neurorehabilitation

BACKGROUND: It is increasingly recognized that treating pain is crucial for effective care within neurological rehabilitation in the setting of the neurological rehabilitation. The Italian Consensus Conference on Pain in Neurorehabilitation was constituted with the purpose identifying best practices for us in this context. Along with drug therapies and physical interventions, psychological treatments have been proven to be some of the most valuable tools that can be used within a multidisciplinary approach for fostering a reduction in pain intensity. However, there is a need to elucidate what forms of psychotherapy could be effectively matched with the specific pathologies that are typically addressed by neurorehabilitation teams. OBJECTIVES: To extensively assess the available evidence which supports the use of psychological therapies for pain reduction in neurological diseases. METHODS: A systematic review of the studies evaluating the effect of psychotherapies on pain intensity in neurological disorders was performed through an electronic search using PUBMED, EMBASE, and the Cochrane Database of Systematic Reviews. Based on the level of evidence of the included studies, recommendations were outlined separately for the different conditions. RESULTS: The literature search yielded 2352 results and the final database included 400 articles. The overall strength of the recommendations was medium/low. The different forms of psychological interventions, including Cognitive-Behavioral Therapy, cognitive or behavioral techniques, Mindfulness, hypnosis, Acceptance and Commitment Therapy (ACT), Brief Interpersonal Therapy, virtual reality interventions, various forms of biofeedback and mirror therapy were found to be effective for pain reduction in pathologies such as musculoskeletal pain, fibromyalgia, Complex Regional Pain Syndrome, Central Post-Stroke pain, Phantom Limb Pain, pain secondary to Spinal Cord Injury, multiple sclerosis and other debilitating syndromes, diabetic neuropathy, Medically Unexplained Symptoms, migraine and headache. CONCLUSIONS: Psychological interventions and psychotherapies are safe and effective treatments that can be used within an integrated approach for patients undergoing neurological rehabilitation for pain. The different interventions can be specifically selected depending on the disease being treated. A table of evidence and recommendations from the Italian Consensus Conference on Pain in Neurorehabilitation is also provided in the final part of the pape

HS1, a Lyn Kinase Substrate, Is Abnormally Expressed in B-Chronic Lymphocytic Leukemia and Correlates with Response to Fludarabine-Based Regimen

In B-Chronic Lymphocytic Leukemia (B-CLL) kinase Lyn is overexpressed, active, abnormally distributed, and part of a cytosolic complex involving hematopoietic lineage cell-specific protein 1 (HS1). These aberrant properties of Lyn could partially explain leukemic cells’ defective apoptosis, directly or through its substrates, for example, HS1 that has been associated to apoptosis in different cell types. To verify the hypothesis of HS1 involvement in Lyn-mediated leukemic cell survival, we investigated HS1 protein in 71 untreated B-CLL patients and 26 healthy controls. We found HS1 overexpressed in leukemic as compared to normal B lymphocytes (1.38±0.54 vs 0.86±0.29, p<0.01), and when HS1 levels were correlated to clinical parameters we found a higher expression of HS1 in poor-prognosis patients. Moreover, HS1 levels significantly decreased in ex vivo leukemic cells of patients responding to a fludarabine-containing regimen. We also observed that HS1 is partially localized in the nucleus of neoplastic B cells. All these data add new information on HS1 study, hypothesizing a pivotal role of HS1 in Lyn-mediated modulation of leukemic cells’ survival and focusing, one more time, the attention on the BCR-Lyn axis as a putative target for new therapeutic strategies in this disorder

Safety and immunogenicity of ChAd63-KH vaccine in post-kala-azar dermal leishmaniasis patients in Sudan

Post-kala-azar dermal leishmaniasis (PKDL) is a chronic, stigmatizing skin condition occurring frequently after apparent clinical cure from visceral leishmaniasis. Given an urgent need for new treatments, we conducted a phase IIa safety and immunogenicity trial of ChAd63-KH vaccine in Sudanese patients with persistent PKDL. LEISH2a (ClinicalTrials.gov: NCT02894008) was an open-label three-phase clinical trial involving sixteen adult and eight adolescent patients with persistent PKDL (median duration, 30 months; range, 6-180 months). Patients received a single intramuscular vaccination of 1 × 1010 viral particles (v.p.; adults only) or 7.5 × 1010 v.p. (adults and adolescents), with primary (safety) and secondary (clinical response and immunogenicity) endpoints evaluated over 42-120 days follow-up. AmBisome was provided to patients with significant remaining disease at their last visit. ChAd63-KH vaccine showed minimal adverse reactions in PKDL patients and induced potent innate and cell-mediated immune responses measured by whole-blood transcriptomics and ELISpot. 7/23 patients (30.4%) monitored to study completion showed >90% clinical improvement, and 5/23 (21.7%) showed partial improvement. A logistic regression model applied to blood transcriptomic data identified immune modules predictive of patients with >90% clinical improvement. A randomized controlled trial to determine whether these clinical responses were vaccine-related and whether ChAd63-KH vaccine has clinical utility is underway