Identifying Changes in Peripheral Lymphocyte Subpopulations in Adult Onset Type 1 Diabetes.

T- and B-lymphocytes play an important role in the pathogenesis of type 1 diabetes (T1D), a chronic disease caused by the autoimmune destruction of the insulin-producing cells in the pancreatic islets. Flow cytometry allows their characterization in peripheral blood, letting to investigate changes in cellular subpopulations that can provide insights in T1D pathophysiology. With this purpose, CD4+ and CD8+ T cells (including naïve, central memory, effector memory and terminally differentiated effector (TEMRA), Th17 and Tregs) and B cells subsets (naïve, unswitched memory, switched memory and transitional B cells) were analysed in peripheral blood of adult T1D patients at disease onset and after ≥2 years using multiparametric flow cytometry. Here we report changes in the percentage of early and late effector memory CD4+ and CD8+ T cells as well as of naïve subsets, regulatory T cells and transitional B cells in peripheral blood of adult patients at onset of T1D when compared with HD. After 2 years follow-up these changes were maintained. Also, we found a decrease in percentage of Th17 and numbers of T cells with baseline. In order to identify potential biomarkers of disease, ROC curves were performed being late EM CD4 T cell subset the most promising candidate. In conclusion, the observed changes in the percentage and/or absolute number of lymphocyte subpopulations of adult T1D patients support the hypothesis that effector cells migrate to the pancreas and this autoimmune process perseveres along the disease. Moreover, multiparametric flow allows to identify those subsets with potential to be considered biomarkers of disease.

In vivo Effects of Romidepsin on T-Cell Activation, Apoptosis and Function in the BCN02 HIV-1 Kick&Kill Clinical Trial.

Romidepsin (RMD) is a well-characterized histone deacetylase inhibitor approved for the treatment of cutaneous T-cell lymphoma. in vitro and in vivo studies have demonstrated that it is able to induce HIV-1 gene expression in latently infected CD4+ T cells from HIV-1+ individuals on suppressive antiretroviral therapy. However, in vitro experiments suggested that RMD could also impair T-cell functionality, particularly of activated T cells. Thus, the usefulness of RMD in HIV-1 kick&kill strategies, that aim to enhance the immune system elimination of infected cells after inducing HIV-1 viral reactivation, may be limited. In order to address whether the in vitro observations are replicated in vivo, we determined the effects of RMD on the total and HIV-1-specific T-cell populations in longitudinal samples from the BCN02 kick&kill clinical trial (NCT02616874). BCN02 was a proof-of-concept study in 15 early treated HIV-1+ individuals that combined MVA.HIVconsv vaccination with three weekly infusions of RMD given as a latency reversing agent. Our results show that RMD induced a transient increase in the frequency of apoptotic T cells and an enhanced activation of vaccine-induced T cells. Although RMD reduced the number of vaccine-elicited T cells secreting multiple cytokines, viral suppressive capacity of CD8+ T cells was preserved over the RMD treatment. These observations have important implications for the design of effective kick&kill strategies for the HIV-1 cure.

Multi-level immune response network in mild-moderate Chronic Obstructive Pulmonary Disease (COPD).

BACKGROUND: Chronic Obstructive Pulmonary Disease (COPD) is associated with an abnormal pulmonary and systemic immune response to tobacco smoking. Yet, how do immune cells relate within and between these two biological compartments, how the pulmonary infiltrate influences the lung transcriptome, and what is the role of active smoking vs. presence of disease is unclear. METHODS: To investigate these questions, we simultaneously collected lung tissue and blood from 65 individuals stratified by smoking habit and presence of the disease. The immune cell composition of both tissues was assessed by flow cytometry, whole lung transcriptome was determined with Affymetrix arrays, and we used Weighted Gene Co-expression Network Analysis (WGCNA) to integrate results. RESULTS: Main results showed that: (1) current smoking and the presence of COPD were both independently associated with a reduction in the proportion of lung T cells and an increase of macrophages, specifically those expressing CD80 + CD163+; (2) changes in the proportion of infiltrating macrophages, smoking status or the level of airflow limitation were associated to different WGCNA modules, which were enriched in iron ion transport, extracellular matrix and cilium organization gene ontologies; and, (3) circulating white blood cells counts were correlated with lung macrophages and T cells. CONCLUSIONS: Mild-moderated COPD lung immune infiltrate is associated with the active smoking status and presence of disease; is associated with changes in whole lung tissue transcriptome and marginally reflected in blood.

Head-to-head comparison of two engineered cardiac grafts for myocardial repair: From scaffold characterization to pre-clinical testing.

Cardiac tissue engineering, which combines cells and supportive scaffolds, is an emerging treatment for restoring cardiac function after myocardial infarction (MI), although, the optimal construct remains a challenge. We developed two engineered cardiac grafts, based on decellularized scaffolds from myocardial and pericardial tissues and repopulated them with adipose tissue mesenchymal stem cells (ATMSCs). The structure, macromechanical and micromechanical scaffold properties were preserved upon the decellularization and recellularization processes, except for recellularized myocardium micromechanics that was ∼2-fold stiffer than native tissue and decellularized scaffolds. Proteome characterization of the two acellular matrices showed enrichment of matrisome proteins and major cardiac extracellular matrix components, considerably higher for the recellularized pericardium. Moreover, the pericardial scaffold demonstrated better cell penetrance and retention, as well as a bigger pore size. Both engineered cardiac grafts were further evaluated in pre-clinical MI swine models. Forty days after graft implantation, swine treated with the engineered cardiac grafts showed significant ventricular function recovery. Irrespective of the scaffold origin or cell recolonization, all scaffolds integrated with the underlying myocardium and showed signs of neovascularization and nerve sprouting. Collectively, engineered cardiac grafts -with pericardial or myocardial scaffolds- were effective in restoring cardiac function post-MI, and pericardial scaffolds showed better structural integrity and recolonization capability.

Location of mammograms ROI's and reduction of false-positive.

BACKGROUND AND OBJECTIVE: There are many work related with segmentation techniques, including nearest neighbor algorithm, fuzzy rules, morphological filters, image entropy, thresholding, machine learning, wavelet analysis, and so on. Such methods carry out the segmentation, but take a lot of processing time by modifying the content of the image or showing discern problems in homogeneous areas, and the segmentation technique is designed to work efficiently only with the techniques used. In this paper a method to segment mammograms in order to separate breast area from pectoral-muscle avoiding bright areas that produce noise and therefore reducing false-positives is presented. METHODS: The proposed methodology is divided into four sections: 1) Pre-processing to acquire image and decreasing its size. 2) Improving the image quality through image thresholding and histogram equalization. 3) Localization of regions of interest (ROI) applying Scale-Invariant Feature Transform to find image's descriptors. Clustering methods were implemented to determine the best number of clusters and which of these represent the most significant breast area. Then found ROI's coordinates are compared with the position of abnormalities diagnosed by the Mammographic Image Analysis Society. 4) Microcalcifications (mcc) detection; wavelet transform is used, and to enhance its performance different high-pass filters and high-frequency emphasis filters are evaluated. Symlet wavelets: Sym8 and Sym16 were used with different decomposition level; images results from both processes are compared and only those elements in common are detected as microcalcifications. RESULTS: Moreover, muscle's remnants in the corners of the regions of interest were removed using fuzzy c-means clustering. The best results in terms of sensitivity (91.27), false-positives per image (80.25), and precision (74.38) are compared with previous work. CONCLUSIONS: Results shows that the breast area can be discriminated from the pectoral-muscle by avoiding to work with brightness areas that produces false positives. Moreover, because the image size is reduced the computer processing time will be decreased. This segmentation stage can be an addition to mammograms analysis broadly, not only to find mcc but abnormalities such as circumscribed masses, speculated masses and architectural distortion. Also is useful to create automatically an unsupervised segmentation in mammograms without stage of training.

Postinfarction Functional Recovery Driven by a Three-Dimensional Engineered Fibrin Patch Composed of Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells.

Considerable research has been dedicated to restoring myocardial cell slippage and limiting ventricular remodeling after myocardial infarction (MI). We examined the ability of a three-dimensional (3D) engineered fibrin patch filled with human umbilical cord blood-derived mesenchymal stem cells (UCBMSCs) to induce recovery of cardiac function after MI. The UCBMSCs were modified to coexpress luciferase and fluorescent protein reporters, mixed with fibrin, and applied as an adhesive, viable construct (fibrin-cell patch) over the infarcted myocardium in mice (MI-UCBMSC group). The patch adhered well to the heart. Noninvasive bioluminescence imaging demonstrated early proliferation and differentiation of UCBMSCs within the construct in the postinfarct mice in the MI-UCBMSC group. The implanted cells also participated in the formation of new, functional microvasculature that connected the fibrin-cell patch to both the subjacent myocardial tissue and the host circulatory system. As revealed by echocardiography, the left ventricular ejection fraction and fractional shortening at sacrifice were improved in MI-UCBMSC mice and were markedly reduced in mice treated with fibrin alone and untreated postinfarction controls. In conclusion, a 3D engineered fibrin patch composed of UCBMSCs attenuated infarct-derived cardiac dysfunction when transplanted locally over a myocardial wound.

Alternative effector-function profiling identifies broad HIV-specific T-cell responses in highly HIV-exposed individuals who remain uninfected.

The characterization of host immune responses to human immunodeficiency virus (HIV) in HIV controllers and individuals with high exposure but seronegativity to HIV (HESN) is needed to guide the development of effective preventive and therapeutic vaccine candidates. However, several technical hurdles severely limit the definition of an effective virus-specific T-cell response. By using a toggle-peptide approach, which takes HIV sequence diversity into account, and a novel, boosted cytokine staining/flow cytometry strategy, we here describe new patterns of T-cell responses to HIV that would be missed by standard assays. Importantly, this approach also allows detection of broad and strong virus-specific T-cell responses in HESN individuals that are characterized by a T-helper type 1 cytokine-like effector profile and produce cytokines that have been associated with potential control of HIV infection, including interleukin 10, interleukin 13, and interleukin 22. These results establish a novel approach to improve the current understanding of HIV-specific T-cell immunity and identify cellular immune responses and individual cytokines as potential markers of relative HIV resistance. As such, the findings also help develop similar strategies for more-comprehensive assessments of host immune responses to other human infections and immune-mediated disorders.

New insights into lipid raft function regulating myocardial vascularization competency in human idiopathic dilated cardiomyopathy.

OBJECTIVE: Idiopathic dilated cardiomyopathy (IDCM) affects myocardial vascularization. Whether a lack of demand for increased myocardial vascularization and/or an impaired response of circulating angiogenic-supportive cells are responsible for the vascular derangements found in IDCM is unknown. METHODS AND RESULTS: Left ventricle (LV) samples obtained at transplant from IDCM hearts were compared to control hearts from non-cardiac decedents. Peripheral colony-forming myeloid cells were extracted from age- and sex-matched IDCM patients and healthy volunteers. At the tissue level, no differences were detected in stromal cell-derived factor (SDF)-1α expression, but integrin-linked kinase (ILK) levels and activity were increased in IDCM. A marked co-localization of SDF-1α and the specific marker of cholesterol-enriched lipid rafts Flotillin (Flot)-1 was found in IDCM. SDF-1α was also highly distributed into IDCM lipid rafts. Non-adherent pro-angiogenic cells from both groups, which were found increased in patients but showed similar surface levels of CXCR-4, equally supported Matrigel-mediated cell network formation. However, SDF-1-mediated migration was reduced in IDCM-derived cells, which also exhibited decreased ILK activity and downstream ERK activation. CONCLUSIONS: Taken together, our results point out that myocardial competency to increase vascularization is not altered in IDCM, but dysfunctional SDF-1-mediated migration by peripheral pro-angiogenic cells through ILK and downstream ERK signaling may compromise endothelial recovery in patients. We provide new insights into lipid raft function in human IDCM and envision more effective treatments.

TLR-activated conventional DCs promote γ-secretase-mediated conditioning of plasmacytoid DCs.

Cooperative events between DC subsets involve cell contact and soluble factors. Upon viral challenge, murine pDCs induce cDC cooperation through CD40-CD40L interactions and IL-15 secretion, whereas in humans, the same effect is mediated by IFN-α. Conversely, during bacterial infections, pDC maturation may be induced by activated cDCs, although no mechanisms had been described so far. Here, we investigate how human pDCs are "conditioned" by cDCs. Blood-borne DC subsets (cDCs and pDCs) were sorted from healthy donors. IL-3-maintained pDCs were cocultured with LPS-activated, poly (I:C)-activated, or control cDCs [cDC(LPS), cDC(P(I:C)), cDC(CTRL)]. Coculture experiments showed that cDC(LPS)-conditioned pDCs up-regulated maturation markers, such as CD25 and CD86, whereas SNs contained higher amounts of IL-6 and CCL19 compared with control conditions. Gene-expression analyses on sorted cDC(LPS) or cDC(P(I:C)) conditioned pDCs confirmed the induction of several genes, including IL-6 and CCL19 and remarkably, several Notch target genes. Further studies using the γ-secretase/Notch inhibitor DAPT and soluble Notch ligands resulted in a significantly reduced expression of canonical Notch target genes in conditioned pDCs. DAPT treatment also hampered the secretion of CCL19 (but not of IL-6) by cDC(LPS) conditioned pDCs. These results reveal the involvement of γ-secretase-mediated mechanisms, including the Notch pathway, in the cell contact-dependent communication between human DC subsets. The resulting partial activation of pDCs after encountering with mature cDCs endows pDCs with an accessory function that may contribute to T cell recruitment and activation.

A modified host-cell reactivation assay to quantify DNA repair capacity in cryopreserved peripheral lymphocytes.

The host-cell reactivation assay (HCRA) is a functional assay that allows the identification of the genes responsible for DNA repair-deficient syndromes, such as Xeroderma pigmentosum, by cross-complementation experiments. It has also been used in molecular epidemiology studies to correlate the low nucleotide excision repair pathway function in peripheral blood lymphocytes with an increased risk of bladder, head and neck, skin and lung cancers. Herein, we present the technical validation of a newly modified HCRA, where nucleofection is used for the transfection of the pmaxGFP plasmid into cryopreserved peripheral blood lymphocytes (PBLs) or lymphoblastoid cell lines. In each sample, 20-24h after transfection, the relative DNA repair capacity (DRC) was quantified by flow cytometry, comparing the transfection efficiency of nucleoporated cells with undamaged plasmid to those transfected with UV-light damaged plasmid in the seven cell lines that were characterized by different DNA repair phenotypes. Dead cells were excluded from the analysis. We observed a high reproducibility of the relative DRC, transfection efficiency and cell viability. The inter-experimental normalization of the flow cytometry resulted in an increased data accuracy and reproducibility. The amount of cells required for each transfection reaction was reduced fourfold, without affecting the final relative DRC. Furthermore, our HCRA demonstrated strong discrimination power in the UV-light dose-response, both in lymphoblastoid cell lines and cryopreserved PBLs. We also observed a strong correlation of the relative DRC data, when samples were measured against two independent batches of both damaged and undamaged plasmid DNA. The relative DRC variable shows a normal distribution when analyzed in the cryopreserved PBLs from a cohort of 35 lung cancer patients and a 5.59-fold variation in the relative DRC is identified among our patients. The mitotic dynamic was discarded as a confounding factor for the relative DRC measurement in this cohort of patients. The results indicate that our method is highly sensitive, reliable and reproducible, and thus, it suitable for population-based studies to quantify in vitro DNA-repair deficiencies.

Expression and function of the IL-2 receptor in activated human plasmacytoid dendritic cells.

Human and mouse plasmacytoid dendritic cells (PDC) express IL-2 mRNA specifically upon TLR stimulation, but not under CD40L stimulation. Even though the expression of the IL-2R by PDC has been described, the functional implications of this expression remain unknown. Here, we investigated the expression and function of the IL-2R in activated human PDC. The IL-2Ralpha chain, CD25, is expressed in both CpG- and CD40L-activated PDC. CD25 expression is a relatively rapid event, as the receptor was detected 6 h after the initial activation signal. Exogenous IL-2 added to CD40L-activated PDC increased the expression of CD25, enhanced the secretion of pro-inflammatory cytokines and promotes PDC survival. CpG-activated PDC cultured in the presence of IL-2R-blocking monoclonal antibodies showed a reduced expression of pro-inflammatory cytokines, especially TNF-alpha. This reduction was dose and time dependent, suggesting a regulatory role of IL-2 in TNF secretion that might occur at the post-transcriptional level. These results indicate that the expression of the IL-2R is relevant to human PDC activation, and that IL-2 may be an important auto- and/or paracrine factor modulating the activation and survival of PDC. Finally, CD25 expression may be considered as a useful early activation marker for human PDC.

Human intestinal alphabeta IEL clones in celiac disease show reduced IL-10 synthesis and enhanced IL-2 production.

Celiac disease is a gluten-induced T-cell mediated autoimmune process that results in the destruction of the intestinal mucosa and is associated with an expansion of CD8(+) CD103(+) TCRalphabeta intraepithelial lymphocytes (IELs) in the damaged epithelium. The role of this IEL population in the pathology is unknown. The aim of this work was to compare the cytokine profile and the cytotoxicity pattern from CD8(+) IEL clones isolated from celiac (CD) and non-celiac (NCD) biopsies. We report that the number of IL-10 producing CD clones was significantly lower (26%) than that obtained from the NCD sample (62%). Instead, IL-2 was produced by more CD (44%) than NCD clones (26%). Cytotoxicity patterns against intestinal epithelial cell lines suggest different functional subsets of CD8(+) IELs. CD clones capable of high cytotoxicity produced IL-2 whereas most cytotoxic NCD IELs produced IL-10. This clonal analysis indicates that an impaired immune regulation in celiac mucosa may be partially attributed to the low generation of regulatory CD8(+) IELs that produce IL-10.

Valoración de APACHE II. Experiencia en dos grupos con patología neumológica / APACHE II assessment. Experience with two groups with pulmonary diseases

Un grupo de pacientes que fueron sujetos a procedimientos quirúrgicos de tórax y uno más con patología toracopulmonar diversa no quirúrgica, fueron estudiados en una unidad de Cuidados Intensivos Respiratorios (UCIR), y valorados con la escala de Evaluación Fisiológica Aguda y Crónica del Estado de Salud (APACHE II, por su siglas en inglés), a su ingreso y salida. Se registraron la mortalidad esperada (Knauss) y la observada. En los quirúrgicos la mortalidad fue de 11.8 por ciento (11/93) y en los no quirúrgicos de 50.9 por ciento (53/104) p< 0.01. Cuando la calificación inicial de APACHE II estaba elevada (más de 10.14), la mortalidad fue mayor. Las condiciones de admisión son determinantes para el pronóstico de sobrevida. APACHE II es una escala práctica para la valoración de los pacientes admitidos en una UCIR