Deep genomic analysis of malignant peripheral nerve sheath tumor cell lines challenges current malignant peripheral nerve sheath tumor diagnosis.

Malignant peripheral nerve sheath tumors (MPNSTs) are soft-tissue sarcomas of the peripheral nervous system that develop either sporadically or in the context of neurofibromatosis type 1 (NF1). MPNST diagnosis can be challenging and treatment outcomes are poor. We present here a resource consisting of the genomic characterization of 9 widely used human MPNST cell lines for their use in translational research. NF1-related cell lines recapitulated primary MPNST copy number profiles, exhibited NF1, CDKN2A, and SUZ12/EED tumor suppressor gene (TSG) inactivation, and presented no gain-of-function mutations. In contrast, sporadic cell lines collectively displayed different TSG inactivation patterns and presented kinase-activating mutations, fusion genes, altered mutational frequencies and COSMIC signatures, and different methylome-based classifications. Cell lines re-classified as melanomas and other sarcomas exhibited a different drug-treatment response. Deep genomic analysis, methylome-based classification, and cell-identity marker expression, challenged the identity of common MPNST cell lines, opening an opportunity to revise MPNST differential diagnosis.

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.

KIF11 and KIF15 mitotic kinesins are potential therapeutic vulnerabilities for malignant peripheral nerve sheath tumors.

BACKGROUND: Malignant peripheral nerve sheath tumor (MPNST) constitutes the leading cause of neurofibromatosis type 1-related mortality. MPNSTs contain highly rearranged hyperploid genomes and exhibit a high division rate and aggressiveness. We have studied in vitro whether the mitotic kinesins KIF11, KIF15, and KIF23 have a functional role in maintaining MPNST cell survival and can represent potential therapeutic vulnerabilities. METHODS: We studied the expression of kinesin mRNAs and proteins in tumors and cell lines and used several in vitro functional assays to analyze the impact of kinesin genetic suppression (KIF15, KIF23) and drug inhibition (KIF11) in MPNST cells. We also performed in vitro combined treatments targeting KIF11 together with other described MPNST targets. RESULTS: The studied kinesins were overexpressed in MPNST samples. KIF15 and KIF23 were required for the survival of MPNST cell lines, which were also more sensitive than benign control fibroblasts to the KIF11 inhibitors ispinesib and ARRY-520. Co-targeting KIF11 and BRD4 with ARRY-520 and JQ1 reduced MPNST cell viability, synergistically killing a much higher proportion of MPNST cells than control fibroblasts. In addition, genetic suppression of KIF15 conferred an increased sensitivity to KIF11 inhibitors alone or in combination with JQ1. CONCLUSIONS: The mitotic spindle kinesins KIF11 and KIF15 and the cytokinetic kinesin KIF23 play a clear role in maintaining MPNST cell survival and may represent potential therapeutic vulnerabilities. Although further in vivo evidences are still mandatory, we propose a simultaneous suppression of KIF11, KIF15, and BRD4 as a potential therapy for MPNSTs.

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.

Type 1 Diabetes Prevention in NOD Mice by Targeting DPPIV/CD26 Is Associated with Changes in CD8⁺T Effector Memory Subset.

CD26 is a T cell activation marker consisting in a type II transmembrane glycoprotein with dipeptidyl peptidase IV (DPPIV) activity in its extracellular domain. It has been described that DPPIV inhibition delays the onset of type 1 diabetes and reverses the disease in non-obese diabetic (NOD) mice. The aim of the present study was to assess the effect of MK626, a DPPIV inhibitor, in type 1 diabetes incidence and in T lymphocyte subsets at central and peripheral compartments. Pre-diabetic NOD mice were treated with MK626. Diabetes incidence, insulitis score, and phenotyping of T lymphocytes in the thymus, spleen and pancreatic lymph nodes were determined after 4 and 6 weeks of treatment, as well as alterations in the expression of genes encoding ß-cell autoantigens in the islets. The effect of MK626 was also assessed in two in vitro assays to determine proliferative and immunosuppressive effects. Results show that MK626 treatment reduces type 1 diabetes incidence and after 6 weeks of treatment reduces insulitis. No differences were observed in the percentage of T lymphocyte subsets from central and peripheral compartments between treated and control mice. MK626 increased the expression of CD26 in CD8+ T effector memory (TEM) from spleen and pancreatic lymph nodes and in CD8+ T cells from islet infiltration. CD8+TEM cells showed an increased proliferation rate and cytokine secretion in the presence of MK626. Moreover, the combination of CD8+ TEM cells and MK626 induces an immunosuppressive response. In conclusion, treatment with the DPPIV inhibitor MK626 prevents experimental type 1 diabetes in association to increase expression of CD26 in the CD8+ TEM lymphocyte subset. In vitro assays suggest an immunoregulatory role of CD8+ TEM cells that may be involved in the protection against autoimmunity to ß pancreatic islets associated to DPPIV inhibitor treatment.

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.

Comparison of lymphocyte isolation methods for endoscopic biopsy specimens from the colonic mucosa.

An ideal method of immune cell isolation should provide maximum cell yield without disturbing functional properties. Intestinal endoscopic biopsies, in contrast to surgical samples, allow the study of all disease stages but have the drawback of a minimum amount of tissue available, making protocol optimization mandatory. We compared for the first time two methods of separation of colonic epithelium and five methods of lamina propria cell isolation for colonic biopsy specimens (mechanical, enzymatic and organ culture protocols). Lymphocyte number, viability and phenotype (CD45+, CD103+, CD3+, CD4+, CD8+, CD19+, CD16-56+) were analyzed by flow cytometry. Neither of the two epithelial detachment protocols achieved proper epithelial separation, though the high intensity ion chelation method was more accurate. Maximum cell yield of lamina propria lymphocytes without phenotypic modification was obtained with overnight smooth enzymatic digestion. High dose collagenase incubation caused a marked decrease in CD4+ lymphocytes of the lamina propria as compared to low enzymatic method (p=0.004). Mechanical and biopsy culture are not advisable methods because of the low cell yield, and phenotypic alterations and high contamination rate, respectively.

Morbilidad materno-perinatal de la rotura prematura de membranas en el embarazo pretérmino en el Hospital Nacional Dos de Mayo. 2011-2012 / Maternal and perinatal morbidity of premature rupture of membranes in preterm pregnancy at the National Hospital Dos de Mayo. 2011-2012

Antecedentes: La morbilidad asociada a la ruptura prematura de membranas puede ser mayor en neonatos pretérmino. Objetivo: Determinar la morbilidad materno-perinatal de la Rotura Prematura de Membranas Pretérmino en el Hospital Nacional Dos de Mayo en el periodo 2011-2012. Métodos: Revisión documentaria de las historias clínicas de las madres y neonatos prematuros que cumplieron criterios de selección, encontrando 170 casos en el periodo de estudio. Se muestran los resultados mediante estadística descriptiva. Resultados: La edad de las madres de neonatos prematuros con RPM fue 25,52 años. La forma de terminación del parto en 34,12 por ciento fue la vía vaginal, y en 65,88 por ciento por cesárea. El tiempo de latencia desde la RPM al parto en 9,41 por ciento fue dentro de las 6 primeras horas, en 21,18 por ciento dentro de las primeras 12 horas, y en 69,41 por ciento luego de las 24horas. Se emplearon corticoides para maduración pulmonar en 46,47 por ciento de casos, todos ellos con dexametasona, y en 27,65 por ciento se completó cuatro cursos. El 47,65 por ciento de neonatos fueron varones y 52,35 por ciento mujeres. En 82,35 por ciento de casos el líquido amniótico fue claro, 15,88 por ciento fue verde claro y en 1,76 por ciento fue purulento. No se usó antibióticos en 10 por ciento de casos, se usó sólo un antibiótico en 48,24 por ciento, dos antibióticos en 24,71 por ciento y tres antibióticos en 17,06 por ciento; el antibiótico más empleado fue la cefazolina. En 45,29 por ciento de madres se presentó endometritis; la duración de la hospitalización en las madres fue 5,73 días. El 14,71 por ciento de neonatos pretérmino fue llevado a alojamiento conjunto con la madre, 29,41 por ciento llegó a cuidados intermedios y 55,88 por ciento pasó a UCI; la indicación de hospitalización en UCI fue la sepsis en 55,79 por ciento, insuficiencia respiratoria en 20 por ciento, prematuridad extrema en 12,63 por ciento. Un 22,35 por ciento de neonatos no requirieron de apoyo...

Background: The morbidity associated with premature rupture of membranes may be higher in preterm infants. Objective: To determine maternal and perinatal morbidity of Premature Rupture of Membranes at the National Hospital Dos de Mayo in the period 2011-2012. Methods: Review of medical records documentary of mothers and preterm infants who met selection criteria, finding 170 cases in the study period. Results are shown using descriptive statistics. Results: The age of the mothers of preterm infants with RPM was 25.52 years. The manner of termination of labor was vaginal in 34.12 per cent, and by cesarean in 65.88 per cent. The latency from the RPM to birth was 9.41 per cent within the first 6 hours, in 21.18 per cent within the first 12 hours, and 69.41 per cent after 24 hours. Corticosteroids were used for 11Ing maturation in 46.47 per cent of cases, all with dexamethasone, and 27.65 per cent completed four courses. The 47.65 per cent of infants were male and 52.35 per cent female. In 82.35 per cent of cases the amniotic fluid was clear, light green in 15.88 per cent and in 1.76 per cent was purulent. No antibiotics were used in 10 per cent of cases, only one antibiotic was used in 48.24 per cent, two antibiotics in 24.71 per cent and three in 17.06 per cent, the most used antibiotic was cefazolin. In 45.29 per cent of mothers showed endometritis, the duration of hospitalization in mothers was 5.73 days. The 14.71 per cent of preterm infants was admitted with mother, intermediate care in 29.41 per cent and 55.88 per cent went to ICU, the indication for hospitalization in ICU was in 55.79 per cent sepsis, respiratory failure in 20 per cent, and in 12.63 per cent extreme prematurity. A 22.35 per cent of neonates did not required ventilatory support, 39.41 per cent required mechanical ventilation and 38.24 per cent used CPAP. In only 5.29 per cent of children there were no complications, a 91.18 per cent of cases had metabolic disorders, 90 per cent developed...

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.

Chemokines determine local lymphoneogenesis and a reduction of circulating CXCR4+ T and CCR7 B and T lymphocytes in thyroid autoimmune diseases.

Chemokines and their corresponding receptors are crucial for the recruitment of lymphocytes into the lymphoid organs and for its organization acting in a multistep process. Tissues affected by autoimmune disease often contain ectopic lymphoid follicles which, in the case of autoimmune thyroid disorders, are highly active and specific for thyroid Ags although its pathogenic role remains unclear. To understand the genesis of these lymphoid follicles, the expression of relevant cytokines and chemokines was assessed by real time PCR, immunohistochemistry and by in vitro assays in autoimmune and nonautoimmune thyroid glands. Lymphotoxin alpha, lymphotoxin beta, C-C chemokine ligand (CCL) 21, CXC chemokine ligand (CXCL) 12, CXCL13, and CCL22 were increased in thyroids from autoimmune patients, whereas CXCL12, CXCL13, and CCL22 levels were significantly higher in autoimmune glands with ectopic secondary lymphoid follicles than in those without follicles. Interestingly, thyroid epithelium produced CXCL12 in response to proinflammatory cytokines providing a possible clue for the understanding of how tissue stress may lead to ectopic follicle formation. The finding of a correlation between chemokines and thyroid autoantibodies further suggests that intrathyroidal germinal centers play a significant role in the autoimmune response. Unexpectedly, the percentage of circulating CXCR4(+) T cells and CCR7(+) B and T cells (but not of CXCR5) was significantly reduced in PBMCs of patients with autoimmune thyroid disease when they were compared with their intrathyroidal lymphocytes. This systemic effect of active intrathyroidal lymphoid tissue emerges as a possible new marker of thyroid autoimmune disease activity.

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