Whole-proteome analysis of mesonephric-derived cancers describes new potential biomarkers.

Mesonephric carcinomas (MEs) and female adnexal tumors of probable Wolffian origin (FATWO) are derived from embryologic remnants of Wolffian/mesonephric ducts. Mesonephric-like carcinomas (MLCs) show identical morphology to ME of the cervix but occur in the uterus and ovary without convincing mesonephric remnants. ME, MLC, and FATWO are challenging to diagnose due to their morphologic similarities to Müllerian/paramesonephric tumors, contributing to a lack of evidence-based and tumor-specific treatments. We performed whole-proteomic analysis on 9 ME/MLC and 56 endometrial carcinomas (ECs) to identify potential diagnostic biomarkers. Although there were no convincing differences between ME and MLC, 543 proteins showed increased expression in ME/MLC relative to EC. From these proteins, euchromatic histone lysine methyltransferase 2 (EHMT2), glutathione S-transferase Mu 3 (GSTM3), eukaryotic translation elongation factor 1 alpha 2 (EEF1A2), and glycogen synthase kinase 3 beta were identified as putative biomarkers. Immunohistochemistry was performed on these candidates and GATA3 in 14 ME/MLC, 8 FATWO, 155 EC, and normal tissues. Of the candidates, only GATA3 and EHMT2 were highly expressed in mesonephric remnants and mesonephric-derived male tissues. GATA3 had the highest sensitivity and specificity for ME/MLC versus EC (93% and 99%) but was absent in FATWO. EHMT2 was 100% sensitive for ME/MLC & FATWO but was not specific (65%). Similarly, EEF1A2 was reasonably sensitive to ME/MLC (92%) and FATWO (88%) but was the least specific (38%). GSTM3 performed intermediately (sensitivity for ME/MLC and FATWO: 83% and 38%, respectively; specificity 67%). Although GATA3 remained the best diagnostic biomarker for ME/MLC, we have identified EHMT2, EEF1A2, and GSTM3 as proteins of interest in these cancers. FATWO's cell of origin is uncertain and remains an area for future research.

Expression of MHC-I and II by Microglia and Lymphocytes in the Brain of Diet-Restricted Mice.

Microglia are immunocompetent cells of the central nervous system whose function is to preserve nervous tissue homeostasis; however, under inflammatory conditions, they are associated with tissue damage. Dietary restriction is a nutritional intervention used to delay the onset of chronic disease and inflammation, in addition to improving the functions of the immune system. The aim of this study was to analyze the effect of dietary restriction on microglial expression of MHC molecules. Adult female and male BALB/c mice were fed ad libitum (controls) or kept under dietary restriction (30% reduction in food intake) for 4 wk. Purified brain mononuclear cells were analyzed by flow cytometry staining for CD45, CD11b, MHC-I and MHC-II. Our results show that female animals under dietary restriction had a significant increase in MHC-I expression in microglia (mean fluorescence intensity=7,854 (control) vs. 10,628 (diet-restricted), arbitrary units; p=0.0108), along with increased frequencies of lymphocytes compared to controls (1.39% (control) vs. 7.85% (diet-restricted); p=0.0175), whereas male animals did not show significant differences between groups. Our data suggest a differential effect for dietary restriction on female and male animals, with this nutritional regimen predominantly affecting females. Increased expression of MHC-I by diet-restricted microglia may play a role in maintaining tolerance in the absence of antigenic stimulation.

FLEXamers: A Double Tag for Universal Generation of Versatile Peptide-MHC Multimers.

Peptide-MHC (pMHC) multimers have become a valuable tool for immunological research, clinical immune monitoring, and immunotherapeutic applications. Biotinylated tetramers, reversible Streptamers, or dye-conjugated pMHC multimers are distinct pMHC reagents tailored for T cell identification, traceless T cell isolation, or TCR characterization, respectively. The specific applicability of each pMHC-based reagent is made possible either through conjugation of probes or reversible multimerization in separate production processes, which is laborious, time-consuming, and prone to variability between the different types of pMHC reagents. This prohibits broad implementation of different types of pMHC reagents as a standard toolbox in routine clinical immune monitoring and immunotherapy. In this article, we describe a novel method for fast and standardized generation of any pMHC multimer reagent from a single precursor ("FLEXamer"). FLEXamers unite reversible multimerization and versatile probe conjugation through a novel double tag (Strep-tag for reversibility and Tub-tag for versatile probe conjugation). We demonstrate that FLEXamers can substitute conventional pMHC reagents in all state-of-the-art applications, considerably accelerating and standardizing production without sacrificing functional performance. Although FLEXamers significantly aid the applicability of pMHC-based reagents in routine workflows, the double tag also provides a universal tool for the investigation of transient molecular interactions in general.

Rotavirus Vaccine Take in Infants Is Associated With Secretor Status.

Rotaviruses bind to enterocytes in a genotype-specific manner via histo-blood group antigens (HBGAs), which are also detectable in saliva. We evaluated antirotavirus immunoglobulin A seroconversion ('vaccine take") among 166 Ghanaian infants after 2-3 doses of G1P[8] rotavirus vaccine during a vaccine trial, by HBGA status from saliva collected at age 4.1 years. Only secretor status was associated with seroconversion: 41% seroconversion for secretors vs 13% for nonsecretors; relative risk, 3.2 (95% confidence interval, 1.2-8.1; P = .016). Neither Lewis antigen nor salivary antigen blood type was associated with seroconversion. Likelihood of "take" for any particular rotavirus vaccine may differ across populations based on HBGAs.

Successful kidney transplantation across a positive complement-dependent cytotoxicity crossmatch by using C1q assay-directed, bortezomib-assisted desensitization: A case report.

RATIONALE: Human leukocyte antigen (HLA) is the major immunologic barrier in kidney transplantation (KT). Various desensitization protocols to overcome the HLA barrier have increased the opportunity for transplantation in sensitized patients. In addition, technological advances in solid-phase assays have permitted more comprehensive assessment of donor-specific antibodies. Although various desensitization therapies and immunologic techniques have been developed, the final transplantation decision is still based on the classic complement-dependent cytotoxicity (CDC) crossmatch (XM) technique. Some patients who fail to achieve negative XM have lost their transplant opportunities, even after receiving sufficient desensitization therapies. PATIENT CONCERNS: A 57-year-old male with end-stage renal disease secondary to chronic glomerulonephritis was scheduled to have a second transplant from his son, but CDC XM was positive. DIAGNOSES: Initial CDC XM (Initial T-AHG 1:32) and flow-cytometry XM were positive. Anti-HLA-B59 donor specific antibody was detected by Luminex single antigen assay. INTERVENTIONS: Herein, we report a successful case of KT across a positive CDC XM (T-AHG 1:8 at the time of transplantation) by using C1q assay-directed, bortezomib-assisted desensitization. After confirming a negative conversion in the C1q donor-specific antibody, we decided to perform KT accepting a positive AHG-CDC XM of 1:8 at the time of transplantation. OUTCOMES: The posttransplant course was uneventful and a protocol biopsy at 3 months showed no evidence of rejection. The patient had excellent graft function at 12 months posttransplant. LESSONS: The results of XM test and solid-phase assay should be interpreted in the context of the individual patient.

Is HLA the cause of the high incidence of type 1 diabetes in the Canary Islands? Results from the Type 1 Diabetes Genetics Consortium (T1DGC) / ¿Es el HLA la causa de la alta incidencia de diabetes tipo 1 en las Islas Canarias? Resultados del Consorcio de Genética de la Diabetes tipo 1 (T1DGC)

Introduction: Incidence of childhood-onset type 1 diabetes mellitus in the Canary Islands is the highest reported so far in Spain, and among the highest worldwide. The HLA region accounts for approximately half the genetic risk of type 1 diabetes. Our aim was to assess distribution of high-risk and protective HLA haplotypes in the Canarian families included in the T1DGC, as compared to the rest of Spain. Methods: The T1DGC study, an international project to study the genetics and pathogenesis of type 1 diabetes, enrolled more than 3000 families with type 1 diabetes worldwide. Spain provided 149 of these families, of whom 42 were from Tenerife and Gran Canaria. HLA was genotyped centrally using a PCR-based, sequence-specific oligonucleotide probe system. Haplotypes were reconstructed using the deterministic algorithm alleHap in the R programming environment. Based on prior T1DGC results in Caucasian population, haplotypes DRB1*0405-DQA1*0301-DQB1*0302, DRB1*0401-DQA1*0301-DQB1*0302, DRB1*0301-DQA1*0501-DQB1*0201, DRB1*0402-DQA1*0301-DQB1*0302 and DRB1*0404-DQA1*0301-DQB1*0302 were considered high-risk. DRB1*0701-DQA1*0201-DQB1*0303, DRB1*1401-DQA1*0101-DQB1*0503, DRB1*1501-DQA1*0102-DQB1*0602, DRB1*1101-DQA1*0501-DQB1*0301, DRB1*1104-DQA1*0501-DQB1*0301, DRB1*1303-DQA1*0501-DQB1*0301, DRB1*1301-DQA1*0103-DQB1*0603 and DRB1*0403-DQA1*0301-DQB1*0302 were considered protective. The distribution of protective, high-risk, and other haplotypes in the (first two) affected siblings and unaffected parents from Canarian and non-Canarian Spanish families was compared (Chi-square test). Results: No significant differences were found between the regions in distribution of the HLA haplotypes in the affected siblings or in the non-affected parents. Conclusions: The high incidence of childhood-onset type 1 diabetes in the Canarian population does not appear to be explained by a greater prevalence of high-risk class II HLA haplotypes in families with the disease. However, sample size limits the differences that can be detected in this study (AU)

Introducción: La incidencia de diabetes tipo 1 infantil en Canarias es la más alta descrita hasta el momento en España y una de las mayores a nivel mundial. La región HLA explica aproximadamente el 50% del riesgo genético de la diabetes tipo 1. Nuestro objetivo fue comparar la frecuencia de haplotipos de HLA de riesgo y protectores en familias españolas canarias y peninsulares incluidas en el T1DGC. Métodos: El T1DGC es un proyecto internacional que estudia la genética y patogenia de la diabetes tipo 1, para el que fueron inluidas más de 3000 familias con la enfermedad. Un total de 149 familias provenían de España, y 42 de ellas, de Tenerife y Gran Canaria. El HLA fue genotipado en un laboratorio central, utilizando un método basado en PCR y sondas específicas de secuencia. Los haplotipos fueron reconstruidos utilizando el algoritmo determinista alleHap en el entorno de programación R. En base a los resultados previos del T1DGC en población caucásica, los haplotipos DRB1*0405-DQA1*0301-DQB1*0302, DRB1*0401-DQA1*0301-DQB1*0302, DRB1*0301-DQA1*0501-DQB1*0201, DRB1*0402-DQA1*0301-DQB1*0302 y DRB1*0404-DQA1*0301-DQB1*0302 fueron definidos como de alto riesgo. DRB1*0701-DQA1*0201-DQB1*0303, DRB1*1401-DQA1*0101-DQB1*0503, DRB1*1501-DQA1*0102-DQB1*0602, DRB1*1101-DQA1*0501-DQB1*0301, DRB1*1104-DQA1*0501-DQB1*0301, DRB1*1303-DQA1*0501-DQB1*0301, DRB1*1301-DQA1*0103-DQB1*0603 y DRB1*0403-DQA1*0301-DQB1*0302 fueron considerados protectores. La distribución de haplotipos de riesgo, protectores y otros en los (dos primeros) hermanos afectos y en los padres no afectos fue comparada entre las familias canarias y no canarias (chi cuadrado). Resultados: No se encontraron diferencias significativas en la distribución de haplotipos HLA entre las regiones estudiadas, ni en los hermanos afectos ni en los padres no afectos. Conclusiones: La alta incidencia de la enfermedad en la población canaria no parece ser explicada por una mayor prevalencia de haplotipos de HLA de clase II de riesgo en los casos con agregación familiar, aunque el tamaño de la muestra limita las diferencias detectables en este estudio (AU)

Fluorescence-based methods for screening writers and readers of histone methyl marks.

The histone methyltransferase (HMT) family of proteins consists of enzymes that methylate lysine or arginine residues on histone tails as well as other proteins. Such modifications affect chromatin structure and play a significant regulatory role in gene expression. Many HMTs have been implicated in tumorigenesis and progression of multiple malignancies and play essential roles in embryonic development and stem cell renewal. Overexpression of some HMTs has been observed and is correlated positively with various types of cancer. Here the authors report development of a continuous fluorescence-based methyltransferase assay in a 384-well format and its application in determining kinetic parameters for EHMT1, G9a, PRMT3, SETD7, and SUV39H2 as well as for screening against libraries of small molecules to identify enzyme inhibitors. They also report the development of a peptide displacement assay using fluorescence polarization in a 384-well format to assay and screen protein peptide interactions such as those of WDR5 and EED, components of MLL and EZH2 methyltransferase complexes. Using these high-throughput screening methods, the authors have identified potent inhibitors and ligands for some of these proteins.

Characterization of MHC/peptide complexes refolded by a one-step ion-exchange chromatography.

The current refolding process of MHC/peptide complexes is low-yield and time-consuming, thereby limiting the wide uses of MHC/peptide multimers. Here the heavy chain protein of MHC/peptide complex (H-2K(b)/TRP2(180-188)) was immobilized onto an ion-exchange chromatography column, and the ß2m or TRP2(180-188)-fused ß2m protein, which renatured previously in refolding buffer, was able to pass through the column for the gradient refolding. This strategy refolds, concentrates and purifies MHC/peptide complexes in a single integrated step, achieving a high level of process simplification and automation. Using this on-column refolding method, MHC/peptide complexes could be prepared within 24h with a refolding yield of over 20%. Anti-H-2K(b) mAb staining and flow cytometric analyses revealed that the on-column refolded H-2K(b)/TRP2(180-188) complexes had conformational characteristics similar to the dilution refolded H-2K(b)/TRP2(180-188) complexes and the commercial ones. Furthermore, H-2K(b)/TRP2(180-188) tetramer staining and the enumeration of TRP2(180-188)-specific T cells and H-2K(b)-alloreactive T cells confirmed that the H-2K(b)/TRP2(180-188) complexes prepared by on-column refolding or dilution refolding had comparable TCR-binding ability. These data demonstrate a novel, simple and efficient refolding strategy for the generation of MHC class I/peptide complexes.

The MHC motif viewer: a visualization tool for MHC binding motifs.

In vertebrates, the onset of cellular immune reactions is controlled by presentation of peptides in complex with major histocompatibility complex (MHC) molecules to T cell receptors. In humans, MHCs are called human leukocyte antigens (HLAs). Different MHC molecules present different subsets of peptides, and knowledge of their binding specificities is important for understanding differences in the immune response between individuals. Algorithms predicting which peptides bind a given MHC molecule have recently been developed with high prediction accuracy. The utility of these algorithms is hampered by the lack of tools for browsing and comparing specificity of these molecules. We have developed a Web server, MHC Motif Viewer, which allows the display of the binding motif for MHC class I proteins for human, chimpanzee, rhesus monkey, mouse, and swine, as well as HLA-DR protein sequences. The binding motif for each MHC molecule is predicted using state-of-the-art, pan-specific peptide-MHC binding-prediction methods, and is visualized as a sequence logo, in a format that allows for a comprehensive interpretation of binding motif anchor positions and amino acid preferences.

EVI-1 interacts with histone methyltransferases SUV39H1 and G9a for transcriptional repression and bone marrow immortalization.

The ecotropic viral integration site-1 (EVI-1) is a nuclear transcription factor and has an essential function in the proliferation/maintenance of haematopoietic stem cells. Aberrant expression of EVI-1 has been frequently found in myeloid leukaemia as well as in several solid tumours, and is associated with a poor patient survival. It was recently shown that EVI-1 associates with two different histone methyltransferases (HMTs), SUV39H1 and G9a. However, the functional roles of these HMTs in EVI-1-mediated leukemogenesis remain unclear. In this study, we showed that EVI-1 physically interacts with SUV39H1 and G9a, but not with Set9. Immunofluorescence analysis revealed that EVI-1 colocalizes with these HMTs in nuclei. We also found that the catalytically inactive form of SUV39H1 abrogates the transcriptional repression mediated by EVI-1, suggesting that SUV39H1 is actively involved in EVI-1-mediated transcriptional repression. Furthermore, RNAi-based knockdown of SUV39H1 or G9a in Evi-1-expressing progenitors significantly reduced their colony-forming activity. In contrast, knockdown of these HMTs did not impair bone marrow immortalization by E2A/HLF. These results indicate that EVI-1 forms higher-order complexes with HMTs, and this association has a role in the transcription repression and bone marrow immortalization. Targeting these HMTs may be of therapeutic benefit in the treatment for EVI-1-related haematological malignancies.

Phenotype and function of monocyte-derived dendritic cells from chinese rhesus macaques.

Dendritic cells (DCs) play a pivotal role in linking the innate immunity and acquired immunity in responses to pathogen. Non-human primates such as Chinese Rhesus Macaque (CRM) are the favorable models for preclinical study of potential therapeutic drugs, vaccines and mechanisms of human diseases. However, the phenotypical characterization of monocyte-derived dendritic cells (MDDCs) from CRM has not been elucidated. Monocytes from CRM were cultured with GM-CSF and IL-4 in RPMI-1640. Six days later, these cells were differentiated with typical dendritical morphology. CD11c and DC-SIGN were highly expressed. The immature MDDCs expressed the low levels of CD25, CD80, CD83, moderate CD40, CD86, and high MHC. After stimulation, the mature MDDCs increased expression of mature molecules CD25 and CD83, co-stimulatory molecules such as CD80, CD86 and CD40, and kept a high level of MHC. The capacity of endocytosis decreased with maturation. The mature MDDCs have strong ability of inducing allogeneic T cell proliferation and producing IL-12. In conclusion, we have characterized the phenotype and ultimate function of MDDCs from CRM for the first time.

Quantifying soluble HLA-G in supernatants of cultured embryos as a marker of implantation potential in an assisted reproduction program

HLA-G desempeña un papel tolerogénico en la interfase maternofetal.En programas de reproducción asistida, en los que se cultivan embrionesin vitro, ha cobrado interés la detección de las isoformas solubles deesta molécula (sHLA-G) en el medio en el que se han crecido los embriones.Aunque la determinación es compleja, tiene interés por su aparenterelación con la idoneidad de dichos embriones. En el presente trabajo,se ha puesto a punto un ensayo ELISA amplificado para medir sHLA-Ga las concentraciones esperables en dichos sobrenadantes. Como modelocomparativo se ha utilizado el cultivo a dilución límite de la línea de coriocarcinomaJEG-3. Con el ELISA desarrollado se han analizado retrospectivamente111 sobrenadantes recogidos a las 44-48 horas de efectuadafecundación mediante inyección intracitoplasmática del espermatozoide,y los datos se han correlacionado con los resultados reproductivos dedichos embriones. En 22 de los sobrenadantes (19.8%) se ha detectadosHLA-G. No se ha encontrado relación entre los niveles de sHLA-G y elgrado morfológico de los embriones. Los resultados reproductivos de losembriones de morfología normal que fueron transferidos al útero de laspacientes (2-3 por caso) fueron los siguientes: en el grupo de mujeres querecibió únicamente embriones sHLA-G negativos, las tasas de embarazoe implantación fueron, respectivamente, 29% (4 embarazos/14 mujeres)y 14% (5 sacos gestacionales/35 embriones transferidos). Por contra,en el grupo que recibió al menos un embrión sHLA-G positivo, las tasasde embarazo e implantación subieron al 60% (3 / 5) y 29% (4/14) respectivamente.En conclusión, es posible cuantificar niveles de sHLA-G ensobrenadantes de embriones y los resultados obtenidos sugieren asociacióncon la probabilidad de embarazo. La detección de sHLA-G, por tanto,podría ser un buen complemento de la selección morfológica de embrionesútil para incrementar la tasa de implantación y reducir la de embarazosmúltiples

HLA-G plays a tolerogenic function at the maternal-fetal interface.Detection of the soluble isoforms of HLA-G (sHLA-G) in culture mediumderived from embryos grown in vitro, although technically complex,has gained interest in assisted reproduction programs because of an apparentrelationship with embryo competence. Here, an amplified ELISA wasdesigned to measure sHLA-G at the concentrations expected in embryosupernatants using a limiting-dilution assay of the choriocarcinoma JEG-3 cell line as a surrogate model. With this ELISA approach, 111 singleembryo culture supernatants, collected 44-48 hours after intracytoplasmicsperm injection, were retrospectively analysed and levels correlatedwith pregnancy results. The presence of sHLA-G was demonstrated in 22(19.8%) of the embryo cultures. There was no relationship between sHLAGlevels and grading of embryo morphology. The reproductive outcomeof the morphologically normal embryos that were transferred to thewomen uterus (2-3 per patient) was as follows: in the group of women inwhich all transferred embryos were sHLA-G negative, the pregnancy andimplantation rates were 29% (4 pregnancies/14 women) and 14% (5 gestationalsacs/35 embryos transferred), respectively. In contrast, in thegroup in which the embryo transfers included at least one sHLA-G positivethe pregnancy and implantation rates increased to 60% (3/5) and29% (4/14) respectively. In conclusion, sHLA-G levels in preimplantationembryo supernatants can be quantified and results suggest positive associationwith pregnancy likelihood. sHLA-G detection seems to be usefulto complement morphology in selecting good quality embryos for increasingimplantation rates and reducing multiple gestations

Colorectal carcinoma rearranges cell surface protein topology and density in CD4+ T cells.

Previously, we described conserved protein clusters including MHC I and II glycoproteins, ICAM-1 adhesion molecules, and interleukin-2 and -15 receptors in lipid rafts of several human cell types. Differential protein-protein interactions can modulate function, thus influence cell fate. Therefore, we analyzed supramolecular clusters of CD4(+) T cells from draining lymph nodes and peripheral blood of colorectal carcinoma patients, and compared these to healthy controls. Superclusters of MHC I and II with IL-2/15 receptors were identified by confocal microscopy on all cell types. Flow-cytometric FRET revealed molecular associations of these proteins with each other and with ICAM-1 as well. In draining lymph nodes expression levels of all these proteins were lower, and interactions, particularly between IL-2/15 receptors and MHC molecules weakened or disappeared as compared to the control. Stimuli/local conditions can rearrange cell surface protein patterns on the same cell type in the same patient, having important implications on further function and cell fate.

Análisis comparativo de un grupo de pacientes con narcolepsia-cataplejía, narcolepsia sin cataplejía e hipersomnia idiopática / Comparative analysis of patients with narcolepsy-cataplexy, narcolepsy without cataplexy and idiopathic hypersomnia

Fundamento y objetivo: Analizar la distribución de variables clínicas, electrofisiológicas y biológicas, así como su relación con los valores de hipocretina 1 (Hcrt-1) en el líquido cefalorraquídeo (LCR), en pacientes con hipersomnia central diagnosticados, según los criterios de la segunda revisión de la Internacional Classification of Sleep Disorders (ICSD-2), como narcolepsia-cataplejía (NC), narcolepsia sin cataplejía (NnC) e hipersomnia idiopática (HI). Pacientes y método: A todos los pacientes se les realizaron una entrevista clínica, un polisomnograma nocturno y un test de latencias múltiples de sueño, tipificación de antígenos de histocompatibilidad (HLA) y análisis de Hcrt-1 en el LCR (valores bajos ¾ 110 pg/ml). Resultados: De un total de 51 pacientes, se diagnosticó a 32 de NC, a 11 de NnC y a 8 de HI, y en 34 (66,7%) se encontraron valores bajos de Hcrt-1 (29 con NC, 3 con NnC y uno con HI). Entre los pacientes con NC, un 96,1% fueron positivos para HLA DQB1*0602 y el 91% presentó valores bajos de Hcrt-1. Las variables más frecuentemente encontradas en pacientes con NC y en aquéllos con valores bajos de Hcrt-1 fueron la cataplejía, el sueño nocturno fragmentado, siestas cortas reparadoras, conductas automáticas, el HLA DQB1*0602 y, en el test de latencias múltiples de sueño, una latencia media de sueño reducida, un número mayor de episodios de sueño REM y una latencia media reducida de éstos. El tiempo de sueño nocturno prolongado o las dificultades en el despertar, 2 variables incorporadas a la ICSD-2 en el diagnóstico de HI, no diferenciaron los distintos grupos. Conclusiones: Las hipersomnias centrales presentan una superposición de diversas características clínicas, electrofisiológicas y biológicas que dificultan su diagnóstico diferencial. La determinación de Hcrt-1 en LCR puede facilitar el diagnóstico en casos con escasa definición clínica y/o electrofisiológica

Backgrund and objective: To evaluate the distribution of clinical, electrophysiological and biological variables, and their relationship with the CSF hypocretin-1 levels, in patients with central hypersomnias diagnosed as narcolepsy-cataplexy (NC), narcolepsy without cataplexy (NnC) and idiopathic hypersomnia (IH) based on the ICSD-2 criteria. Patients and method: We performed in all patients a clinical interview, a nocturnal polysomnogram and a multiple sleep latency test (MSLT), HLA analysis and measurement of CSF Hcrt-1 levels (low ¾ 110 pg/mL). Results: Out of 51 patients, 31 were classified as NC, 11 as NnC and 8 as IH. 34 patients (66.7%) had low CSF Hcrt-1 levels (29 NC, 3 NnC and 1 IH). In the NC group, 96.1% were HLA DQB1*0602 positive and 91% had low CSF Hcrt-1 levels. The most frequent variables found in NC patients and in those with a low CSF Hcrt-1 levels were cataplexy, fragmented nocturnal sleep, short refreshing naps, automatic behavior, HLA DQB1*0602, and, in the MSLT, a short mean sleep latency, a higher number of REM sleep episodes and a short mean latency of REM sleep episodes. A long nocturnal sleep time and morning sleep drunkenness, 2 variables used in the ICSD-2 for the diagnosis of IH, were not different among the three groups of hypersomnias. Conclusions: Central hypersomnias have a superposition of several clinical, electrophysiological and biological variables that makes sometimes difficult the differential diagnosis. The measurement of CSF Hcrt-1 levels may help in the diagnosis of those patients with unclear clinical or electrophysiological forms

Antígenos de histocompatibilidad e infarto de miocardio / Histocompatibility antigens and myocardial infarction

Introducción. Los polimorfismos genéticos están considerados como los nuevos factores de riesgo para la enfermedad coronaria. Dentro de estos genes podría incluirse al complejo mayor de histocompatibilidad, dada la naturaleza inflamatoria de esta patología. El objetivo de este estudio fue conocer si existe asociación entre el complejo mayor de histocompatibilidad y el infarto de miocardio. Métodos. Se realizó la determinación de los antígenos de histocompatibilidad por el método de microlinfocitotoxicidad en 2 pasos modificado para doble fluorescencia, en 60 pacientes masculinos con antecedentes de infarto de miocardio y 142 donantes sanos, y se estudió la relación con algunos factores de riesgo cardiovasculares. Resultados. Se encontró asociación positiva con los antígenos DR4 (odds ratio [OR] = 3,75; p < 0,025; intervalo de confianza [IC] del 95%, 0,60-2,45) y B8 (OR = 4,37; p < 0,025; IC del 95%, 1,69-3,11) para el infarto de miocardio en comparación con los controles. El antígeno B15 (1,66 frente al 16,66%, p < 0,001; OR = 0,14; p < 0,007; IC del 95%, 0,06-0,63) resultó el menos frecuente en el grupo de pacientes, pudiendo ser útil como marcador de protección. Asimismo, la frecuencia de los haplotipos B8-DR4 (1,2 frente al 9,0%, p < 0,001; OR = 7,50; p < 0,0003; IC del 95%, 2,15-4,17) y B8-DR3 (8,5 frente al 2,3%, p < 0,001; OR = 3,70; p < 0,004; IC del 95%, 2,50-5,12) fue significativamente mayor. Los pacientes DR4 positivos mostraron mayor predisposición para desarrollar múltiples infartos, mientras que los B8 tuvieron mayores antecedentes familiares de cardiopatía isquémica, sin que se encontraran diferencias significativas con los otros factores de riesgo estudiados. Conclusiones. Se encontró asociación entre el sistema mayor de histocompatibilidad y el infarto de miocardio, así como evidencias sobre la posible naturaleza autoinmune de dicho episodio (AU)

Introduction. Genetic polymorphisms have recently been identified as a risk factor for coronary heart disease. Because of the inflammatory nature of this disease, major histocompatibility complex may be included among these genes. The aim of this study was to investigate whether major histocompatibility complex is involved in conferring resistance or susceptibility to myocardial infarction. Methods. Selected human leukocyte antigens (HLA) were studied by the classical two-step microlymphocytotoxicity assay modified for double staining in 60 male patients with a history of myocardial infarction and 142 healthy donors. Several risk factors were analyzed. Results. Positive associations with HLA-DR4 (odds ratio [OR] = 3.75; p < 0.025; 95% confidence interval [CI], 0.60-2.45) and B8 (OR = 4.37; p < 0.025; 95% CI, 1.69-3.11) antigens were found for myocardial infarction compared with healthy controls. The frequency of HLA-B15 antigen was lower in the patient group (1.66% vs. 16.66%, p < 0.001; OR = 0.14; p < 0.007; 95% CI, 0.06-0.63), which would suggest that this antigen is probably a protective factor against myocardial infarction. The frequency of haplotypes B8-DR4 (1.2% vs. 9.0, p < 0.001; OR = 7.50; p < 0.0003; 95% CI, 2.15-4.17) and B8-DR3 (8.5% vs. 2.3%, p < 0.001; OR = 3.70; p < 0.004; 95% CI, 2.50-5.12) was significantly increased in the patient group. DR4-positive patients showed a strong predisposition to developing multiple infarctions, while HLA-B8 positive patients more frequently had a familial history of coronary heart disease. No significant differences were observed between the presence of HLA-DR4 and B8 and the risk factors analyzed. Conclusions. An association was found between major histocompatibility complex and myocardial infarction, as well as evidence of the possible autoimmune nature of this event (AU)

Intrinsic ability of GM+IL-4 but not Flt3L-induced rat dendritic cells to promote allogeneic T cell hyporesponsiveness.

The influence of GM+IL-4 and Flt3 ligand (FL) on phenotype and function of BM-derived DC from Lewis rats was investigated. GM+IL-4-induced DC, despite expression of CD80/CD86, were less stimulatory than FL-induced DC that expressed low CD80/CD86 and were efficient stimulators of allogeneic T cells. GM+IL-4 DC were CD11b+ OX62lo, whereas FL DC were CD11blo OX62+. Following activation, GM+IL-4 DC produced IL-10 and IL-6, but no IL-12p70, and were resistant to further maturation. FL DC produced IL-12p70, IFN-alpha/beta, IL-10 and IL-6 and underwent maturation. Repeated stimulation of T cells with GM+IL-4 DC inhibited proliferation, cytokine production and induced early T cell apoptosis. FL DC-activated T cells produced large amounts of IFN-gamma/IL-10 and exhibited late T cell apoptosis/necrosis. In vivo, GM+IL-4 DC induced alloAg-specific hyporesponsiveness following T cell restimulation. These results demonstrate that GM+IL-4 DC display intrinsic regulatory properties, inducing passive-cell-death in T cells with potential for inactivation/regulation of alloreactive T cells in transplantation.

Undifferentiated murine embryonic stem cells cannot induce portal tolerance but may possess immune privilege secondary to reduced major histocompatibility complex antigen expression.

Induction of donor-specific tolerance using embryonic stem (ES) cells followed by transplantation of ES cell-derived tissues from the same allogeneic strain could theoretically engender successful transplantation without immunosuppression. We sought to induce tolerance using bona fide murine ES cells in immunocompetent mice. ES cells were evaluated for the expression of markers restricted to undifferentiated cells [stage-specific embryonic antigen-1 (SSEA-1) and OCT-4] and the ability to form teratomas in immunodeficient mice. BALB/cByJ mice underwent intraportal inoculation with YC5-EYFP ES cells (129 strain; R1-derived) or saline followed by transplantation with 129X1/SvJ, CBA/J, or BALB/cByJ nonvascularized, neonatal cardiac grafts. Mice were sacrificed at graft failure and underwent histologic evaluation of transplanted grafts and lymphoid organs. ES cells and early differentiated progeny underwent real time (RT)-PCR and fluorescence-activated cell sorting (FACS) analysis to detect major histocompatibility complex (MHC) gene transcription and antigen expression. ES cells expressed markers restricted to undifferentiated cells while maintaining the ability to form teratomas in immunodeficient mice. No prolongation of allograft survival or evidence of lymphoid chimerism was observed in immunocompetent recipient mice despite hepatic teratoma formation. MHC class I, class II, and nonclassical antigens were undetectable on ES cells and early differentiated progeny despite the presence of mRNA transcripts. Class I expression was strongly upregulated upon exposure to gamma-interferon. Intraportal inoculation with murine ES cells does not produce lymphoid chimerism or induce donor-specific unresponsiveness to neonatal cardiac grafts in unmanipulated immunocompetent hosts. However, specific differentiated cell types such as ES cellderived dendritic cells, or alternate routes of ES cell administration, may be effective. ES cells appear to have immune privilege, allowing them to form teratomas in immunocompetent mice.

Which complement assays and typings are necessary for the diagnosis of complement deficiency in patients with lupus erythematosus? A study of 25 patients.

INTRODUCTION: Deficiencies in components of the classical pathway of complement activation are strong risk factors for lupus erythematosus (LE).Yet, it has not been addressed whether the conventional measurements of the serum hemolytic CH50 activity and antigenic concentrations of C3 and C4 are sufficient to asses a deficiency in C4A, C4B or C2 components, the most common deficiencies associated with LE. PATIENTS AND METHODS: In a retrospective series, we performed complement analyses in 35 patients with LE who were systematically screened for a complement deficiency. The majority of patients had cutaneous LE with mild systemic involvement and no complement consumption. Of 25 patients (72%) with complement deficiency we found 13 with a partial C4A deficiency, 2 with a complete C4A deficiency, 6 with a partial C4B deficiency, 2 with a complete C4B deficiency and 2 with a combined partial C2 and C4A deficiency. RESULTS: The total complement activity (CH50) was decreased in only one out of two patients with complete C4B deficiency. CH50 level was found to be low-normal (35-38 U/ml(-1)) in one patient with partial C4B deficiency, one patient with complete C4B deficiency and both patients with combined partial C4A and C2 deficiency. Total C4 levels were normal in 9 out of 13 the patients with a partial C4A deficiency and in 2 out of 6 patients with a complete C4B deficiency. The antigenic concentration of C3 was low in only 1 patients with a complete C4B deficiency and within the normal range in all the others patients. Overall, 50% of the patients had normal or elevated C3, C4, and CH50 levels. DISCUSSION: This study emphasizes that the usual measurements of CH50, C3 and C4 levels are not adequate to detect a C4 and/or C2 deficiency in patients with LE. In epidemiologic or investigative studies addressing the prevalence of complement deficiency, more elaborated diagnostic tests, such as C4 protein allotyping, C2 level measurement and genetic screening for type I C2 deficiency should also be performed.

Human NK cells can lyse porcine endothelial cells independent of their expression of Galalpha(1,3)-Gal and killing is enhanced by activation of either effector or target cells.

BACKGROUND: Xenotransplantation of pig organs may provide an approach to alleviate the severe shortage of human organs. Natural antibodies against Galalpha(1,3)-Gal (alphaGal) epitopes cause hyperacute rejection of pig organs in primates. However, evidence for the role of alphaGal in the natural killer (NK) cell-mediated xenoresponse has been contradictory. METHODS: We investigated the recognition of alphaGal by human NK cells using endo-beta-galactosidase C, an enzyme that cleaves alphaGal, and endothelial cells (EC) from alpha1,3-galactosyltransferase null pigs that do not synthesize alphaGal. Endo-beta-galactosidase C treatment variably reduced the susceptibility of porcine EC to lysis by fresh human NK cells. RESULTS: Removal of alphaGal from porcine EC using endo-beta-galactosidase C, produced variable results, i.e. cytotoxicity was decreased in half of the human NK cell donors tested. The two EC strains from alphaGal-/- pigs were marginally, and not significantly, less susceptible to lysis by naïve human NK cells compared with alphaGal-expressing cells obtained from animals from the same herd, but these differences were not statistically significant (P > 0.10). Treatment of porcine EC with recombinant human tumor necrosis factor (TNF)-alpha, which is known to activate porcine EC, enhanced the susceptibility of all target cells to lysis by fresh human NK cells. Surface expression of MHC or adhesion molecules on alphaGal-/- cells, compared with wild type cells, showed no consistent difference in either MHC or adhesion molecules CD106 (VCAM-1), CD31 (PECAM) or CD62E (E-selectin), either with or without TNF-alpha stimulation, that could explain the differential susceptibility to lysis. Strikingly, all alphaGal-/- and wild type EC exhibited similar susceptibility to human NK cells that had been cultured for 5 days with or without interleukin-2. CONCLUSIONS: These findings demonstrate that human NK cells can kill porcine targets in the absence of alphaGal, and donor variability plays a major role in whether alphaGal has a role in determining susceptibility of porcine EC to lysis. Moreover, susceptibility to lysis of alphaGal null EC is enhanced to the level of wild type EC by activation of either effector or target cells. Elimination of alphaGal alone from source pigs will be insufficient to circumvent the NK cell mediated destruction of porcine EC.

[HLA typing, analysis methods, and clinical applications]. / Typage HLA, méthodes d'analyses et applications cliniques.

The polygenic HLA system is a well known region of the human genome. Its main function is to present antigenic peptides to the immune system and thereby regulate induction of immune responses. Extensive genetic polymorphisms characterize some HLA genes. Initially, genetic variations were analyzed by a serological typing technique (microlymphocytotoxicity). The introduction of polymerase chain reaction (PCR) in the mid-1980s led the development of a variety of methods that use molecular biology. An international nomenclature, regularly updated, governs the names of HLA antigens defined by serology as well as of HLA alleles. Knowledge of the specific polymorphisms of individuals is essential in organ and stem cell transplantation and highly useful in disease association studies.