Restricting datasets to classifiable samples augments discovery of immune disease biomarkers

Immunological diseases are typically heterogeneous in clinical presentation, severity and response to therapy. Biomarkers of immune diseases often reflect this variability, especially compared to their regulated behaviour in health. This leads to a common difficulty that frustrates biomarker discovery and interpretation – namely, unequal dispersion of immune disease biomarker expression between patient classes necessarily limits a biomarker’s informative range. To solve this problem, we introduce dataset restriction, a procedure that splits datasets into classifiable and unclassifiable samples. Applied to synthetic flow cytometry data, restriction identifies biomarkers that are otherwise disregarded. In advanced melanoma, restriction finds biomarkers of immune-related adverse event risk after immunotherapy and enables us to build multivariate models that accurately predict immunotherapy-related hepatitis. Hence, dataset restriction augments discovery of immune disease biomarkers, increases predictive certainty for classifiable samples and improves multivariate models incorporating biomarkers with a limited informative range. This principle can be directly extended to any classification task

External validation of biomarkers for immune-related adverse events after immune checkpoint inhibition

Immune checkpoint inhibitors have revolutionized treatment of advanced melanoma, but commonly cause serious immune-mediated complications. The clinical ambition of reserving more aggressive therapies for patients least likely to experience immune-related adverse events (irAE) has driven an extensive search for predictive biomarkers. Here, we externally validate the performance of 59 previously reported markers of irAE risk in a new cohort of 110 patients receiving Nivolumab (anti-PD1) and Ipilimumab (anti-CTLA-4) therapy. Alone or combined, the discriminatory value of these routine clinical parameters and flow cytometry biomarkers was poor. Unsupervised clustering of flow cytometry data returned four T cell subsets with higher discriminatory capacity for colitis than previously reported populations, but they cannot be considered as reliable classifiers. Although mechanisms predisposing some patients to particular irAEs have been described, we are presently unable to capture adequate information from pre-therapy flow cytometry and clinical data to reliably predict risk of irAE in most cases

The occupation of Benzu Cave (Ceuta) by Neolithic and Bronze Age Societies

Abstract This study presents the results of the archaeological investigation in Benzú Cave, located on the North African shore of the Strait of Gibraltar. The archaeological deposits, approximately 1 m deep, belong to two occupational levels dated to theNeolithic and the Bronze Age. Awide range of artifacts was found, and this led to an interdisciplinary study that led us to reach new conclusions concerning the material life, subsistence economy, and environment of the Neolithic and Bronze Age societies in northern Africa and the southern Iberian Peninsula.Résumé Cette étude présente les résultats des recherches archéologiques menées dans la grotte de Benzú, située sur la rive nord-africaine du détroit de Gibraltar. Les gisements archéologiques, d'environ un mètre de profondeur, appartiennent à deux niveaux d'occupation datant du néolithique et de l'âge du bronze. Une large gamme d'artefacts ont été trouvés, ce qui a conduit à une étude interdisciplinaire qui nous a amenés à tirer de nouvelles conclusions concernant la vie matérielle, l'économie de subsistance et l'environnement des sociétés du Néolithique et de l'Age du Bronze en Afrique du Nord et au sud de la Péninsule Ibérique

Soluble CD46 as a diagnostic marker of hepatic steatosis

Background The increasing prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD) incurs substantial morbidity, mortality and healthcare costs. Detection and clinical intervention at early stages of disease improves prognosis; however, we are currently limited by a lack of reliable diagnostic tests for population screening and monitoring responses to therapy. To address this unmet need, we investigated human invariant Natural Killer T cell (iNKT) activation by fat-loaded hepatocytes, leading to the discovery that circulating soluble CD46 (sCD46) levels accurately predict hepatic steatosis. Methods sCD46 in plasma was measured using a newly developed immuno-competition assay in two independent cohorts: Prospective living liver donors (n = 156; male = 66, female = 90) and patients with liver tumours (n = 91; male = 58, female = 33). sCD46 levels were statistically evaluated as a predictor of hepatic steatosis. Findings Interleukin-4-secreting (IL-4+) iNKT cells were over-represented amongst intrahepatic lymphocytes isolated from resected human liver samples. IL-4+ iNKT cells preferentially developed in cocultures with a fat-loaded, hepatocyte-like cell line, HepaRG. This was attributed to induction of matrix metalloproteases (MMP) in fat-loaded HepaRG cells and primary human liver organoids, which led to indiscriminate cleavage of immune receptors. Loss of cell-surface CD46 resulted in unrepressed differentiation of IL-4+ iNKT cells. sCD46 levels were elevated in patients with hepatic steatosis. Discriminatory cut-off values for plasma sCD46 were found that accurately classified patients according to histological steatosis grade. Interpretation sCD46 is a reliable clinical marker of hepatic steatosis, which can be conveniently and non-invasively measured in serum and plasma samples, raising the possibility of using sCD46 levels as a diagnostic method for detecting or grading hepatic steatosis

TIGIT+ iTregs elicited by human regulatory macrophages control T cell immunity

Human regulatory macrophages (Mreg) have shown early clinical promise as a cell-based adjunct immunosuppressive therapy in solid organ transplantation. It is hypothesised that recipient CD4(+) T cell responses are actively regulated through direct allorecognition of donor-derived Mregs. Here we show that human Mregs convert allogeneic CD4(+) T cells to IL-10-producing, TIGIT(+) FoxP3(+)-induced regulatory T cells that non-specifically suppress bystander T cells and inhibit dendritic cell maturation. Differentiation of Mreg-induced Tregs relies on multiple non-redundant mechanisms that are not exclusive to interaction of Mregs and T cells, including signals mediated by indoleamine 2,3-dioxygenase, TGF-beta, retinoic acid, Notch and progestagen-associated endometrial protein. Preoperative administration of donor-derived Mregs to living-donor kidney transplant recipients results in an acute increase in circulating TIGIT(+) Tregs. These results suggest a feed-forward mechanism by which Mreg treatment promotes allograft acceptance through rapid induction of direct-pathway Tregs

DC-SIGN(+) Macrophages Control the Induction of Transplantation Tolerance

Tissue effector cells of the monocyte lineage can differentiate into different cell types with specific cell function depending on their environment. The phenotype, developmental requirements, and functional mechanisms of immune protective macrophages that mediate the induction of transplantation tolerance remain elusive. Here, we demonstrate that costimulatory blockade favored accumulation of DC-SIGN-expressing macrophages that inhibited CD8(+) T cell immunity and promoted CD4(+)Foxp3(+) Treg cell expansion in numbers. Mechanistically, that simultaneous DC-SIGN engagement by fucosylated ligands and TLR4 signaling was required for production of immunoregulatory IL-10 associated with prolonged allograft survival. Deletion of DC-SIGN-expressing macrophages in vivo, interfering with their CSF1-dependent development, or preventing the DC-SIGN signaling pathway abrogated tolerance. Together, the results provide new insights into the tolerogenic effects of costimulatory blockade and identify DC-SIGN(+) suppressive macrophages as crucial mediators of immunological tolerance with the concomitant therapeutic implications in the clinic.This work was supported by the COST Action BM1305: Action to Focus and Accelerate Cell Tolerogenic Therapies (A FACTT), the Mount Sinai Recanati/Miller Transplantation Institute developmental funds, AST/Pfizer Basic Science Faculty Development Grant, Ministerio de Educacióny Ciencia SAF2010-15062, SAF2013-48834-R, and Fundación Mutua Madrileñ a grants to J.O. A portion of this work appears as part of the doctoral thesis of P.C.S

Complement component C4 structural variation and quantitative traits contribute to sex-biased vulnerability in systemic sclerosis

Altres ajuts: Fondo Europeo de Desarrollo Regional (FEDER), "A way of making Europe".Copy number (CN) polymorphisms of complement C4 play distinct roles in many conditions, including immune-mediated diseases. We investigated the association of C4 CN with systemic sclerosis (SSc) risk. Imputed total C4, C4A, C4B, and HERV-K CN were analyzed in 26,633 individuals and validated in an independent cohort. Our results showed that higher C4 CN confers protection to SSc, and deviations from CN parity of C4A and C4B augmented risk. The protection contributed per copy of C4A and C4B differed by sex. Stronger protection was afforded by C4A in men and by C4B in women. C4 CN correlated well with its gene expression and serum protein levels, and less C4 was detected for both in SSc patients. Conditioned analysis suggests that C4 genetics strongly contributes to the SSc association within the major histocompatibility complex locus and highlights classical alleles and amino acid variants of HLA-DRB1 and HLA-DPB1 as C4-independent signals

Therapeutic and biotechnological applications of serum-deactivated macrophages

Los macrófagos humanos derivados de monocitos adquieren propiedades específicas cuando se cultivan con altas concentraciones de suero humano, incluyendo la capacidad supresora de linfocitos T y la expresión de algunas características típicas de células epiteliales. Los macrófagos desactivados por suero humano se diferencian de otros tipos de macrófago descritos en cuanto a antígenos de superficie celular y actividad in vitro. Además, este grupo de macrófagos se deriva de un modo diferente, que depende de la inmunoglobulina presente en suero humano. Un tipo de macrófago desactivado por suero y estimulado con IFN-gamma, llamado macrófago regulador o M reg, es capaz de inhibir la proliferación de linfocitos T estimulados con PHA y de polarizar linfocitos T hacia respuestas reguladoras. DHRS9, una enzima retinol deshidrogenasa de la familia SDR, se ha identificado como un nuevo marcador de M regs. En contra de publicaciones previas, se ha demostrado que otro tipo de macrófago desactivado por suero, llamado PCMO, no es una célula progenitora multipotente. Sin embargo, derivados de PCMO expresan características de hepatocitos y de células pancreáticas como parte de un programa normal de diferenciación macrofágica, lo que significa que los NeoHepatocitos podrían ser usados en aplicaciones toxicológicas específicas. La caracterización fenotípica y funcional de M regs, PCMO, NeoHepatocitos y NeoIslotes permite clasificarlos en el contexto de estados de activación de macrófagos, y sugieren que los macrófagos desactivados por suero humano presentan un fenotipo similar al de macrófagos residentes en tejidos. Además en este trabajo se han discutido las aplicaciones potenciales de macrófagos desactivados por suero humano en terapias de reparación de tejidos y de inducción de tolerancia inmunológica.Peer Reviewe

Anti-HLA antibodies in pooled human serum do not prevent development of human regulatory macrophages from monocytes in culture

Background: A GMP-compliant process for manufacturing a medicinal cellbased product, known as Mreg_UKR, containing human regulatory macrophages (Mreg) has been established. Mreg_UKR is currently being investigated in a Phase-I/II trial as a means to safely reduce maintenance immunosuppression in kidney transplant recipients. Partway into this study, manufacturing failure occurred in 3 independent batches. These failures could not be attributed to procedural errors or donor-related factors. Therefore, a root-cause analysis was undertaken, including characterising current and previous lots of pooled, heat-inactivated, recalcified plasma-derived, male-only human AB serum that were used in the manufacturing process. Methods: Single Ag bead arrays and CDC-crossmatches were performed by an accredited laboratory. Results: Throughout process-development and clinical production, 3 separate charges of serum from the same supplier were used. Under researchconditions, all 3 sera supported normal development of Mregs from monocytes.To test whether an tibodies in the sera could explain idiosyncratic manufacturing failures, the 3 sera were screened by single-antigen bead array for anti-HLA. All 3 charges contained HLA Clas s I- and II-reactive antibodies at above- threshold levels. To test whether these antibodies fixed complement, mixed- and B cell-crossmatches were performed using cells from a DR4-positive donor. Despite the presence of anti-DR4 antibody, no complement-dependent cytotoxicity was observed. A low concentration of anti-HLA antibody in our AB serum is the most likely explanation for this discrepancy; however, we cannot formally exclude false-positive signals from the bead array or false-negative results from crossmatching. Conclusion: The unexpected presence of anti-HLA antibodies in certain commercially available human AB sera argues for substituting human AB serum with platelet lysate or serum -free medium wherever feasible

First report of Phytophthora cactorum causing trunk and stem rot on loquat (Eriobotrya japonica) in Chile

Montealegre, J.; Riquelme, D.; Ramírez, M.; León Santana, M.; Abad Campos, P.; Pérez, LM. (2016). First report of Phytophthora cactorum causing trunk and stem rot on loquat (Eriobotrya japonica) in Chile. Plant Disease. 100(12):2537-2537. doi:10.1094/PDIS-05-16-0740-PDNS253725371001