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Endometrial cancer is the most common gynecologic cancer in the U.S., but metastasis to the brain is rare, and diagnosis can be challenging. Traditional tools for determining if a tumor is a primary or metastatic lesion include pan-imaging, histopathologic studies, and immunohistochemistry. Molecular testing with next-generation sequencing has been increasingly used to augment these tests. We present a case of a patient who initially presented with a brain lesion diagnosed as glioblastoma on histology and immunohistochemistry, but whose diagnosis was later changed to metastasis from an endometrial primary based on molecular findings. The two tumors shared a common microsatellite instability signature and 51 DNA variants, including oncogenic driver mutations KRAS p.G13D, PIK3CA p.E545A, and PTEN p.I135V and p.K267Rfs*9. This highlights the power of molecular analysis in making the diagnosis in cases of rare metastases. Key Points Brain metastasis from endometrial primary is rare, and histopathological features may be augmented with molecular analysis to aid in diagnosis. Comparison of the molecular makeup of the primary endometrial lesion with the metastatic lesion may reveal high-risk molecular features that may be indicative of metastatic potential.

Mucinous ovarian tumors rarely harbor mural nodules, which have historically been classified as sarcoma-like, anaplastic carcinomatous, or sarcomatous on the basis of predominant morphologic features. The molecular relationship between mural nodules and associated mucinous ovarian tumors remains poorly characterized, as does the molecular pathogenesis of these mural nodules. Thus, we analyzed the morphological, immunohistochemical, and genetic features of 13 mucinous ovarian tumors and associated mural nodule(s). Three harbored sarcoma-like mural nodules and ten contained anaplastic carcinomatous nodules, including 1 tumor with spatially discrete anaplastic carcinomatous and sarcomatous nodules. Twelve of 13 cases showed genetic evidence of clonality between the mural nodule(s) and associated mucinous ovarian tumor, including all three tumors with sarcoma-like morphology. Mural nodules were genetically identical in the five cases in which there were multiple discrete mural nodules that were sequenced separately. MTAP and p53 immunohistochemistry confirmed the distribution of neoplastic cells in a subset of sarcoma-like and anaplastic carcinomatous nodules. No single recurrent genetic alteration was associated with mural nodule development. No recurrent genetic differences were identified between mural nodules with sarcoma-like, anaplastic carcinomatous, and sarcomatous morphology. Of 11 patients with clinical follow-up, three died of disease 3, 8, and 9 months after diagnosis, but no recurrent genetic events were associated with poor outcome. These molecular data suggest that sarcoma-like, anaplastic carcinomatous, and sarcomatous nodules represent a morphologic spectrum of clonal neoplasms arising in mucinous ovarian tumors rather than three discrete biological entities.

HIV-1 sequence variations impact binding of inhibitory killer cell immunoglobulin-like receptors (KIRs) to human leukocyte antigen class I (HLA-I) molecules modulating natural killer cell function. HIV-1 strains encoding amino acids that mediate binding of inhibitory KIRs might therefore have a selective benefit in individuals expressing the respective KIR/HLA genotypes. Here, we demonstrate that HIV-1 clade C avoids a p24 Gag mutation that abolishes binding of KIR2DL2 to HLA-C03:04 and disinhibits natural killer cells in individual encoding for this genotype.

Background Alemtuzumab can induce secondary autoimmunity affecting multiple organs. While kidney involvement is uncommon, it can be associated with devastating forms of glomerulonephritis (GN). Case presentation A 32-year-old African American woman presented with hypertension, proteinuria, and progressive renal failure. Her medical history was remarkable for secondary progressive multiple sclerosis (SPMS). She had received her first induction dose of alemtuzumab 1 year prior to presentation. Upon evaluation, she had scanning speech, multidirectional nystagmus, and mild edema. Her serum creatinine was 2 mg/dL. Urine studies revealed proteinuria and microscopic hematuria. Her serologic tests were positive for c-antineutrophil cytoplasmic antibodies (> 1 : 640). In addition, she was found to have new-onset severe thyroid dysfunction with antibodies against thyroglobulin and thyroid peroxidase. Kidney biopsy was diagnostic for pauci-immune crescentic GN. The patient was treated with methylprednisolone and rituximab with subsequent renal, thyroid, and neurological recovery. Conclusion This is an atypical case of GN following therapy with alemtuzumab. We hypothesize that immune reconstitution may be a potential mechanism. Alemtuzumab is a new treatment for SPMS that can be associated with GN. Practice guidelines should address the management of its renal complications.

Mesenchymal Stem cells (MSCs) has fascinating immunomodulatory effect in vitro and in vivo. This effect has been documented by interesting researches worldwide. However the protocols of MSCs culture are different. This difference is translated on different results regarding the mediators as well as on potency of immunomodulatory effect in both vitro and in vivo. In this article we will analyze and discussing the effect of culture environment on MSC immunomodulatory effect.

In pair bonding animals, coordinated behavior between partners is required for the pair to accomplish shared goals such as raising young. Despite this, experimental designs rarely assess the behavior of both partners within a bonded pair. Thus, we lack an understanding of the interdependent behavioral dynamics between partners that likely facilitate relationship success. To identify intra-pair behavioral correlates of pair bonding, we used socially monogamous prairie voles, a species in which females and males exhibit both overlapping and distinct pair bond behaviors. We tested both partners using social choice and non-choice tests at short- and long-term pairing timepoints. Females developed a preference for their partner more rapidly than males, with preference driven by different behaviors in each sex. Further, as bonds matured, intra-pair behavioral sex differences and coordinated behavior emerged – females consistently huddled more with their partner than males did, and partner huddle time became correlated between partners. When animals were allowed to freely interact with a partner or a novel in sequential free interaction tests, pairs spent more time interacting together than either animal did with a novel. Pair interaction was correlated with female, but not male, behavior. Via a social operant paradigm, we found that pair-bonded females, but not males, are more motivated to access and huddle with their partner than a novel vole. Together, our data indicate that as pair bonds mature, sex differences and coordinated behavior emerge, and that these intra-pair behavioral changes are likely organized and driven by the female animal.

Background In this study, we investigated whether G9a related DNA methylation and histone modification is involved in the transcription alteration of NR2B following peripheral nerve injury and subsequently contributes to pain facilitation of G9a inhibition. Methods After approval by the institutional ethical committee on pain research in conscious animals, C57BL/6 mice were used to induce neuropathic pain with spared nerve injury (SNI). G9a/Glp expression, GRIN2B gene 5’-regulatory region CpG sites methylation profile, as well as NR2B expression in the spinal dorsal horn following SNI was detected with immunofluorescence, bisulfite sequencing, and western blot, respectively. For mechanism study, threshold doses of G9a/Glp inhibitors BIX01294/UNC0638 or direct DNA demethylation agent 5-Aza was intrathecal injected through the pre-buried catheter daily in bolus for 3 days, G9a/Glp and its enzymatic substrate H3K9me2/H3K9me3 expression, GRIN2B gene methylation alteration, as well as NR2B expression were observed. Nociceptive behavior was depicted in response to von Frey filaments following SNI with or without intrathecal BIX/UNC and 5-Aza treatments. Results Ipsilateral mechanical withdrawal threshold rather than thermal withdrawal latency prominently decreased and peaked at day 7 to 14 post SNI. Besides, nerve injury consistently increased G9a/Glp, H3K9me2 expression, GRIN2B gene 5’-regulatory region CpG sites methylation, as well as NR2B expression in the spinal dorsal horn at day 7 post SNI. Furthermore, either G9a/Glp inhibition by BIX/UNC or H3K9me2 blockade by 5-Aza independently reversed GRIN2B gene high methylation, followed with disinhibition of NR2B transcription inhibition. Consistent with molecular changes, either BIX/UNC or 5-Aza further worsens nerve injury-induced allodynia. Conclusion G9a/Glp contributes to the pathogenesis of neuropathic pain via methylating GRIN2B gene affecting NR2B transcription. G9a/Glp at an elevated setpoint may prevent the over-sensitization following peripheral nerve injury.

BACKGROUND: Despite routine use of echocardiographic parameters to evaluate the severity of cardiac amyloidosis (CA), this methodology has not been well validated. We developed a histopathologic schema for quantifying CA burden and evaluated its relationship with clinical outcomes. Additionally, echocardiographic parameters were tested as potential noninvasive indices of CA burden. METHODS: We retrospectively studied 59 patients with CA (17 light chain, 42 transthyretin) who underwent endomyocardial biopsies. Light microscopy with staining was used to categorize CA burden as mild-to-moderate (

Pet rabbits have increased their popularity in a lot of countries. However, most of the laboratory profiles in rabbit medicine come from the observations made in rabbit as biomodels or meat production. So that further researches are necessary to obtain reference values for hematology and biochemical profiles in pet rabbits and the different breeds, especially, in relation to acid-base balance. The aim of this report was to offer the mean values of the main parameters connected with acid-base profile in Netherland Dwarf breed. Thirty-five healthy rabbits (15 males and 20 females) were studied. Venous blood sample from lateral saphenous vein was analyzed to measure: haematocrit, haemoglobin, blood urea nitrogen, glucose, blood pH, partial pressure of CO2 (pCO2), total CO2, ions bicarbonate, chloride, sodium, potassium, base excess and anion Gap. Results showed a shorter range that those reported by different researchers. Moreover, differences between genders were showed in pCO2, its values were higher in males. It may be associated with a greater cellular metabolism. Values obtained in this research should be taken into account by veterinary clinicians for this breed in their clinical assessments. Besides, these values provide new results in parameters with few reference values. Resumo A popularidade de coelhos como animais de estimação aumentou em muitos países. No entanto, a maioria dos perfis de laboratório em medicina de coelhos advém das observações de biomodelos animais ou da produção de carne. Assim, são necessárias pesquisas adicionais para obter valores de referência para hematologia e perfis bioquímicos em coelhos de estimação, e das diferentes raças, especialmente, em relação ao equilíbrio ácido-base. O objetivo deste relatório foi oferecer os valores médios dos principais parâmetros ligados ao perfil ácido-base na raça Anã Holandês. Trinta e cinco coelhos saudáveis (15 machos e 20 fêmeas) foram estudados. A amostra de sangue venoso da veia safena lateral foi analisada para mensuração: hematócrito, hemoglobina, nitrogênio ureico sanguíneo, glicose, pH sanguíneo, pressão parcial de CO2 (pCO2), CO2 total, íons bicarbonato, cloreto, sódio, potássio, excesso de base e ânion Gap. Os resultados apresentaram um intervalo menor do que aqueles relatados por diferentes pesquisadores. Além disso, as diferenças entre os gêneros foram mostradas na pCO2, seus valores foram maiores no sexo masculino. Pode estar associado a um maior metabolismo celular. Os valores obtidos nesta pesquisa devem ser levados em consideração pelos clínicos veterinários para esta raça em suas avaliações clínicas. Além disso, esses valores fornecem novos resultados em parâmetros com poucos valores de referencia.

Aims Methylthioadenosine phosphorylase (MTAP) immunohistochemical expression is a specific marker of CDKN2A deletion in malignant mesothelioma. However, the relationship of MTAP expression with MTAP copy number remains unexplored. Methods and results Forty malignant pleural mesotheliomas were characterised by targeted next-generation sequencing (29), single-nucleotide polymorphism microarray (seven), or both (four). MTAP and CDKN2A copy numbers were correlated with MTAP expression. Twenty-seven (68%) tumours showed CDKN2A deletion (14 heterozygous; 13 homozygous), of which 20 (74%) showed MTAP codeletion (15 heterozygous; five homozygous). No tumours showed MTAP deletion without CDKN2A codeletion. Loss of MTAP expression was seen in 16 (40%) tumours, and was 75% sensitive and 95% specific for MTAP deletion, and 59% sensitive and 100% specific for CDKN2A deletion. Nine of 40 (23%) tumours showed heterogeneous MTAP staining, and the percentage of tumour cells with MTAP loss correlated with molecular detection of MTAP deletion. Conclusions MTAP is frequently codeleted with CDKN2A in pleural mesothelioma. However, homozygous deletion of both genes occurs in a minority of tumours (5/40; 13%); CDKN2A deletion often co-occurs with heterozygous MTAP deletion or neutral MTAP copy number; and MTAP expression correlates inconsistently with heterozygous MTAP deletion. Correspondingly, MTAP immunohistochemistry is a highly specific but only moderately sensitive assay for CDKN2A deletion.

Phenylalanine hydroxylase (PAH) deficiency is an inborn error of phenylalanine (Phe) metabolism that results in the buildup of dietary Phe to potentially toxic levels. Poorly controlled Phe levels in women of childbearing age are particularly worrisome due to the toxic effect of elevated Phe on fetal development. Pegvaliase was recently approved as an enzyme substitution therapy to reduce Phe concentrations in adult patients with PAH deficiency who have suboptimal Phe control on existing management. During the pegvaliase clinical trials pregnant patients were excluded from participation, but the approved label does not contraindicate its use during pregnancy. This case report describes the outcome of the first PAH deficient patient who elected to continue treatment with pegvaliase during pregnancy and reviews the lessons learned and future considerations.

Absence of 'live' residual poliovirus in Inactivated Poliovirus Vaccine (IPV) is routinely checked using Primary Monkey Kidney Cells (PMKC). However, the increasing demand for IPV and the ethical, technical and safety issues associated with the use of non-human primates in research and quality control, has made the replacement of primary cells with an established cell line a priority, in line with the principles of the 3Rs (Replacement, Reduction and Refinement in animal testing). As an alternative to PMKC, we evaluated the L20B cell line; a mouse cell-line genetically engineered to express human poliovirus receptor, CD155. L20B is already used for the detection and diagnosis of poliovirus in clinical samples. We demonstrate the stability of L20B cells in terms of CD155 gene and receptor expression, and permissivity to polioviruses for at least 16 sequential passages. In addition, the L20B cell line was found to be at least as sensitive as PMKC in detecting the presence of 'live' poliovirus in IPV samples. Equivalence or superiority of L20B cells versus PMKCs was demonstrated for assessing the presence of residual 'live' poliovirus in formaldehyde-inactivated preparations for the three poliovirus serotypes. These results demonstrate that the L20B cell line is a suitable alternative to PMKC in IPV inactivation testing.

The potential use of stem cell-based therapies for the repair and regeneration of different tissues and organs offers a paradigm shift that will give different therapeutic solutions for variety of diseases. The use of either embryonic stem cells (ESCs) or elicited pluripotent stem cells in clinical scenarios is restricted because of cell inability to divide and to technical and ethical concerns within the genetic manipulation of human ESCs, although these cells are, on paper, extremely helpful. Mesenchymal stem cells appear to be a perfect population of stem cells for sensible regenerative medicine, as they are not subjected to a similar kind of restrictions. Recent basic analyses and clinical studies have shown that the utilization of adipose-derived stem cells (ASCs) harvested from animal fat tissue in regenerative medication is not restricted to mesoblastic tissue, but extends to each germ layer and endodermal tissues and organs. Supported by this information, main purpose of this brief review is to summarize and describe the underlying biology of ASCs and their proliferation and differentiation capacities, alongside current diagnostic and clinical knowledge from a range of medical fields relating to the utilization of ASCs in regenerative drugs.

The cholesterol ester transfer protein (CETP) inhibitor dalcetrapib has been under evaluation for its potential to prevent cardiovascular (CV) events for almost two decades. The current clinical development program, representing new advances in precision medicine and focused on a genetically defined population with acute coronary syndrome (ACS), is supported by a large body of pharmacokinetic and pharmacodynamic data as well as substantial clinical experience in over 13,000 patients and volunteers. Dalcetrapib treatment of 600 mg/day produces significant inhibition of CETP activity, and has been utilized in phase II and III studies, including CV endpoint trials. Numerous studies have investigated the interactions between dalcetrapib and most drugs commonly prescribed to CV patients and have not demonstrated any clinically significant effects. Evaluations in patients with renal and hepatic impairment demonstrate a greater exposure to dalcetrapib than in the non-impaired population, but long-term clinical studies including patients with mild to moderate hepatic and renal dysfunction demonstrate no increase in adverse events. Safety pharmacology and toxicology studies as well as the clinical safety experience support the continuing development of dalcetrapib as an adjunct to 'standard of care' for the ACS population. This article provides a full review of the pharmacokinetics, as well as pharmacodynamics and pharmacology, of dalcetrapib in the context of a large clinical program.

[EN] The aim of this study was to provide reference values for a single, popular breed of pet rabbit. Moreover, additional objectives were to determine whether sex, body position or age alter Netherland Dwarf rabbit electrocardiographic variables and whether the use of electrocardiographic filters affects those variables. Forty Netherland Dwarf rabbits were examined clinically and standard six-lead electrocardiograms (ECGs) were recorded in sternal and then dorsal recumbency. At first power-line and anti-drift filters were used and then they were disabled. The following variables were measured in lead II: heart rate; P wave duration and amplitude; P-R interval; QRS duration; R wave amplitude (with and without filters); Q-T interval; T wave duration and amplitude; S-T segment; J-T duration; and mean electrical axis (MEA) (with and without filters). MEA was determined by 3 different methods. After statistical processing of the data, our results showed that there were no significant differences between both recumbencies, with the exception of the J-T duration, which was higher in dorsal recumbency. The R wave amplitude using electrocardiographic filters showed significant differences between males (0.083 mV) and females (0.115 mV; P, J. M. Chapel held a research fellowship (Ref. Plan I2C) from the organization Xunta de Galicia (Spain). The authors thank C.R. Hughes for his grammar review.

The study objective was to characterize effects of early gestation in utero heat stress (IUHS) on postnatal fasting heat production (FHP) and blood biomarkers associated with metabolism in growing pigs. Based on previous observation of increased postnatal core body temperature set point in IUHS pigs, we hypothesized that FHP would be altered during postnatal life because of IUHS. Pregnant first-parity gilts were exposed to thermoneutral (TN; = 4; 17.8 ± 0.1°C) or heat stress (HS; = 4; cyclical 28 to 38°C) conditions from d 30 to 60 of gestation. At weaning (21 d of age), 2 median-weight male pigs (1 barrow and 1 boar) were selected from each litter ( = 8 in utero TN [IUTN] and 8 IUHS pigs) and then housed in TN conditions based on age. Blood samples were collected at 8, 9, and 10 wk of age when pigs were in a fed state to analyze thyroxine (T4) and triiodothyronine (T3) concentrations. Pigs were trained to enter an indirect calorimeter from wk 8 through 10 of life and then acclimated over a 24-h period 1 wk prior to testing. At 12 wk of age, pigs were fasted for 24 h, and then indirect calorimetry was performed on individual pigs over a 23-h testing period to determine FHP and the respiratory quotient in 3 intervals (0900 to 1700 h, 1700 to 0000 h, and 0000 to 0800 h). Body weight was determined before and after testing and was similar for all pigs ( = 0.77; 37.0 ± 0.5 kg BW). Data were analyzed using PROC MIXED in SAS 9.4. No boar vs. barrow differences were observed with any analysis. Overall, FHP per kilogram BW was greater ( = 0.03; 12.1%) in IUHS pigs compared with IUTN pigs. Fasting heat production per kilogram BW was greater ( < 0.01; 19.8%) from 0900 to 1700 h compared with 1700 to 0000 h and 0000 to 0800 h and was greater (10.9%) from 1700 to 0000 h compared with 0000 to 0800 h. The RQ did not differ by in utero treatment ( = 0.51; 0.72 ± 0.01); however, the RQ was increased ( < 0.01; 13.0%) from 1700 to 0000 h compared with 0900 to 1700 h and 0000 to 0800 h. No other FHP and RQ differences were detected. Although no in utero treatment differences were observed for T4 ( = 0.11; 52.2 ± 6.2 ng/mL), T3 was greater overall ( = 0.04; 19.5%) in IUHS pigs than in IUTN pigs. In summary, FHP and circulating T3 were increased in IUHS pigs, and this may have implications for postnatal production efficiency in pigs gestated during hot summer months.

BACKGROUND Autoantibodies against Factor VIII (FVIII) define the rare but life-threatening bleeding disorder acquired hemophilia A (AHA). Correction of FVIII deficiency and eradication of the factor inhibitor are the ultimate therapeutic goals in this disorder. Bypassing agents such as recombinant factor VIIa (rFVIIa) or FVIII inhibitor bypassing agent are often used to control coagulopathy before the inhibitor is eradicated. Bypassing agents carry a risk of thrombosis, however. CASE REPORT We report a patient with newly diagnosed AHA and thalamic bleed who additionally had active atrial fibrillation and developed a segmental pulmonary embolism, limiting tolerable rFVIIa dosage. This patient with very-high-risk brain bleed and concurrent thrombosis on bypass agent represented a significant management dilemma for which we successfully utilized therapeutic plasma exchange (TPE) to reduce the inhibitor titer. RESULTS FVIII inhibitor was undetectable and FVIII level was above the lower limit of normal within 12.5 days from starting TPE. While the patient ultimately died 24 days from admission for reasons unrelated to bleeding, his intracerebral hemorrhage was unchanged in size and no other bleeding morbidity was observed. CONCLUSION This patient achieved eradication of FVIII inhibitor and did not have bleed expansion while receiving multimodal therapy including corticosteroids, rituximab, and TPE. We discuss the periprocedural risks of TPE in an acquired hemophilia patient and our multiteam management of that risk.

Background High performance anti-hepatitis E virus (HEV) IgG assays are crucial for epidemiology. Objective To evaluate the performance of 2 prototypes developed for the VIDAS® automatic system for detecting anti-HEV IgG, one based on the ORF2 antigen (ORF2 prototype) and the other on the ORF2 and ORF3 antigens (ORF2/3 prototype), with reference to the Wantai anti-HEV IgG assay. Study design The sensitivity of each assay was determined by testing 113 blood samples, 63 from immunocompetent and 50 from immunocompromised patients, with a proven HEV infection defined by detecting HEV RNA. Their specificity was assessed with 103 blood samples that the Wantai assay indicated was negative for anti-HEV IgM and IgG, and negative for HEV-RNA. Cross reactivity was assessed using samples that were positive for hepatitis A virus IgG (n = 16), hepatitis C virus antibodies (n = 15), hepatitis B virus antigen and anti-HBc antibodies (n = 16), rheumatoid factor (n = 14), and negative for anti-HEV IgG with the Wantai assay. Results The sensitivities in immunocompetent patients were: 95.2% (ORF2), 96.8% (ORF2/3), and 93.6% (Wantai); in immunocompromised patients they were: 66% (ORF2), 72% (ORF2/3), and 68% (Wantai). Both VIDAS prototypes detected low concentrations of anti-HEV IgG. The overall specificity was 100% (ORF2 prototype) and 98.1% (ORF2/3 prototype). Both VIDAS prototypes cross-reacted in five samples (9.6%), mainly those containing HCV antibodies or rheumatoid factor. Conclusion Both VIDAS® prototypes performed very well and appear to be suitable for routine detection of anti-HEV IgG.

In allergic airway inflammation, VEGFR-3-mediated lymphangiogenesis occurs in humans and mouse models, yet its immunological roles, particularly in adaptive immunity, are poorly understood. Here, we explored how pro-lymphangiogenic signaling affects the allergic response to house dust mite (HDM). In the acute inflammatory phase, the lungs of mice treated with blocking antibodies against VEGFR-3 (mF4-31C1) displayed less inflammation overall, with dramatically reduced innate and T cell numbers and reduced inflammatory chemokine levels. However, when inflammation was allowed to resolve and memory recall was induced 2 months later, mice treated with mF4-31C1 as well as VEGF-C/-D knockout models showed exacerbated type 2 memory response to HDM, with increased Th2 cells, eosinophils, type 2 chemokines, and pathological inflammation scores. This was associated with lower CCL21 and decreased TRegs in the lymph nodes. Together, our data imply that VEGFR-3 activation in allergic airways both help initiate the acute inflammatory response as well as regulate the adaptive (memory) response, possibly in part by shifting the TReg/Th2 balance. This introduces new immunomodulatory roles for pro-lymphangiogenic VEGFR-3 signaling in allergic airway inflammation and suggests that airway lymphatics may be a novel target for treating allergic responses.

International audience; Natural killer (NK) cells can recognize virus-infected and stressed cells1 using activating and inhibitory receptors, many of which interact with HLA class I. Although early studies also suggested a functional impact of HLA class II on NK cell activity2,3, the NK cell receptors that specifically recognize HLA class II molecules have never been identified. We investigated whether two major families of NK cell receptors, killer-cell immunoglobulin-like receptors (KIRs) and natural cytotoxicity receptors (NCRs), contained receptors that bound to HLA class II, and identified a direct interaction between the NK cell receptor NKp44 and a subset of HLA-DP molecules, including HLA-DP401, one of the most frequent class II allotypes in white populations4. Using NKp44ζ+ reporter cells and primary human NKp44+ NK cells, we demonstrated that interactions between NKp44 and HLA-DP401 trigger functional NK cell responses. This interaction between a subset of HLA-DP molecules and NKp44 implicates HLA class II as a component of the innate immune response, much like HLA class I. It also provides a potential mechanism for the described associations between HLA-DP subtypes and several disease outcomes, including hepatitis B virus infection5-7, graft-versus-host disease8 and inflammatory bowel disease9,10.

Atypical hyperplasia/endometrial intraepithelial neoplasia is an accepted precursor to endometrioid-type endometrial carcinoma. Mismatch repair-deficient endometrial carcinomas are also known to be a biologically and clinically distinct subset of tumors. However, the development of microsatellite instability in endometrial carcinogenesis has not yet been evaluated by novel next-generation sequencing-based methods. We examined 17 mismatch repair-deficient endometrioid endometrial carcinomas and their paired atypical hyperplasia/endometrial intraepithelial neoplasia precursors using a next-generation sequencing panel with quantitative microsatellite instability detection at 336 loci. Findings were compared to histological features, polymerase chain reaction-based microsatellite instability testing, immunohistochemical expression of mismatch repair proteins, and tumor mutational burden calculations. All 17 endometrial carcinomas and 8/17 atypical hyperplasia/endometrial intraepithelial neoplasia showed microsatellite instability by next-generation sequencing-based testing. Endometrial carcinoma specimens showed significantly more unstable microsatellite loci than paired atypical hyperplasia/endometrial intraepithelial neoplasia (mean: 40.0% vs 19.9 unstable loci, respectively). Out of nine microsatellite-stable atypical hyperplasia/endometrial intraepithelial neoplasia specimens, four showed mismatch repair loss by immunohistochemistry. All atypical hyperplasia/endometrial intraepithelial neoplasia and endometrial carcinoma specimens with microsatellite instability were also mismatch repair-deficient by immunohistochemistry. Tumor mutational burden was significantly greater in endometrial carcinoma than in paired atypical hyperplasia/endometrial intraepithelial neoplasia specimens, and tumor mutational burden was significantly correlated with percent unstable microsatellite loci. Paired atypical hyperplasia/endometrial intraepithelial neoplasia and endometrial carcinoma specimens show progressive accumulation of unstable microsatellite loci following loss of mismatch repair protein expression. Comprehensive next-generation sequencing-based testing of endometrial carcinomas offers new insights into endometrial carcinogenesis and opportunities for improved tumor surveillance, diagnosis, and management.

Symptomatic primary antibody deficiency syndromes (PADS) range from severe forms, such as common variable immunodeficiency disorders and X-linked agammaglobulinemia, to partial antibody deficiencies. All patients with failure of antibody production suffer from chest infections with common organisms, which should prompt diagnosis. Patients with proven antibody failure require replacement immunoglobulin therapy.

Transmission of HIV across mucosal barriers accounts for the majority of HIV infections worldwide. Thus, efforts aimed at enhancing protective immunity at these sites are a top priority, including increasing virus-specific antibodies (Abs) and antiviral activity at mucosal sites. Mucin proteins, including the largest cell-associated mucin, mucin 16 (MUC16), help form mucus to provide a physical barrier to incoming pathogens. Here, we describe a natural interaction between Abs and MUC16 that is enhanced in specific disease settings such as chronic HIV infection. Binding to MUC16 was independent of IgG subclass, but strongly associated with shorter Ab glycan profiles, with agalactosylated (G0) Abs demonstrating the highest binding to MUC16. Binding of Abs to epithelial cells was diminished following MUC16 knockdown, and the MUC16 N-linked glycans were critical for binding. Further, agalactosylated VRC01 captured HIV more efficiently in MUC16. These data point to a novel opportunity to enrich Abs at mucosal sites by targeting Abs to MUC16 through changes in Fc glycosylation, potentially blocking viral movement and sequestering the virus far from the epithelial border. Thus, next-generation vaccines or monoclonal therapeutics may enhance protective immunity by tuning Ab glycosylation to promote the enrichment of Abs at mucosal barriers.

Background & Aims Both natural killer (NK) cells and human leukocyte antigen (HLA)/killer cell immunoglobulin like receptor (KIR) interactions have been shown to play an important role in the control, clearance and progression of hepatitis C virus (HCV) disease. Here we aimed at elucidating the effects of viral peptides derived from HCV on HLA stabilization, changes in KIR binding and primary NK cell function. Methods Transporter for antigen presentation-deficient 722.221 cells stably transfected with HLA-C∗03:04 were used to screen 200 overlapping peptides, covering the non-structural protein 3 (NS3) and core protein of HCV genotype 1, for their ability to bind and stabilize HLA-C∗03:04. Binding of KIR2DL3 to the HLA-peptide complex was assessed using a KIR2DL3-IgG fusion construct. Primary NK cells were isolated from healthy donors to investigate the effects of identified peptides on KIR2DL3 + NK cell function. Results Thirty-one peptides able to stabilize HLA-C∗03:04 were identified. One 9mer peptide, YIPLVGAPL, resulted in significantly higher KIR2DL3 binding to HLA-C∗03:04 + 722.221 cells and suppression of primary KIR2DL3 + NK cell function. Interestingly this sequence exhibited a high frequency of mutations in different HCV genotypes. These genotype-specific peptides showed lower HLA-C∗03:04 stabilization, decreased binding of the inhibitory KIR2DL3 and lower inhibition of NK cell function. Conclusions Taken together we show that a viral peptide derived from the core protein of HCV genotype 1 binding to HLA-C∗03:04 results in a sequence-dependent engagement of the inhibitory NK cell receptor KIR2DL3, while the large majority of the remaining 30 HLA-C∗03:04 binding HCV core peptides did not. These data show that sequence variations within HCV can modulate NK cell function, providing potential pathways for viral escape. Lay summary We identified a HCV peptide that dampens NK cell responses, and thereby possibly prevents killing of infected cells through this part of the innate immune system. This is facilitated via presentation of the viral peptide on HLA∗03:04 to the inhibitory KIR receptor KIR2DL3 on NK cells. Naturally occurring sequence mutations in the peptide alter these interactions making the inhibition less efficient.

Summary Chronic mucocutaneous candidiasis (CMC) is characterized by recurrent and persistent superficial infections, with Candida albicans affecting the mucous membranes, skin and nails. It can be acquired or caused by primary immune deficiencies, particularly those that impair interleukin (IL)−17 and IL-22 immunity. We describe a single kindred with CMC and the identification of a STAT1 GOF mutation by whole exome sequencing (WES). We show how detailed clinical and immunological phenotyping of this family in the context of WES has enabled revision of disease status and clinical management. Together with analysis of other CMC cases within our cohort of patients, we used knowledge arising from the characterization of this family to develop a rapid ex-vivo screening assay for the detection of T helper type 17 (Th17) deficiency better suited to the routine diagnostic setting than established in-vitro techniques, such as intracellular cytokine staining and enzyme-linked immunosorbent assay (ELISA) using cell culture supernatants. We demonstrate that cell surface staining of unstimulated whole blood for CCR6+CXCR3–CCR4+CD161+ T helper cells generates results that correlate with intracellular cytokine staining for IL-17A, and is able to discriminate between patients with molecularly defined CMC and healthy controls with 100% sensitivity and specificity within the cohort tested. Furthermore, removal of CCR4 and CD161 from the antibody staining panel did not affect assay performance, suggesting that the enumeration of CCR6+CXCR3–CD4+ T cells is sufficient for screening for Th17 deficiency in patients with CMC and could be used to guide further investigation aimed at identifying the underlying molecular cause.

Loss-of-function mutations in DOCK8 are linked to hyper-IgE syndrome. Patients typically present with recurrent sinopulmonary infections, severe cutaneous viral infections, food allergies and elevated serum IgE. Although patients may present with a spectrum of disease-related symptoms, molecular mechanisms explaining phenotypic variability in patients are poorly defined. Here we characterized a novel compound heterozygous mutation in DOCK8 in a patient diagnosed with primary combined immunodeficiency which was not typical of classical DOCK8 deficiency. In contrast to previously identified mutations in DOCK8 which result in complete loss of function, the newly identified single nucleotide insertion results in expression of a truncated DOCK8 protein. Functional evaluation of the truncated DOCK8 protein revealed its hypomorphic function. In addition we found somatic reversion of DOCK8 predominantly in T cells. The combination of somatic reversion and hypomorphic DOCK8 function explains the milder and atypical phenotype of the patient and further broadens the spectrum of DOCK8-associated disease., Highlights • Whole exome sequencing identified the underlying defect in a patient with combined immunodeficiency. • A novel compound heterozygous DOCK8 mutation was identified. • Expression of a truncated DOCK8 protein with hypomorphic function was identified. • Somatic reversion of DOCK8 mainly in T cells was identified. • DOCK8 deficiency may present without severe viral infections and increased serum IgE levels.

Summary Primary antibody deficiencies (PAD) constitute the majority of all primary immunodeficiency diseases (PID) and immunoglobulin replacement forms the mainstay of therapy for many patients in this category. Secondary antibody deficiencies (SAD) represent a larger and expanding number of patients resulting from the use of a wide range of immunosuppressive therapies, in particular those targeting B cells, and may also result from renal or gastrointestinal immunoglobulin losses. While there are clear similarities between primary and secondary antibody deficiencies, there are also significant differences. This review describes a practical approach to the clinical, laboratory and radiological assessment of patients with antibody deficiency, focusing on the factors that determine whether or not immunoglobulin replacement should be used. The decision to treat is more straightforward when defined diagnostic criteria for some of the major PADs, such as common variable immunodeficiency disorders (CVID) or X-linked agammaglobulinaemia (XLA), are fulfilled or, indeed, when there is a very low level of immunoglobulin production in association with an increased frequency of severe or recurrent infections in SAD. However, the presentation of many patients is less clear-cut and represents a considerable challenge in terms of the decision whether or not to treat and the best way in which to assess the outcome of therapy. This decision is important, not least to improve individual quality of life and reduce the morbidity and mortality associated with recurrent infections but also to avoid inappropriate exposure to blood products and to ensure that immunoglobulin, a costly and limited resource, is used to maximal benefit.

BACKGROUND Given the high heterogeneity of sub-Saharan populations especially between nonpygmoids and pygmoids, differences are expected during investigation of the DO/ART4 gene. STUDY DESIGN AND METHODS Using genomic DNA extracted from blood samples collected from 77 Tswa pygmoids and 39 Teke and seven San nonpygmoids, DO coding regions were amplified and sequenced. A tetra-primer amplification refractory mutation system-polymerase chain reaction method was developed to specifically detect the DO*B-SH-Gln149Lys variant. Membrane expression of newly identified variant alleles in K562-transduced cells was studied by flow cytometry. RESULTS Extensive polymorphism was confirmed in Teke or San nonpygmoid and Tswa pygmoids with, respectively, 12, zero, and 24 DO*A; 54, 10, and 115 DO*B or DO*B-WL; five, zero, and 14 DO*HY; and six DO*JO alleles in Teke only. The DO*B-SH-Gln149Lys variant was observed as the third most frequent after the DO*HY and DO*JO alleles. Two novel DO*B alleles were identified in the San samples, that is, DO*B-Ile5Thr and DO*B-Trp266Arg. Study of K562-transduced cells showed that compared to the DO*B allele, DO*B-Ile5Thr was expressed more strongly while DO*B-Trp266Arg variant was expressed to a lesser extent and was not recognized by MIMA-123 monoclonal antibodies. CONCLUSION Sequencing analysis showed more allelic combinations in nonpygmoids than in pygmoids with high frequencies of DO*HY, DO*JO, and DO*B-SH-Gln149Lys variant alleles. This finding underlines the importance of including DO*HY and DO*JO single-nucleotide polymorphisms in genotyping tests to improve transfusion safety. Characterization of two novel DO*B alleles highlights the value of testing selected ethnic groups in understanding DO allele diversity.

Introductory paragraphThe successful production of Hematopoietic Stem and Progenitor Cells (HSPCs) from human pluripotent sources is conditioned by transgene delivery1-5. We describe here a dedicated and tractable one step, GMP-grade, transgene-free and stroma-free protocol to produce HSPCs from human induced pluripotent stem cells (hiPSCs). This procedure, applied to several sources of hiPSCs with equal efficiency, is based on a directed differentiation with morphogens and cytokines to generate a cell population close to nascent HSPCs or their immediate forerunners i.e., hemogenic endothelial cells6-9. Following engraftment into immunocompromised recipients, this cell population was proved capable of a robust myeloid, lymphoid and definitive red blood cell production in sequential recipients for at least 40 weeks. Further identification of the repopulating cells show that they express the G protein–coupled receptor APELIN (APLNR) and the homing receptor CXCR4. This demonstrates that the generation of bona fide HSPCs from hiPSCs without transgenes is possible and passes through an early endo-hematopoietic intermediate. This work opens the way to the generation of clinical grade HSPCs for the treatment of hematological diseases and holds promise for the analysis of HSPC development in the human species.

The activating NK cell receptor KIR2DS1 has been shown to be involved in many disorders including autoimmune diseases, malignancies and pregnancy outcomes. However, the precise ligands and functions of this receptor remain unclear. We aimed to gain a better understanding of the factors involved in the binding of KIR2DS1 and its inhibitory counterpart KIR2DL1 to HLA class I molecules, and the consequences for KIR2DS1+ NK-cell function. A systematic screen that assessed binding to 97 HLA-I proteins confirmed that KIR2DS1-binding was narrowly restricted to HLA-C group 2 complexes, while KIR2DL1 showed a broader binding specificity. Using KIR2DS1ζ+ Jurkat reporter-cells and peptide-pulsed 721.221.TAP1KO-HLA-C*06:02 cells, we identified the synthetic peptide SRGPVHHLL presented by HLA-C*06:02 that strongly engaged KIR2DS1- and KIR2DL1-binding. Functional analysis showed that this HLA-C*06:02-presented peptide can furthermore activate primary KIR2DS1(+) NK cell clones. Thus, we demonstrated peptide-dependent binding of the activating NK cell receptor KIR2DS1, providing new insights into the underlying mechanisms involved in KIR2DS1-related disorders.

Autoimmune diseases (AIDs) are polygenic diseases affecting 7–10% of the population in the Western Hemisphere with few effective therapies. Here, we quantify the heritability of paediatric AIDs (pAIDs), including JIA, SLE, CEL, T1D, UC, CD, PS, SPA and CVID, attributable to common genomic variations (SNP-h2). SNP-h2 estimates are most significant for T1D (0.863±s.e. 0.07) and JIA (0.727±s.e. 0.037), more modest for UC (0.386±s.e. 0.04) and CD (0.454±0.025), largely consistent with population estimates and are generally greater than that previously reported by adult GWAS. On pairwise analysis, we observed that the diseases UC-CD (0.69±s.e. 0.07) and JIA-CVID (0.343±s.e. 0.13) are the most strongly correlated. Variations across the MHC strongly contribute to SNP-h2 in T1D and JIA, but does not significantly contribute to the pairwise rG. Together, our results partition contributions of shared versus disease-specific genomic variations to pAID heritability, identifying pAIDs with unexpected risk sharing, while recapitulating known associations between autoimmune diseases previously reported in adult cohorts., Autoimmune diseases are genetically complex disorders that affect up to 10% of the Western population. Here Li et al. quantify the heritability of a range of autoimmune diseases in the largest paediatric cohort examined to date, illustrating that genetic and non-genetic components variably contribute to the susceptibility of each disease.

Summary Treatment of common variable immunodeficiency disorders (CVID) is based on replacement therapy using intravenous (i.v.) or subcutaneous (s.c.) immunoglobulin (Ig)G. Interindividual variation of IgG dose is common. A total of 380 CVID patients on stable IgG replacement from two prospective cohorts were analysed. An ‘efficiency’ index was defined as the ratio of serum IgG trough level minus IgG residual to the average weekly dose of IgG infusion. A reduced efficiency of IgG was associated independently with the i.v. route (P

The transcription factor X-box binding protein 1 (XBP1) represents a key component of the endoplasmatic reticulum (ER) stress response and is required for the production of several pro-inflammatory cytokines. XBP1 is furthermore essential for the development and survival of plasmacytoid dendritic cells (pDCs), and has recently been suggested to be involved in toll-like receptor (TLR) 2/4 signaling. Activation of TLR7 on pDCs results in an upregulation of pro-inflammatory cytokines, such as type I interferons (IFN-I), and has been implicated in several autoimmune and inflammatory diseases, but the role of XBP1 in this process remains unknown. Here, we show that signaling via TLR7 leads to an upregulation of XBP1 and IFNα-production. XBP1 mRNA expression levels positively correlated with the production of IFNα, while blocking of XBP1 mRNA splicing significantly reduced mRNA transcripts of IFNα. In conclusion, these data suggest a central role of XBP1 in TLR7-induced IFNα production and identify XBP1 as a potential novel therapeutic target in IFNα-driven autoimmune and inflammatory diseases.