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The investigation of the bone marrow and the bone marrow biopsy is one of the most valuable diagnostic tools for evaluating hematologic disorders. Furthermore, bone marrow analysis has great importance in the evaluation of non-hematologic conditions. Various chemical toxins, including minerals, as well as radiation may severely affect the bone marrow cells and stroma. In patients with fever of unknown origin (FUO), among others in patients with autoimmune deficiency syndrome (AIDS), the marrow may reveal various microorganisms, such as leishmaniosis, tuberculosis, and fungal as well as viral infections.

AIM To investigate the potential effect of inhibitors of phosphodiesterase-5 (PDE-5) for therapy of portal hypertension in liver cirrhosis. METHODS In the rat model of thioacetamide-induced liver fibrosis/cirrhosis the nitric oxide-cyclic guanosine monophosphate (NO-cGMP) pathway was investigated. Expression and localization of PDE-5, the enzyme that converts vasodilating cGMP into inactive 5’-GMP, was in the focus of the study. Hepatic gene expression of key components of the NO-cGMP pathway was determined by qRT-PCR: Endothelial NO synthase (eNOS), inducible NO synthase (iNOS), soluble guanylate cyclase subunits α1 and β1 (sGCa1, sGCb1), and PDE-5. Hepatic PDE-5 protein expression and localization were detected by immunohistochemistry. Serum cGMP concentrations were measured using ELISA. Acute effects of the PDE-5 inhibitor Sildenafil (0.1 mg/kg or 1.0 mg/kg) on portal and systemic hemodynamics were investigated using pressure transducers. RESULTS Hepatic gene expression of eNOS (2.2-fold; P = 0.003), sGCa1 (1.7-fold; P = 0.003), sGCb1 (3.0-fold; P = 0.003), and PDE-5 (11-fold; P = 0.003) was increased in cirrhotic livers compared to healthy livers. Overexpression of PDE-5 (7.7-fold; P = 0.006) was less pronounced in fibrotic livers. iNOS expression was only detected in fibrotic and cirrhotic livers. In healthy liver, PDE-5 protein was localized primarily in zone 3 hepatocytes and to a lesser extent in perisinusoidal cells. This zonation was disturbed in cirrhosis: PDE-5 protein expression in perisinusoidal cells was induced approximately 8-fold. In addition, PDE-5-expressing cells were also found in fibrous septa. Serum cGMP concentrations were reduced in rats with cirrhotic livers by approximately 40%. Inhibition of PDE-5 by Sildenafil caused a significant increase in serum cGMP concentrations [+ 64% in healthy rats (P = 0.024), + 85% in cirrhotic rats (P = 0.018)]. Concomitantly, the portal venous pressure was reduced by 19% in rats with liver cirrhosis. CONCLUSION Overexpression and abrogated zonation of PDE-5 likely contribute to the pathogenesis of cirrhotic portal hypertension. PDE-5 inhibition may therefore be a reasonable therapeutic approach for portal hypertension.

Context Maturity-onset diabetes of the young type 5 (MODY5) is caused by mutations of the hepatocyte nuclear factor 1 homeobox β gene (HNF1B). Although clinical characteristics and therapeutic management of MODY5 are increasingly better defined, adequate consideration of the frequent association of MODY5 with 17q12 deletion syndrome is often missing. Evidence Acquisition We report two cases of patients with 17q12 deletion syndrome who presented to our clinic. Furthermore, we reviewed the existing literature to improve systematic diagnostic and therapeutic approaches. A PubMed search using the terms 17q12 deletion syndrome, diabetes mellitus type MODY5, and/or HNF1B was performed. Evidence Synthesis Three hundred sixty-one cases of postnatal 17q12 deletion syndrome were assessed, and details on clinical manifestations, diagnostic approaches, and therapeutic management were reviewed and compared with the two cases at our clinic. Furthermore, data on pathogenic mechanisms and their clinical implications were evaluated. Conclusion The 17q12 deletion syndrome usually comprises MODY5, structural or functional abnormalities of the kidneys, and neurodevelopmental or neuropsychiatric disorders. A complete deletion of HNF1B can be found in about 50% of patients with MODY5. A wide variety of additional clinical features, including genital and brain malformations, has been reported. Because HNF1B deletions are virtually always part of a 17q12 deletion syndrome and common genetic analyses for evaluation of MODY5 are unable to detect the deletion of a 1.4-Mb chromosomal region, initial attention to the syndromal features at the stage of diagnosis is of considerable importance for establishing correct diagnosis, subsequent therapy, and interdisciplinary patient care.

Background Chlamydial infection frequently causes damage to the female genital tract. The precise mechanisms of chlamydial clearance and tissue damage are unknown, but studies suggest immunopathology with a particular role of neutrophils. The goal of this study was to understand the contribution of the immune system, in particular neutrophils. Methods Using Chlamydia muridarum, we infected mice with a prolonged immune response due to expression of B-cell lymphoma 2 (Bcl-2) in hematopoietic cells (Bcl-2 mice), and mice where mature neutrophils are lacking due to the deletion of Myeloid cell leukemia 1 (Mcl-1) in myeloid cells (LysM-cre-mcl-1-flox mice; Mcl-1 mice). We monitored bacterial clearance, cellular infiltrate, and long-term tissue damage. Results Both mutant strains showed slightly delayed clearance of the acute infection. Bcl-2 mice had a strongly increased inflammatory infiltrate concerning almost all cell lineages. The infection of Bcl-2 mice caused increased tissue damage. The loss of neutrophils in Mcl-1 mice was associated with substantial quantitative and qualitative alterations of the inflammatory infiltrate. Mcl-1 mice had higher chlamydial burden and reduced tissue damage, including lower incidence of hydrosalpinx and less uterine dilation. Conclusions Inhibition of apoptosis in the hematopoietic system increases inflammation and tissue damage. Neutrophils have broad functions, including a role in chlamydial clearance and in tissue destruction.

Paragangliomas are predominantly benign tumors, but in some cases invasive growth and also metastasis are observed. Given the limited number of nonsurgical treatment options, novel target structures for diagnostics and therapy of this tumor entity are urgently needed. In the present study, expression of all five somatostatin receptor (SST) subtypes, chemokine receptor CXCR4 and endothelin receptor type A (ETA) was assessed by means of immunohistochemistry in a total of 66 paraffin-embedded paraganglioma samples from 55 patients. The stainings were rated by means of the Immunoreactive Score and correlated to clinical data and to succinate dehydrogenase subunit B (SDHB) expression. SST2A was by far the most prominent receptor in the paragangliomas investigated. It was present in 89% of the tumors at a high intensity, followed by SST5, SST3, SST1 and SST4, which were detected in 47%, 35%, 35% and 13% of the samples, respectively. SDHB positive tumors exhibited significantly higher SST2A and SST3 expression as compared to SDHB negative cases. There was no correlation between SST and Ki-67 expression or grading of the tumors and no difference in SST expression between primary tumors and metastases. Cell surface expression of CXCR4 and ETA was detected only in few samples. On tumor capillaries, however, exceptionally strong staining for these two receptors was noticed in the vast majority of the tumors. In conclusion, paragangliomas are well suited for SST2A-based diagnostics and treatment modalities. An indirect targeting of these highly vascularized tumors via CXCR4 or ETA may also represent a promising future strategy.

// Hana Andrlova 1 , Justin Mastroianni 1 , Josef Madl 2, 3 , Johannes S. Kern 4 , Wolfgang Melchinger 1 , Heide Dierbach 1 , Florian Wernet 1 , Marie Follo 1 , Kristin Technau-Hafsi 4 , Cristina Has 4 , Venugopal Rao Mittapalli 4 , Marco Idzko 5 , Ricarda Herr 6 , Tilman Brummer 6 , Hendrik Ungefroren 7 , Hauke Busch 7, 8, 9, 15 , Melanie Boerries 6, 8, 15 , Andreas Narr 10, 11 , Gabriele Ihorst 12 , Claire Vennin 13 , Annette Schmitt-Graeff 14 , Susana Minguet 10, 11 , Paul Timpson 13 , Justus Duyster 1 , Frank Meiss 4 , Winfried Romer 2, 3 and Robert Zeiser 1, 3 1 Department of Hematology and Oncology, University Medical Center, Faculty of Medicine, Freiburg, Germany 2 Faculty of Biology, Albert Ludwigs University, Freiburg, Germany 3 BIOSS Centre for Biological Signalling Studies, Albert Ludwigs University Freiburg, Freiburg, Germany 4 Department of Dermatology and Venereology, University Medical Center, Freiburg, Germany 5 Department of Pneumology, University Medical Center, Freiburg, Germany 6 Institut fur Molekulare Medizin und Zellforschung, University Medical Center, Freiburg, Germany 7 First Department of Medicine, University of Lubeck, Lubeck, Germany 8 German Cancer Consortium (DKTK), Freiburg, Germany 9 Institute of Experimental Dermatology, University of Lubeck, Lubeck, Germany 10 Department of Immunology, BIOSS Center for Biological Signaling Studies, Faculty of Biology, Albert-Ludwigs-University of Freiburg, Freiburg, Germany 11 Center of Chronic Immunodeficiency CCI, University Clinics and Medical Faculty, Freiburg, Germany 12 Clinical Trials Unit, University Medical Center, Freiburg, Germany 13 The Garvan Institute of Medical Research and The Kinghorn Cancer Centre, Sydney, Australia 14 Department of Pathology, University Medical Center, Faculty of Medicine, Freiburg, Germany 15 German Cancer Research Center (DKFZ), Heidelberg, Germany Correspondence to: Robert Zeiser, email: robert.zeiser@uniklinik-freiburg.de Keywords: biglycan, melanoma, microenvironment, tissue stiffness, integrin-β1 Received: October 04, 2016 Accepted: March 14, 2017 Published: April 17, 2017 ABSTRACT Novel targeted and immunotherapeutic approaches have revolutionized the treatment of metastatic melanoma. A better understanding of the melanoma-microenvironment, in particular the interaction of cells with extracellular matrix molecules, may help to further improve these new therapeutic strategies. We observed that the extracellular matrix molecule biglycan (Bgn) was expressed in certain human melanoma cells and primary fibroblasts when evaluated by microarray-based gene expression analysis. Bgn expression in the melanoma tissues correlated with low overall-survival and low progression-free-survival in patients. To understand the functional role of Bgn we used gene-targeted mice lacking functional Bgn. Here we observed that melanoma growth, metastasis-formation and tumor-related death were reduced in Bgn -/- mice compared to Bgn +/+ mice. In vitro invasion of melanoma cells into organotypic-matrices derived from Bgn -/- fibroblasts was reduced compared to melanoma invasion into Bgn -proficient matrices. Tissue stiffness as determined by atomic-force-microscopy was reduced in Bgn -/- matrices. Isolation of melanoma cells and fibroblasts from the stiffer Bgn +/+ matrices revealed an increase in integrin-β1 expression compared to the Bgn -/- fibroblast matrices. Overexpression of integrin-β1 in B16-melanoma cells abolished the survival benefit seen in Bgn -/- mice. Consistent with the studies performed in mice, the abundance of Bgn-expression in human melanoma samples positively correlated with the expression of integrin-β1, which is in agreement with results from the organotypic invasion-assay and the in vivo mouse studies. This study describes a novel role for Bgn-related tissue stiffness in the melanoma-microenvironment via regulation of integrin-β1 expression by melanoma cells in both mice and humans.

Microbial invasion into the intestinal mucosa after allogeneic hematopoietic cell transplantation (allo-HCT) triggers neutrophil activation and requires antibiotic interventions to prevent sepsis. However, antibiotics lead to a loss of microbiota diversity, which is connected to a higher incidence of acute graft-versus-host disease (aGVHD). Antimicrobial therapies that eliminate invading bacteria and reduce neutrophil-mediated damage without reducing the diversity of the microbiota are therefore highly desirable. A potential solution would be the use of antimicrobial antibodies that target invading pathogens, ultimately leading to their elimination by innate immune cells. In a mouse model of aGVHD, we investigated the potency of active and passive immunization against the conserved microbial surface polysaccharide poly-N-acetylglucosamine (PNAG) that is expressed on numerous pathogens. Treatment with monoclonal or polyclonal antibodies to PNAG (anti-PNAG) or vaccination against PNAG reduced aGVHD-related mortality. Anti-PNAG treatment did not change the intestinal microbial diversity as determined by 16S ribosomal DNA sequencing. Anti-PNAG treatment reduced myeloperoxidase activation and proliferation of neutrophil granulocytes (neutrophils) in the ileum of mice developing GVHD. In vitro, anti-PNAG treatment showed high antimicrobial activity. The functional role of neutrophils was confirmed by using neutrophil-deficient LysM(cre) Mcl1(fl/fl) mice that had no survival advantage under anti-PNAG treatment. In summary, the control of invading bacteria by anti-PNAG treatment could be a novel approach to reduce the uncontrolled neutrophil activation that promotes early GVHD and opens a new avenue to interfere with aGVHD without affecting commensal intestinal microbial diversity.

Oncogenic Ras mutations occur in various leukemias. It was unclear if, besides the direct transforming effect via constant RAS/MEK/ERK signaling, an inflammation-related effect of KRAS contributes to the disease. Here, we identify a functional link between oncogenic KrasG12D and NLRP3 inflammasome activation in murine and human cells. Mice expressing active KrasG12D in the hematopoietic system developed myeloproliferation and cytopenia, which is reversed in KrasG12D mice lacking NLRP3 in the hematopoietic system. Therapeutic IL-1-receptor blockade or NLRP3-inhibition reduces myeloproliferation and improves hematopoiesis. Mechanistically, KrasG12D-RAC1 activation induces reactive oxygen species (ROS) production causing NLRP3 inflammasome-activation. In agreement with our observations in mice, patient-derived myeloid leukemia cells exhibit KRAS/RAC1/ROS/NLRP3/IL-1β axis activity. Our findings indicate that oncogenic KRAS not only act via its canonical oncogenic driver function, but also enhances the activation of the pro-inflammatory RAC1/ROS/NLRP3/IL-1β axis. This paves the way for a therapeutic approach based on immune modulation via NLRP3 blockade in KRAS-mutant myeloid malignancies., Oncogenic Ras mutations are common drivers in myeloid leukemia. Here, the authors show in patient cells and in mice that oncogenic K-Ras activates NLRP3 inflammasome to drive myeloproliferation, which can be reversed by genetic or pharmacologic NLRP3 blockade.

Activating mutations in FMS-like tyrosine kinase receptor-3 (FLT3) and Nucleophosmin-1 (NPM1) are most frequent alterations in acute myeloid leukemia (AML), and are often coincidental. The mutational status of NPM1 has strong prognostic relevance to patients with point mutations of the FLT3 tyrosine kinase domain (TKD), but the biological mechanism underlying this effect remains unclear. In the present study, we investigated the effect of the coincidence of NPM1c and FLT3-TKD. Although expression of FLT3-TKD is not sufficient to induce a disease in mice, coexpression with NPM1c rapidly leads to an aggressive myeloproliferative disease in mice with a latency of 31.5 days. Mechanistically, we could show that FLT3-TKD is able to activate the downstream effector molecule signal transducer and activator of transcription 5 (STAT5) exclusively in the presence of mutated NPM1c. Moreover, NPM1c alters the cellular localization of FLT3-TKD from the cell surface to the endoplasmic reticulum, which might thereby lead to the aberrant STAT5 activation. Importantly, aberrant STAT5 activation occurs not only in primary murine cells but also in patients with AML with combined FLT3-TKD and NPM1c mutations. Thus, our data indicate a new mechanism, how NPM1c mislocalizes FLT3-TKD and changes its signal transduction ability.

Background & Aims: In hepatocellular carcinoma (HCC), CD8+ T-cell responses targeting tumor-associated antigens (TAA) are considered to be beneficial. However, the molecular profile of TAA-specific CD8+ T cells in HCC is not well defined due to their low frequency.\ud\udResults: In this study, we demonstrate that TAA-specific CD8+ T-cell responses are not efficiently induced in the peripheral blood of HCC patients as supported by the following observations: First, in HCC patients, frequencies of TAA-specific CD8+ T cells were not increased compared to healthy donors (HD) or patients with liver cirrhosis. Second, a remarkable proportion of TAA-specific CD8+ T cells were naïve despite the presence of antigen within the tumor tissue. Third, antigen-experienced TAA-specific CD8+ T cells lack the characteristic transcriptional regulation of exhausted CD8+ T cells, namely EomeshiTbetdim, and express inhibitory receptors only on a minor proportion of cells. This suggests restricted antigen recognition and further supports the hypothesis of inefficient induction and activation.\ud\udMethods: By applying peptide/MHCI tetramer-based enrichment, a method of high sensitivity, we now could define the heterogeneity of circulating TAA-specific CD8+ T cells targeting glypican-3, NY-ESO-1, MAGE-A1 and MAGE-A3. We focused on therapy-naïve HCC patients of which the majority underwent transarterial chemoembolization (TACE).\ud\udConclusion: Our analysis reveals that circulating TAA-specific CD8+ T cells targeting 4 different immunodominant epitopes are not properly induced in therapy-naïve HCC patients thereby unravelling new and unexpected insights into TAA-specific CD8+ T-cell biology in HCC. This clearly highlights severe limitations of these potentially anti-tumoral T cells that may hamper their biological and clinical relevance in HCC.

Graft-versus-host disease (GVHD) is among the most challenging complications in unrelated donor hematopoietic cell transplantation (HCT). The highly polymorphic MHC class I chain-related gene A, MICA, encodes a stress-induced glycoprotein expressed primarily on epithelia. MICA interacts with the invariant activating receptor NKG2D, expressed by cytotoxic lymphocytes, and is located in the MHC, next to HLA-B. Hence, MICA has the requisite attributes of a bona fide transplantation antigen. Using high-resolution sequence-based genotyping of MICA, we retrospectively analyzed the clinical effect of MICA mismatches in a multicenter cohort of 922 unrelated donor HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 10/10 allele-matched HCT pairs. Among the 922 pairs, 113 (12.3%) were mismatched in MICA. MICA mismatches were significantly associated with an increased incidence of grade III-IV acute GVHD (hazard ratio [HR], 1.83; 95% confidence interval [CI], 1.50-2.23; P < .001), chronic GVHD (HR, 1.50; 95% CI, 1.45-1.55; P < .001), and nonelapse mortality (HR, 1.35; 95% CI, 1.24-1.46; P < .001). The increased risk for GVHD was mirrored by a lower risk for relapse (HR, 0.50; 95% CI, 0.43-0.59; P < .001), indicating a possible graft-versus-leukemia effect. In conclusion, when possible, selecting a MICA-matched donor significantly influences key clinical outcomes of HCT in which a marked reduction of GVHD is paramount. The tight linkage disequilibrium between MICA and HLA-B renders identifying a MICA-matched donor readily feasible in clinical practice.

A patient with autoimmune lymphoproliferative disorder (ALPS) developed IgG4-related disease. In retrospect, he had high levels of serum IgG4 for several years prior to presenting with IgG4-related pancreatitis. These high IgG4 levels were masked by hypergammaglobulinemia, a common feature of ALPS. We next screened 18 ALPS patients; four of them displayed increased levels of IgG4. Hence, IgG4-related disease should be considered in ALPS patients, especially in those manifesting lymphocytic organ infiltration or excessive hypergammaglobulinaemia. Screening of IgG4-related disease patients for ALPS-associated mutations would provide further information on whether this disease could be a late-onset atypical presentation of ALPS.

Recent evidence has revealed that oncogenic mutations may confer immune escape. A better understanding of how an oncogenic mutation affects immunosuppressive programmed death ligand 1 (PD-L1) expression may help in developing new therapeutic strategies. We show that oncogenic JAK2 (Janus kinase 2) activity caused STAT3 (signal transducer and activator of transcription 3) and STAT5 phosphorylation, which enhanced PD-L1 promoter activity and PD-L1 protein expression in JAK2V617F-mutant cells, whereas blockade of JAK2 reduced PD-L1 expression in myeloid JAK2V617F-mutant cells. PD-L1 expression was higher on primary cells isolated from patients with JAK2V617F–myeloproliferative neoplasms (MPNs) compared to healthy individuals and declined upon JAK2 inhibition. JAK2V617F mutational burden, pSTAT3, and PD-L1 expression were highest in primary MPN patient–derived monocytes, megakaryocytes, and platelets. PD-1 (programmed death receptor 1) inhibition prolonged survival in human MPN xenograft and primary murine MPN models. This effect was dependent on T cells. Mechanistically, PD-L1 surface expression in JAK2V617F-mutant cells affected metabolism and cell cycle progression of T cells. In summary, we report that in MPN, constitutive JAK2/STAT3/STAT5 activation, mainly in monocytes, megakaryocytes, and platelets, caused PD-L1–mediated immune escape by reducing T cell activation, metabolic activity, and cell cycle progression. The susceptibility of JAK2V617F-mutant MPN to PD-1 targeting paves the way for immunomodulatory approaches relying on PD-1 inhibition.

Contains fulltext : 190745.pdf (Publisher’s version ) (Closed access) Individuals with acute myeloid leukemia (AML) harboring an internal tandem duplication (ITD) in the gene encoding Fms-related tyrosine kinase 3 (FLT3) who relapse after allogeneic hematopoietic cell transplantation (allo-HCT) have a 1-year survival rate below 20%. We observed that sorafenib, a multitargeted tyrosine kinase inhibitor, increased IL-15 production by FLT3-ITD(+) leukemia cells. This synergized with the allogeneic CD8(+) T cell response, leading to long-term survival in six mouse models of FLT3-ITD(+) AML. Sorafenib-related IL-15 production caused an increase in CD8(+)CD107a(+)IFN-gamma(+) T cells with features of longevity (high levels of Bcl-2 and reduced PD-1 levels), which eradicated leukemia in secondary recipients. Mechanistically, sorafenib reduced expression of the transcription factor ATF4, thereby blocking negative regulation of interferon regulatory factor 7 (IRF7) activation, which enhanced IL-15 transcription. Both IRF7 knockdown and ATF4 overexpression in leukemia cells antagonized sorafenib-induced IL-15 production in vitro. Human FLT3-ITD(+) AML cells obtained from sorafenib responders following sorafenib therapy showed increased levels of IL-15, phosphorylated IRF7, and a transcriptionally active IRF7 chromatin state. The mitochondrial spare respiratory capacity and glycolytic capacity of CD8(+) T cells increased upon sorafenib treatment in sorafenib responders but not in nonresponders. Our findings indicate that the synergism of T cells and sorafenib is mediated via reduced ATF4 expression, causing activation of the IRF7-IL-15 axis in leukemia cells and thereby leading to metabolic reprogramming of leukemia-reactive T cells in humans. Therefore, sorafenib treatment has the potential to contribute to an immune-mediated cure of FLT3-ITD-mutant AML relapse, an otherwise fatal complication after allo-HCT.

Item does not contain fulltext BACKGROUND: Cytotoxic T-lymphocyte antigen 4 (CTLA-4) is a negative immune regulator. Heterozygous CTLA4 germline mutations can cause a complex immune dysregulation syndrome in human subjects. OBJECTIVE: We sought to characterize the penetrance, clinical features, and best treatment options in 133 CTLA4 mutation carriers. METHODS: Genetics, clinical features, laboratory values, and outcomes of treatment options were assessed in a worldwide cohort of CTLA4 mutation carriers. RESULTS: We identified 133 subjects from 54 unrelated families carrying 45 different heterozygous CTLA4 mutations, including 28 previously undescribed mutations. Ninety mutation carriers were considered affected, suggesting a clinical penetrance of at least 67%; median age of onset was 11 years, and the mortality rate within affected mutation carriers was 16% (n = 15). Main clinical manifestations included hypogammaglobulinemia (84%), lymphoproliferation (73%), autoimmune cytopenia (62%), and respiratory (68%), gastrointestinal (59%), or neurological features (29%). Eight affected mutation carriers had lymphoma, and 3 had gastric cancer. An EBV association was found in 6 patients with malignancies. CTLA4 mutations were associated with lymphopenia and decreased T-, B-, and natural killer (NK) cell counts. Successful targeted therapies included application of CTLA-4 fusion proteins, mechanistic target of rapamycin inhibitors, and hematopoietic stem cell transplantation. EBV reactivation occurred in 2 affected mutation carriers after immunosuppression. CONCLUSIONS: Affected mutation carriers with CTLA-4 insufficiency can present in any medical specialty. Family members should be counseled because disease manifestation can occur as late as 50 years of age. EBV- and cytomegalovirus-associated complications must be closely monitored. Treatment interventions should be coordinated in clinical trials.

Background Most patients with MHC class I (MHC-I) deficiency carry genetic defects in transporter associated with antigen processing 1 (TAP1) or TAP2 . The clinical presentation can vary, and about half of the patients have severe skin disease. Previously, one report described β 2 -microglobulin (β 2 m) deficiency as another monogenetic cause of MHC-I deficiency, but no further immunologic evaluation was performed. Objective We sought to describe the molecular and immunologic features of β 2 m deficiency in 2 Turkish siblings with new diagnoses. Methods Based on clinical and serologic findings, the genetic defect was detected by means of candidate gene analysis. The immunologic characterization comprises flow cytometry, ELISA, functional assays, and immunohistochemistry. Results Here we provide the first extensive clinical and immunologic description of β 2 m deficiency in 2 siblings. The sister had recurrent respiratory tract infections and severe skin disease, whereas the brother was fairly asymptomatic but had bronchiectasis. Not only polymorphic MHC-I but also the related CD1a, CD1b, CD1c, and neonatal Fc receptor molecules were absent from the surfaces of β 2 m-deficient cells. Absent neonatal Fc receptor surface expression led to low serum IgG and albumin levels in both siblings, whereas the heterozygous parents had normal results for all tested parameters except β 2 m mRNA (B2M) expression. Similar to TAP deficiency in the absence of a regular CD8 T-cell compartment, CD8 + γδ T cells were strongly expanded. Natural killer cells were normal in number but not "licensed to kill." Conclusion The clinical presentation of patients with β 2 m deficiency resembles that of patients with other forms of MHC-I deficiency, but because of the missing stabilizing effect of β 2 m on other members of the MHC-I family, the immunologic defect is more extensive than in patients with TAP deficiency.

The successful treatment of acute leukemias with allogeneic hematopoietic cell transplantation (allo-HCT) is limited by acute graft-versus-host disease (GVHD). Because microRNA-155 (miR-155) regulates activation of the innate immune system, we aimed to determine its function in dendritic cells (DCs) during GVHD in an experimental model. We observed that miR-155 deficiency of the recipient led to improved survival, reduced serum levels of proinflammatory cytokines, and lower GVHD histopathology scores. In addition, miR-155(-/-) bone marrow chimeric mice receiving allo-HCT and miR-155(-/-) DCs showed that miR-155 deficiency in the DC compartment was responsible for protection from GVHD. Activated miR-155(-/-) DCs displayed lower expression of various purinergic receptors and impaired migration toward adenosine triphosphate (ATP). Microarray analysis of lipopolysaccharide/ATP-stimulated miR-155(-/-) DCs revealed mitogen-activated protein kinase pathway dysregulation and reduced inflammasome-associated gene expression. Consistent with this gene expression data, we observed reduced ERK activation, caspase-1 cleavage, and IL-1β production in miR-155(-/-) DCs. The connection between miR-155 and inflammasome activation was supported by the fact that Nlrp3/miR-155 double-knockout allo-HCT recipient mice had no increased protection from GVHD compared with Nlrp3(-/-) recipients. This study indicates that during GVHD, miR-155 promotes DC migration toward sites of ATP release accompanied by inflammasome activation. Inhibiting proinflammatory miR-155 by antagomir treatment could help reduce this complication of allo-HCT.

Members of the B7 family have been shown to be important for regulating immune responses by providing either positive or negative costimulatory signals. The function of B7-H3 has been controversial. We show that B7-H3 is upregulated in graft-versus-host disease (GVHD) target organs, including the colon, liver, and lung. Infusion of allogeneic donor T cells into B7-H3(-/-) vs wild-type (WT) recipients resulted in increased GVHD lethality associated with increased T-cell proliferation, colonic inflammatory cytokines, and destruction of epithelial barriers. Allogeneic B7-H3(-/-) vs WT donor T cells also had increased T-cell proliferation and GVHD lethality associated with increased proliferation and cytokine secretion in the spleen, intraepithelial lymphocyte inflammatory cytokines, and intestinal permeability. Both resting and activated regulatory T cells (Tregs) lack B7-H3 messenger RNA. Consistent with these data, GVHD was augmented in recipients of B7-H3(-/-) Treg-depleted grafts. In two delayed lymphocyte infusion (DLI) models, T cells lacking B7-H3 are capable of providing graft-versus-leukemia (GVL) effects. We conclude that B7-H3 is responsible for providing a negative costimulatory signal. Our studies provide support for developing and testing new therapies directed toward the B7-H3 pathway, including approaches to augment host B7-H3 early after bone marrow transplantation to prevent GVHD and to develop potent antagonistic antibodies later after transplant to facilitate DLI-mediated GVL without GVHD complications.

[Figure][1] A 73-year-old man presented with abdominal pain 4 months after allogeneic hematopoietic cell transplantation for complex karyotype acute myeloid leukemia (AML). Endoscopy revealed a polypoid mass lesion in the gastric body. Neoplastic cells distorted mucosa and submucosa (

Recent evidence has revealed that oncogenic mutations may confer immune escape. A better understanding of how an oncogenic mutation affects immunosuppressive PD-L1 expression may help in developing new therapeutic strategies. Here, we show that oncogenic JAK2 activity caused STAT3 and STAT5 phosphorylation, which enhanced PD-L1 promoter activity and PD-L1 protein expression in JAK2(V617F)-mutant cells, whereas blockade of JAK2 reduced PD-L1 expression in myeloid JAK2(V617F)-mutant cells. PD-L1 expression was higher on primary cells isolated from patients with JAK2(V617F)-myeloproliferative neoplasms (MPN) compared to healthy individuals and declined upon JAK2 inhibition. JAK2(V617F) mutational burden, pSTAT3, and PD-L1 expression were highest in primary MPN patient-derived monocytes, megakaryocytes, and platelets. PD-1 inhibition prolonged survival in human MPN xenograft and primary murine MPN models. This effect was dependent on T cells. Mechanistically, PD-L1 surface expression in JAK2(V617F)-mutant cells affected metabolism and cell cycle progression of T cells. In summary, we report that in MPN, constitutive JAK2/STAT3/STAT5 activation, mainly in monocytes, megakaryocytes, and platelets, caused PD-L1-mediated immune escape by reducing T cell activation, metabolic activity, and cell cycle progression. The susceptibility of JAK2(V617F)-mutant MPN to PD-1 targeting paves the way for immunomodulatory approaches relying on PD-1 inhibition.

Acute Graft-versus-host disease (GVHD) is a major immunological complication after allogeneic hematopoietic cell transplantation and a better understanding of the molecular regulation of the disease could help to develop novel targeted therapies. Here we found that a G/C polymorphism within the human microRNA-146a (miR-146a) gene of transplant recipients, which causes reduced miR-146a levels, was strongly associated with the risk of developing severe acute GVHD (n=289). In mice, deficiency of miR-146a in the hematopoietic system or transfer of recipient-type miR-146a-/- dendritic cells (DCs) enhanced GVHD, while miR-146a mimic-transfected DCs ameliorated disease. Mechanistically, lack of miR-146a enhanced JAK2-STAT1 pathway activity, which led to higher expression of class II-transactivator (CIITA) and consecutively increased MHCII-levels on DCs. Inhibition of JAK1/2 or CIITA knockdown in DCs prevented miR-146a-/- DC-induced GVHD exacerbation. Consistent with our findings in mice, patients with the miR-146a polymorphism rs2910164 in hematopoietic cells displayed higher MHCII levels on monocytes, which could be targeted by JAK1/2 inhibition. Our findings indicate that the miR-146a polymorphism rs2910164 identifies patients at high risk for GVHD before allo-HCT. Functionally we show that miR-146a acts as a central regulator of recipient-type DC activation during GVHD by dampening the pro-inflammatory JAK-STAT/CIITA/MHCII axis, which provides a scientific rationale for early JAK1/2 inhibition in selected patients.

Acute graft-versus-host disease (GVHD) limits the success of allogeneic hematopoietic cell transplantation (allo-HCT); therefore, a better understanding of its biology may improve therapeutic options. We observed miR-146a up-regulation in T cells of mice developing acute GVHD compared with untreated mice. Transplanting miR-146a(-/-) T cells caused increased GVHD severity, elevated tumor necrosis factor (TNF) serum levels, and reduced survival. TNF receptor-associated factor 6 (TRAF6), a verified target of miR-146a, was up-regulated in miR-146a(-/-) T cells following alloantigen stimulation. Higher TRAF6 levels translated into increased nuclear factor-κB activity and TNF production in miR-146a(-/-) T cells. Conversely, the detrimental effect of miR-146a deficiency in T cells was antagonized by TNF blockade, whereas phytochemical induction of miR-146a or its overexpression using a miR-146a mimic reduced GVHD severity. In humans, the minor genotype of the single nucleotide polymorphism rs2910164 in HCT donors, which reduces expression of miR-146a, was associated with severe acute GVHD (grade III/IV). We show that miR-146a functions as a negative regulator of donor T cells in GVHD by targeting TRAF6, leading to reduced TNF transcription. Because miR-146a expression can be exogenously enhanced, our results provide a novel targeted molecular approach to mitigate GVHD.

The diagnosis of bone marrow (BM) infiltration by Waldenström macroglobulinemia (WM)/lymphoplasmacytic lymphoma (LPL) poses a diagnostic challenge in hematopathology. No definitive morphology or immunophenotype is able to distinguish between infiltration of paraffin-embedded BM sections by WM/LPL and other indolent lymphomas, in particular those of the splenic marginal zone (SMZL) which may also show plasmacytic maturation. An oncogenic gain-of-function mutation (L265P) in the human MYD88 gene has been found to be present in most cases of WM/LPL, yet is absent in most other cases of B-cell chronic lymphoproliferative disorders (LPD), including SMZL. Here, we compare two newly developed diagnostic protocols for detection of this mutation in paraffin-embedded archival tissues which are particularly applicable to decalcified BM biopsies. Sanger sequencing can easily detect levels of BM infiltration above 15% by WM lymphoplasmacytic cells, while the allele-specific PCR can detect the L265P mutation in BM infiltrations below 1% of lymphoma cells. We show that these methods are easily applicable to archival BM specimens and markedly improve diagnostic accuracy of BM infiltrations by indolent B-cell lymphomas.

Graft-versus-host-disease (GVHD) is a severe complication of allogeneic hematopoietic cell transplantation (allo-HCT) characterized by the production of high levels of proinflammatory cytokines. Activated Janus kinases (JAKs) are required for T-effector cell responses in different inflammatory diseases, and their blockade could potently reduce acute GVHD. We observed that inhibition of JAK1/2 signaling resulted in reduced proliferation of effector T cells and suppression of proinflammatory cytokine production in response to alloantigen in mice. In vivo JAK 1/2 inhibition improved survival of mice developing acute GVHD and reduced histopathological GVHD grading, serum levels of proinflammatory cytokines, and expansion of alloreactive luc-transgenic T cells. Mechanistically, we could show that ruxolitinib impaired differentiation of CD4(+) T cells into IFN-γ- and IL17A-producing cells, and that both T-cell phenotypes are linked to GVHD. Conversely, ruxolitinib treatment in allo-HCT recipients increased FoxP3(+) regulatory T cells, which are linked to immunologic tolerance. Based on these results, we treated 6 patients with steroid-refractory GVHD with ruxolitinib. All patients responded with respect to clinical GVHD symptoms and serum levels of proinflammatory cytokines. In summary, ruxolitinib represents a novel targeted approach in GVHD by suppression of proinflammatory signaling that mediates tissue damage and by promotion of tolerogenic Treg cells.

Given the severely reduced numbers of circulating class-switched memory B cells and plasmablasts in patients with common variable immunodeficiency (CVID) the germinal center (GC) reaction as the source of both populations is expected to be disturbed in many CVID patients. Therefore immunohistochemical studies were performed on lymph node (LN) biopsies from ten CVID patients with benign lymphoproliferation. According to the Sander classification the majority of patients presented with reactive lymphoid hyperplasia (7/10), 6/10 showed granulomatous inflammation. All cases showed some normal GCs but in 9/10 these concurred to a varying degree with hyperplastic, ill-defined GCs in the same LN. The percentage of ill-defined GCs correlated significantly with the percentage of circulating CD21(low) B cells suggesting a common origin of both immune reactions. In 9/10 CVID LNs significantly higher numbers of infiltrating CD8+ T cells were found in GCs of CVID patients compared to controls, but no HHV-8 and only in 2/10 LNs EBV infection was detected. Class switched plasma cells (PCs) were severely reduced in 8/10 LNs and if present, rarely found in the medulla of the LN. Based on the presence of large GCs in all examined patients, the reduction of circulating memory B cells and PCs points towards a failure of GC output rather than GC formation in CVID patients with lymphadenopathy.

Introduction Enhancer of Zeste Homolog 2 (EZH2), the catalytic domain of Polycomb Repressive Complex 2 (PRC2), mediates the repressive mark of trimethylation of histone H3 lysine 27 (H3K27me3). The EZH2 gene is located on chromosome (chr.) 7q36.1 (frequently deleted in AML/MDS). Its role in tumorigenesis appears to be context-dependent since both EZH2 overexpression and loss of function are associated with different types of cancer. In patients (pts) with MDS or MDS/MPN, loss-of-function EZH2 mutations (mut) are recurrently found, associated with a poor prognosis, and EZH2 is considered a tumor suppressor gene (TSG). In AML, the incidence of EZH2-mut is lower and less well-studied. We wished to determine the effects of EZH2-mut and copy number (CN) on EZH2 expression, and the consequences for H3K27me3 levels in vivo. Methods Fifty-eight pts (51 AML, 3 MDS, 4 MDS/MPN), median age 63.5 years (yr, range 20-86, almost all diagnosed between 2015 and 2017), with available sequencing data, EZH2 CN status and EZH2 expression data were studied. Mutation status was determined by next-generation sequencing (NGS) including a panel of 54 genes (Illumina Myeloid NGS panel). Metaphase cytogenetics and/or fluorescence in situ hybridization and single nucleotide polymorphism arrays (selected pts) were conducted to determine EZH2 CN status. Protein expression of EZH2 and H3K27me3 was assessed semi-quantitatively by immunohistochemistry (IHC) on formalin-fixed EDTA-decalcified paraffin-embedded bone marrow (BM) core biopsies. The Kruskal-Wallis test and Dunn's multiple comparison test were used to address whether EZH2 protein expression was associated with EZH2-mut and CN. Results The EZH2 gene showed mutations (mostly missense or nonsense, median variant allele frequency (VAF) 45%, range 7-63%) in 13/58 pts and was unmutated in 45/58 pts. EZH2-mut pts had a median of 4 mutations. Additional mutations were most frequently found in ASXL1 (10/13; median VAF 35%, range 19-48%), less frequently in TET2, RUNX1, STAG2 (3/13 each), NRAS (2/13), and DNMT3A (1/13). In contrast, EZH2-wt pts had a median of 2 mutations, most frequently in DNMT3A (12/45), followed by NRAS (10/45), IDH1 (9/45), FLT3 (8/45), and NPM1 (7/45). Regarding chr. 7, 43 pts had no detectable deletion, 15 had 7q-/-7. Notably, the incidence of EZH2-mut was similar in pts with 7q-/-7 lesions (3/15, i.e. 20%) and pts with normal chr. 7 (10/43, 23%). EZH2 expression in neoplastic BM cells ranked from no (score 0) to strong expression (score 3). While the hematopoietic BM cells of healthy donors usually showed a moderate EZH2 expression (score 2), in our cohort the score was 0 in 3, 1 in 13, 2 in 23, and 3 in 19 pts, respectively. We next asked whether EZH2 protein expression differed between pts depending on EZH2-mut and chr. 7 status. In Figure 1, 4 subgroups are depicted, showing highest expression in pts with EZH2-wt and either no chr. 7 abnormalities (group A, n=33) or 7q-/-7 (group C, n=12). In comparison, expression was significantly reduced in pts with EZH2-mut and no chr. 7 abnormalities (group B, n=10), and lowest in pts with EZH2-mut and chr. 7 abnormalities (group D, n=3), p Since functional EZH2 protein is necessary for the trimethylation of H3K27, the presence of this histone mark was also determined semi-quantitatively by IHC in 40 pts. H3K27me3 levels were variable, and a test for a possible association between EZH2 and H3K27me3 levels by linear regression analysis did not show an association (R²=0.13). Sixty-two % of EZH2-mut (median age 71 yr) and 51% of EZH2-wt pts (median age 61 yr) received allografting. Median overall survival in EZH2-mut pts was 16.1 months compared to 23.5 in EZH2-wt pts (p=0.16). Conclusions Inactivating EZH2 mutations are infrequent events in AML. In our study, they were strongly associated with mutations of ASXL1 (also involved in PRC2 function). We did not observe overrepresentation of EZH2-mut in 7q-/-7 pts, in line with Bejar et al. (N Engl J Med 2011); this is in contrast to the frequent cooperative events between TSG mutations and deletions, e.g. of TP53. Functionally, EZH2 mutations appeared to have a stronger effect on decreased EZH2 expression than hemizygosity. Global H3K27me3 levels were not altered by EZH2 reduction, which could be due to compensatory upregulation of EZH1, supporting the clinical development of EZH1/2 inhibition. Disclosures No relevant conflicts of interest to declare.

Inherited bone marrow failure syndromes (IBMFS) are a heterogeneous group of disorders characterized by impaired stem cell function resulting in pancytopenia. Diagnosis of IBMFS presents a major challenge due to limited diagnostic tests and overlapping phenotypes. For that reason, novel and clinical relevant biomarkers and possible targets are urgently needed. Our study defines NIPA as an IBMFS gene, which is significantly downregulated in a distinct subset of MDS-type refractory cytopenia of childhood patients. Mechanistically, NIPA binds FANCD2 and regulates its nuclear abundance. The stabilization of both non- and monoubiquitinated FANCD2 identifies NIPA as an essential player in the Fanconi Anemia (FA) pathway. NIPA thereby prevents MMC hypersensitivity visualized by increased numbers of chromosome radials and reduced cell survival after induction of interstrand crosslinks. To provide proof of principle, re-expression of FANCD2 in Nipa deficient cells restores MMC sensitivity. In a knockout mouse model, Nipa deficiency leads to major cell intrinsic long-term repopulation defects of hematopoietic stem cells (HSCs), with impaired self-renewal in serial transplantations and a bias towards myeloid differentiation. Unresolved DNA damage in Nipa deficient HSCs causes increased sensitivity to cell death and leads to progressive, age-related loss of the HSC pool. Induction of replication stress triggers the phenotypic reduction and functional decline of murine HSCs, resulting in complete bone marrow failure and death of the mice thereby mimicing Fanconi Anemia. Taken together, our study adds NIPA to the short list of FA-associated proteins being essential for a functional DNA repair/FA/BRCA axis and thereby emphasizing its impact as potential diagnostic marker and/or possible target in bone marrow failure syndromes. Disclosures Niemeyer: Celgene: Consultancy.

Background Chronic eosinophilic leukemia (CEL) is a myeloproliferative neoplasm characterized by expansion of neoplastic eosinophils, tissue infiltration, and organ damage. In a subset of these patients, the FIP1L1/PDGFRA (F/P) oncoprotein is detectable. F/P exhibits constitutive tyrosine kinase activity and activates a number of signaling pathways. So far, however, little is known about the role of F/P-dependent proteins in the pathogenesis of CEL. Methods A screen for F/P-dependent cytokines was performed in growth factor-dependent human cell lines lentivirally transduced with F/P. Signal transduction pathways were characterized in Ba/F3 cells with doxycycline-inducible expression of F/P and in EOL-1 cells. Cytokine expression was confirmed in patients' material by immunohistochemistry, immunofluorescence, and confocal microscopy. Gene expression analysis, proliferation assays, and chemotaxis assays were used to elucidate paracrine interactions between neoplastic eosinophils and stromal cells. Results We show that F/P upregulates expression of oncostatin M (OSM) in various cell line models in a STAT5-dependent manner. Correspondingly, neoplastic eosinophils in the bone marrow were found to overexpress OSM. OSM derived from F/P + cells stimulated proliferation of stromal cells. Moreover, OSM-containing supernatants from F/P + cells were found to upregulate production of stromal cell-derived factor-1 (SDF-1)/CXCL12 in human fibroblasts. SDF-1, in turn, induced migration of EOL-1 cells in a dose-dependent manner. Conclusions We have identified a F/P-driven paracrine interaction between neoplastic eosinophils and stromal cells that may contribute to tissue fibrosis and accumulation of neoplastic eosinophils in CEL.

Myelodysplastische Syndrome (MDS) umfassen ein Spektrum klonaler hamatopoetischer Stammzellerkrankungen, die nach den Kriterien der 2008 revidierten Klassifikation der WHO eingeordnet werden. Eine Weiterentwicklung ist zu erwarten. Das klinische Krankheitsbild ist durch ungeklarte periphere Zytopenien gekennzeichnet, die zu Anamie, Blutungsereignissen und vermehrter Infektanfalligkeit fuhren. Das meist hyper-, selten hypozellulare, gelegentlich auch fibrosierte Knochenmark zeigt Dysplasien in ≥ 10% der Zellen zumindest einer oder aber mehrerer hamatopoetischer Zellreihen. Diese Veranderungen sowie eine gesteigerte Apoptoserate, Stammzellseneszenz und eine immunologische Dysregulation fuhren zu einer ineffektiven Hamatopoese. Die diagnostische Aufarbeitung sollte komplementare hamatologische, morphologische und zytogenetische/molekulare Parameter berucksichtigen. Das Methodenspektrum umfasst zytologische Untersuchungen von Blut und Knochenmark, Zytogenetik, Fluoreszenz-in-situ-Hybridisierung (FISH), Beckenkammtrepanate, Immunphanotypisierung und Evaluation molekularer Marker durch etablierte und neue Techniken. Mutationen, die Wachstumsfaktorrezeptoren, Regulatoren von Zellzyklus und Apoptose, intrazellulare Signalwege, Transkriptionsfaktoren, epigenetische Regulationsmechanismen und das Splicosom betreffen, sind an der MDS-Pathogenese und -Progression beteiligt.

Syntaxin-11 (Stx11), an atypical member of the SNARE protein family, is part of the cytolytic machinery of T and NK cells and involved in the fusion of lytic granules with the plasmamembrane. Functional loss of syntaxin-11 in humans causes defective degranulation and impaired cytolytic activity of T and NK cells. Furthermore, patients with STX11 deficiency develop familial hemophagocytic lymphohistiocytosis type 4 (FHL4), a life-threatening disease of severe hyperinflammation. We established Stx11-deficient mice as an animal model for FHL4. Stx11-deficient mice exhibited severely reduced degranulation and cytolytic activity of CTL and NK cells and developed all clinical symptoms of hemophagocytic lymphohistiocytosis (HLH) after infection with lymphocytic choriomeningitis virus (LCMV). The HLH phenotype was further characterized by hyperactive CD8 T cells and continuous IFN-γ production. However, in contrast to perforin-deficient mice, which represent a model for FHL2, progression of HLH was not fatal. Survival of Stx11-deficient mice was determined by exhaustion of antigen-specific T cells, characterized by expression of inhibitory receptors, sequential loss of effector functions, and finally T-cell deletion. Blockade of inhibitory receptors on T cells in Stx11-deficient mice converted nonfatal disease course into fatal HLH, identifying T-cell exhaustion as an important factor for determination of disease severity in HLH.

The immune cell composition of the follicular lymphoma (FL) tumor microenvironment is increasingly recognized as an important determinant for clinical outcome. Here, we explored frequency and distribution of dendritic cell (DC) subtypes in relation to regulatory T cells (Treg) by immunohistochemistry in lymph node biopsies from patients with de novo FL. We found that neoplastic follicles contained lower DC and higher Treg frequencies than hyperplastic follicles in control lymph nodes. Treg numbers particularly correlated with the subset of conventional CD11c(+ )DCs. Additionally, both a high intra- to interfollicular ratio of CD11c(+ )DCs and increased intrafollicular Treg frequencies were associated with decreased overall survival. This suggests that functional interactions between these cells may be relevant for FL progression/recurrence. The presence of CD11c(+ )DCs in the tumor microenvironment may assist tumor infiltration by Tregs, thus contributing to the suppression of an otherwise beneficial T-cell-dominated FL microenvironment.

In B-cells, activation-induced cytidine deaminase (AID) is required for somatic hypermutation (SHM) and class switch recombination (CSR) of immunoglobulin genes. AID introduces mutations in immunoglobulin variable regions (IGV) during B-cell receptor affinity maturation, but may also introduce aberrant mutations into non-immunoglobulin genes, most commonly BCL6. Follicular lymphoma (FL) B-cells constitutively express AID and undergo CSR, SHM and aberrant SHM. We have studied AID expression, the presence of SHM mutations, CSR, and aberrant SHM in BCL6 in a cohort of 75 FL patients. Whereas IgM-expressing (non-switched) FL were characterized by an expected positive correlation between AID and IGV and BCL6 mutations, isotype-switched FL showed dissociation between AID expression and aberrant SHM, and inverse correlation between SHM and AID expression. Our results unveil two manifest biological subgroups of FL and indicate that the specific dissociation between AID and SHM after isotype switch may correlate with the clinical outcome of this heterogeneous disease.

Item does not contain fulltext BACKGROUND: LPS-responsive beige-like anchor protein (LRBA) deficiency is a primary immunodeficiency caused by biallelic mutations in LRBA that abolish LRBA protein expression. OBJECTIVE: We sought to report the extended phenotype of LRBA deficiency in a cohort of 22 LRBA-deficient patients. METHODS: Clinical criteria, protein detection, and genetic sequencing were applied to diagnose LRBA deficiency. RESULTS: Ninety-three patients met the inclusion criteria and were considered to have possible LRBA deficiency. Twenty-four patients did not express LRBA protein and were labeled as having probable LRBA deficiency, whereas 22 were genetically confirmed as having definitive LRBA deficiency, with biallelic mutations in LRBA. Seventeen of these were novel and included homozygous or compound heterozygous mutations. Immune dysregulation (95%), organomegaly (86%), recurrent infections (71%), and hypogammaglobulinemia (57%) were the main clinical complications observed in LRBA-deficient patients. Although 81% of LRBA-deficient patients had normal T-cell counts, 73% had reduced regulatory T (Treg) cell numbers. Most LRBA-deficient patients had low B-cell subset counts, mainly in switched memory B cells (80%) and plasmablasts (92%), with a defective specific antibody response in 67%. Of the 22 patients, 3 are deceased, 2 were treated successfully with hematopoietic stem cell transplantation, 7 are receiving immunoglobulin replacement, and 15 are receiving immunosuppressive treatment with systemic corticosteroids alone or in combination with steroid-sparing agents. CONCLUSION: This report describes the largest cohort of patients with LRBA deficiency and offers guidelines for physicians to identify LRBA deficiency, supporting appropriate clinical management.

Stromal interaction molecule 1 (STIM1) deficiency is a rare genetic disorder of store-operated calcium entry, associated with a complex syndrome including immunodeficiency and immune dysregulation. The link from the molecular defect to these clinical manifestations is incompletely understood. We report two patients with a homozygous R429C point mutation in STIM1 completely abolishing store-operated calcium entry in T cells. Immunological analysis of one patient revealed that despite the expected defect of T cell proliferation and cytokine production in vitro, significant antiviral T cell populations were generated in vivo. These T cells proliferated in response to viral Ags and showed normal antiviral cytotoxicity. However, antiviral immunity was insufficient to prevent chronic CMV and EBV infections with a possible contribution of impaired NK cell function and a lack of NKT cells. Furthermore, autoimmune cytopenia, eczema, and intermittent diarrhea suggested impaired immune regulation. FOXP3-positive regulatory T (Treg) cells were present but showed an abnormal phenotype. The suppressive function of STIM1-deficient Treg cells in vitro, however, was normal. Given these partial defects in cytotoxic and Treg cell function, impairment of other immune cell populations probably contributes more to the pathogenesis of immunodeficiency and autoimmunity in STIM1 deficiency than previously appreciated.

Diagnosis of acute intestinal GVHD (aGVHD) following allogeneic hematopoietic cell transplantation is based on clinical symptoms and histological lesions. This retrospective analysis aimed to validate the ‘Freiburg Criteria' for the endoscopic grading of intestinal aGVHD. Grade 1: no clear-cut criteria; grade 2: spotted erythema; grade 3: aphthous lesions; and grade 4: confluent defects, ulcers, denudation of the mucosa. Having excluded patients with infectious diarrhea, we evaluated 175 consecutive patients between January 2001 and June 2009. Setting a cutoff between grade 1 (no change in therapy) and grade 2 (intensification of immunosuppression), macroscopy had a sensitivity of 89.2% (95% confidence interval (CI): 80.4–94.9%), a specificity of 79.4% (95% CI: 69.6–87.1%), a positive-predictive value of 79.6% (95% CI: 70.0–87.2%) and a negative-predictive value of 89.0% (95% CI: 80.2–94.9%). In all, 20% of patients with aGVHD in the lower gastrointestinal tract (GIT) had lesions only in the terminal ileum. In all patients with aGVHD ⩾2 of the upper GIT, typical lesions were also found in the lower GIT. Ileo-colonoscopy showed the highest diagnostic yield for aGVHD. In conclusion, the ‘Freiburg Criteria' for macroscopic diagnosis of intestinal aGVHD provide high accuracy for identifying aGVHD ⩾2.

Myeloproliferative neoplasms (MPNs) and related chronic disorders constitute a subgroup of myeloid malignancies which are defined according to clinical, morphological and molecular features by the actual World Health Organization classification of tumors of the haematopietic system. Screening procedures for a BCR-ABL fusion gene, JAK2, thrombopoietin receptor and KIT mutations are formally included in the diagnostic approach. Myelodysplastic/MPN overlap syndromes include rare entities such as refractory anemia with ringed sideroblasts characterized by a high proportion of JAK2V617F mutated cases. The paradigm of targeted treatment of chronic myeloid leukemia with imatinib has now been extended to eosinophilia-associated myeloid neoplasms with PDGFRA, PDGFRB or FGFR1 gene mutations. Pegylated interferon-alpha has convincingly been proved to reduce the JAK2 allele burden. JAK2 inhibitor drugs are currently being tested in clinical trials. The development of pathogenesis-targeted diagnostic and therapeutic approaches to the various MPNs will continue in the future.

This study evaluated the efficacy and safety of imatinib in chronic eosinophilic leukaemia (CEL, n = 23) and hypereosinophilic syndrome (HES, n = 13). In CEL with FIP1L1-PDGFRA (n = 16) or various PDGFRB fusion genes (n = 5), complete haematological remission (CHR) was achieved in 95% (20/21) after 3 months. Complete molecular remission (CMR) was seen in 75% (12/16) of cases with FIP1L1-PDGFRA positive CEL by 6 months, and in 87% (13/15) after 12 months. CMR was achieved in three of five PDGFRB fusion positive patients after 3, 9 and 18 months respectively. All patients are currently on imatinib (100 mg; n = 13, 400 mg; n = 8) and no molecular relapse has yet been observed (median 26.7 months; range, 6.9-39.9). Imatinib was less effective in HES and CEL without known molecular aberration (n = 15); CHR was observed in 40% (6/15) of patients, two patients relapsed after 4.8 and 24.5 months. Three patients died due to imatinib-resistant progressive CEL (n = 2) or myocardial infarction (n = 1) unrelated to study treatment. Overall, imatinib was well tolerated with a low incidence of grade III/IV toxicities. These data confirmed the long-term efficacy of imatinib for PDGFR-rearranged CEL patients, and also showed that a minority of HES cases without known molecular aberrations may benefit from imatinib.

Herpes simplex virus (HSV) is an infrequent cause of viral hepatitis, accounting for 1.4% [1] of all cases with acute liver failure, and is associated with a high mortality in adults. HSV 1 and 2 can both cause fulminant acute hepatitis that occurs mainly in immunocompromised patients. Symptoms may be unspecific and include fever, abdominal pain and flulike symptoms. Therapeutic options include intravenous acyclovir therapy and liver transplantation [2–6]. Nevertheless, prognosis for fulminant HSV hepatitis is poor and in a recent French report, all five patients with hepatic failure due to HSV died [1], two of them prior to transplantation, two up to 1 month after transplantation; one patient survived for one year. In contrast, a recent publication suggests that the spectrum and severity of HSV hepatitis reported in the literature over-represents fulminant presentations, and that HSV hepatitis in immunocompromised host occurs with a higher frequency and lower mortality than described so far [7]. The following report describes a kidney and pancreas graft recipient with fulminant HSV2 hepatitis, who underwent successful liver transplantation in combination with acyclovir and foscarnet therapy. Case report

Background and Objectives We sought to identify new fusion genes with involvement of the platelet-derived growth factor receptor β gene ( PDGFRB ) in three patients presenting with various subtypes of chronic myeloproliferative disorders associated with chromosomal aberrations involving chromosome bands 5q31–33. Design and Methods We performed 5′ rapid amplification of cDNA ends (5′-RACE)-polymerase chain reaction (PCR) with RNA/cDNA derived from a patient (case #1) with a t(5;12)(q31–33;q24) and a second patient (case #2) with a complex rearrangement involving chromosomes 1, 5 and 11. A newly developed DNA-based ‘longdistance inverse PCR’ (LDI-PCR) was performed on a third patient (case #3) with a t(4;5;5)(q23;q31;q33). Results In cases #1 and #2, we identified mRNA fusions between GIT2 exon 12 and GPIAP1 exon 7, respectively, and PDGFRB exon 11. In case #3, LDI-PCR revealed a fusion between PRKG2 exon 5 and a truncated PDGFRB exon 12. The region encoding the catalytic domain of PDGFRβ is retained in all three cases, with the partner contributing a coiled-coil domain (GPIAP1, PRKG2) or an ankyrin protein interaction motif (GIT2) that may potentially lead to dimerization and constitutive activation of the fusion proteins. Treatment with imatinib (400 mg/day) has led to sustained complete hematologic remission in all three patients. Interpretation and Conclusions These data provide further evidence that numerous partner genes fuse to PDGFRB in BCR-ABL negative chronic myeloproliferative disorders. Although these fusion genes occur rarely, their identification is essential in order to detect patients in whom targeted treatment with tyrosine kinase inhibitors is likely to be successful.

Omenn syndrome (OS) is a severe immunodeficiency associated with erythroderma, lymphoproliferation, elevated IgE, and hyperactive oligoclonal T cells. A restricted T-cell repertoire caused by defective thymic T-cell development and selection, lymphopenia with homeostatic proliferation, and lack of regulatory T cells are considered key factors in OS pathogenesis. We report 2 siblings presenting with cytomegalovirus (CMV) and Pneumocystis jirovecii infections and recurrent sepsis; one developed all clinical features of OS. Both carried homozygous germline mutations in CARD11 (p.Cys150*), impairing NF-κB signaling and IL-2 production. A somatic second-site mutation reverting the stop codon to a missense mutation (p.Cys150Leu) was detected in tissue-infiltrating T cells of the OS patient. Expression of p.Cys150Leu in CARD11-deficient T cells largely reconstituted NF-κB signaling. The reversion likely occurred in a prethymic T-cell precursor, leading to a chimeric T-cell repertoire. We speculate that in our patient the functional advantage of the revertant T cells in the context of persistent CMV infection, combined with lack of regulatory T cells, may have been sufficient to favor OS. This first observation of OS in a patient with a T-cell activation defect suggests that severely defective T-cell development or homeostatic proliferation in a lymphopenic environment are not required for this severe immunopathology.

Aims In the era of potentially disease-modifying agents such as Janus kinase inhibitors, accurate grading and differentiation of bone marrow (BM) fibrosis has become more relevant to assess staging of disease and therapeutic effects. However, different fibrosis grading models have been used in the past without uniformity, including the proposal by the World Health Organization. Current scoring systems are based only on reticulin fibrosis. Therefore, additional assessment of collagen and the grade of osteosclerosis appear to be essential to discriminate all components of the complex BM fibrous matrix. Methods and results We evaluated problems and pitfalls regarding staining techniques and the interpretation of reticulin fibrosis on a total of 352 samples. Furthermore, we propose a minor modification of the current grading and separate scoring for collagen deposition and osteosclerosis. Reproducibility of gradings was tested among 11 haematopathologists in a blinded assessment. Overall, the inter-rater reliability of all three grading systems ranged between 0.898 and 0.926. Conclusions A standardized assessment of BM fibrosis with differentiation between reticulin, collagen and osteosclerosis is recommended to evaluate the various components of the fibrous matrix which may be delinked after therapy. In this regard, quality of staining and application of laboratory standards enable a highly reproducible scoring.

The FIP1L1-PDGFRA (FP) fusion gene is identified in a substantial proportion of patients with eosinophilia-associated myeloproliferative neoplasms (MPN-eo) who subsequently achieve rapid and durable remissions on imatinib. In the initial diagnostic work-up of hypereosinophilia (HE), histologic and immunohistochemical evaluation of a bone marrow (BM) core biopsy is considered essential for the differentiation between reactive hypereosinophilia (HER), MPN-eo and hypereosinophilic syndrome (HES). We therefore retrospectively analysed the initial reports of BM core biopsies from 116 patients who were subsequently identified as FP positive (FP+, n = 56) or FP negative/corticosteroid-responsive HER or HES (n = 60). Compared to HER or HES, detection of FP was more frequently associated with increased numbers of blasts (11/56 vs. 2/60, p = 0.007) and mast cells (23/33 vs. 7/23, p = 0.006; with expression of CD25 [11/18 vs. 2/13, p = 0.025]), and/or fibrosis (25/35 vs. 1/23, p