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Neutrophils are critical mediators of host defense in pathogen-induced and sterile inflammation. Excessive neutrophil activation has been associated with increased host pathology through collateral organ damage. The beneficial aspects of neutrophil activation, particularly in sterile inflammation, are less well defined. We observed accumulation of nuclear debris in the lungs of neutropenic mice exposed to acid-induced injury compared with wild type. Size analysis of DNA debris showed that neutropenic mice were unable to degrade extracellular DNA fragments. In addition, we found that neutrophils are able to differentially express DNA-degrading and repair-associated genes and proteins. Once neutrophils are at sites of lung inflammation, they are able to phagocytose and degrade extracellular DNA. This neutrophil-dependent DNA degradation occurs in a MyD88-dependent pathway. The increased DNA debris in neutropenic mice was associated with dysregulated alveolar repair and the phenotype is rescued by intratracheal administration of DNase I. Thus, we show a novel mechanism as part of the inflammatory response, in which neutrophils engulf and degrade extracellular DNA fragments and allow for optimal organ repair.

Inherited bone marrow failure (IBMF) syndromes are rare blood disorders characterized by hematopoietic cell dysfunction and predisposition to hematologic malignancies. Despite advances in the understanding of molecular pathogenesis of these heterogeneous diseases, genetic variant interpretation, genotype–phenotype correlation, and outcome prognostication remain difficult. As new IBMF and other myelodysplastic syndrome (MDS) predisposition genes continue to be discovered (frequently in small kindred studies), there is an increasing need for a systematic framework to evaluate penetrance and prevalence of mutations in genes associated with IBMF phenotypes. To address this need, we analyzed population-based genomic data from >125 000 individuals in the Genome Aggregation Database for loss-of-function (LoF) variants in 100 genes associated with IBMF. LoF variants in genes associated with IBMF/MDS were present in 0.426% of individuals. Heterozygous LoF variants in genes in which haploinsufficiency is associated with IBMF/MDS were identified in 0.422% of the population; homozygous LoF variants associated with autosomal recessive IBMF/MDS diseases were identified in only .004% of the cohort. Using age distribution of LoF variants and 2 measures of mutational constraint, LOEUF (“loss-of-function observed/expected upper bound fraction”) and pLI (“probability of being loss-of-function intolerance”), we evaluated the pathogenicity, tolerance, and age-related penetrance of LoF mutations in specific genes associated with IBMF syndromes. This analysis led to insights into rare IBMF diseases, including syndromes associated with DHX34, MDM4, RAD51, SRP54, and WIPF1. Our results provide an important population-based framework for the interpretation of LoF variant pathogenicity in rare and emerging IBMF syndromes.

The advent of chimeric antigen receptor T (CAR-T) and the burgeoning field of cellular therapy has revolutionized the treatment of relapsed/refractory leukemia and lymphoma. This personalized “living therapy” is highly effective against a number of malignancies, but this efficacy is tempered by side effects relatively unique to immunotherapies, including CAR-T. The overwhelming release of cytokines and chemokines by activated CAR-T and other secondarily activated immune effector cells can lead to cytokine release syndrome (CRS), which can have clinical and pathophysiology similarities to systemic inflammatory response syndrome and macrophage activating syndrome/hemophagocytic lymphohistiocytosis. Tocilizumab, an anti-IL6 receptor antibody, was recently FDA approved for treatment of CRS after CAR-T based on its ability to mitigate CRS in many patients. Unfortunately, some patients are refractory and additional therapies are needed. Patients treated with CAR-T can also develop neurotoxicity and, as the biology is poorly understood, current therapeutic interventions are limited to supportive care. Nevertheless, a number of recent studies have shed new light on the pathophysiology of CAR-T-related neurotoxicity, which will hopefully lead to effective treatments. In this review we discuss some of the mechanistic contributions intrinsic to the CAR-T construct, the tumor being treated, and the individual patient that impact the development and severity of CRS and neurotoxicity. As CAR-T and cellular therapy have redefined the concept of personalized medicine, so too will personalization be necessary in managing the unique side effects of these therapies.

Unrelated donor hematopoietic stem cell transplantation (HSCT) is increasingly being used to cure nonmalignant hematologic diseases (NMHD) in patients who lack HLA matched related donors. Both graft rejection and graft-versus-host disease (GVHD) remain major barriers to safe and effective transplant for these patients requiring unrelated donors. Partial T cell depletion combined with peripheral stem cell transplantation (pTCD-PSCT) has the potential advantages of providing a high stem cell dose to facilitate rapid engraftment, maintaining cells that may facilitate engraftment, and decreasing GVHD risk compared with T cell–replete HSCT. Here, we report a single-institution, retrospective experience of unrelated donor pTCD-PSCT for pediatric patients with NMHD. From 2014 to 2017, 12 pediatric patients with transfusion-dependent NMHD underwent matched unrelated donor (MUD) or mismatched unrelated donor (MMUD) pTCD HSCT in our center using disease-specific conditioning. Donor PSCs underwent CD3+ T cell and CD19+ B cell depletion using CliniMACS, followed by a targeted addback of 1 × 105 CD3+ T cells/kg to the graft before infusion. All 12 patients demonstrated rapid trilinear engraftment. At a median follow-up of 740days (range, 279 to 1466), all patients were alive with over 92% total peripheral blood donor chimerism and without transfusion dependence or recurrence of their underlying hematologic disease. Immune reconstitution was rapid and comparable with T cell–replete HSCT. No patients developed severe acute GVHD (grades III to IV) or chronic extensive GVHD, and all patients had discontinued systemic immune suppression. Viral reactivations were common, but no patient developed symptoms of life-threatening infectious disease. Our data indicate that MUD and MMUD pTCD-PSCTs are safe and effective approaches that enable rapid engraftment and immune reconstitution, prevent severe GVHD, and expand availability of HSCT to any patients with NMHD who have closely MUDs.

The congenital sideroblastic anemias (CSAs) are a heterogeneous group of inherited disorders of erythropoiesis characterized by pathologic deposits of iron in the mitochondria of developing erythroblasts. Mutations in the mitochondrial glycine carrier SLC25A38 cause the most common recessive form of CSA. Nonetheless, the disease is still rare, there being fewer than 70 reported families. Here we describe the clinical phenotype and genotypes of 31 individuals from 24 families, including 11 novel mutations. We also review the spectrum of reported mutations and genotypes associated with the disease, describe the unique localization of missense mutations in transmembrane domains and account for the reoccurrence of several alleles in different populations.

Alveolar epithelial regeneration is essential for recovery from devastating lung diseases. This process occurs when type II alveolar pneumocytes (AT2 cells) proliferate and transdifferentiate into type I alveolar pneumocytes (AT1 cells). We used genome-wide analysis of chromatin accessibility and gene expression following acute lung injury to elucidate repair mechanisms. AT2 chromatin accessibility changed substantially following injury to reveal STAT3 binding motifs adjacent to genes that regulate essential regenerative pathways. Single-cell transcriptome analysis identified brain-derived neurotrophic factor (Bdnf) as a STAT3 target gene with newly accessible chromatin in a unique population of regenerating AT2 cells. Furthermore, the BDNF receptor tropomyosin receptor kinase B (TrkB) was enriched on mesenchymal alveolar niche cells (MANCs). Loss or blockade of AT2-specific Stat3, Bdnf or mesenchyme-specific TrkB compromised repair and reduced Fgf7 expression by niche cells. A TrkB agonist improved outcomes in vivo following lung injury. These data highlight the biological and therapeutic importance of the STAT3-BDNF-TrkB axis in orchestrating alveolar epithelial regeneration.

Background: Pediatric immune thrombocytopenia purpura (ITP) is the most common cause of autoimmune cytopenias in children and results in autoimmune destruction of platelets, leading to significantly decreased circulating platelets, increased bleeding risk and fatigue. The underlying mechanism of action is thought to be auto-antibody driven, though auto-reactive T cells have also been implicated in some cases. Given the proximity of many cases of ITP to either recent viral infection or vaccine administration, a leading hypothesis is that the environmental exposure mimics a platelet produced epitope causing platelet clearance. Despite the correlation of viral infection and/or inflammatory cascades in the propagation of ITP, the gut microbiome in newly diagnosed pediatric patients has not previously been interrogated. The gut microbiome has previously been shown to modulate the host inflammatory milieu and dysbiosis has been associated with other auto-immune disorders. Thus, we assessed the gut microbiome in newly diagnosed pediatric ITP patients to determine if a similar dysbiosis is present. Methods: Stool samples were collected from 32 pediatric patients (0-18 years) at the Children's Hospital of Philadelphia. 17 patients were newly diagnosed with ITP, 7 patients had ITP for greater than 3 months at time of stool collection and 8 patients had newly diagnosed severe acquired aplastic anemia. Stool samples were kept on ice until processing at our PENN/CHOP Microbiome Core (not longer than 24 hours/sample). Age matched healthy control samples were provided by the Microbiome Core. Shotgun libraries were generated from 0.5 ng DNA using the Nextera XT Library Prep kit and libraries were sequenced on an Illumina HiSeq 2500 in High Output mode to produce paired-end 125 bp sequence reads. Shotgun metagenomic data were analyzed using Sunbeam, a user-extendable bioinformatics pipeline that we developed for this purpose.Diversity within samples were assessed by the number of OTUs at a rarefaction level of 1,000 sequences and the Shannon index. Sample similarity were assessed by Bray-Curtis and Jaccard distances, which were then visualized using principle coordinates analysis. Results: Subjects included 17 patients with newly diagnosed ITP (i.e. stool sample collected < 3 months from diagnosis), 7 patients with ITP > 3 months and 8 patients with newly diagnosed severe acquired aplastic anemia. Patient samples were assessed for gut microbial diversity and richness. As seen in figure 1a, gut microbial diversity in newly diagnosed pediatric patients with ITP was significantly decreased (p = 0.024) while gut microbial richness trended to a concomitant decrease as well (p = 0.093). Interestingly, healthy controls had similar gut microbiomes to one another than newly diagnosed ITP patients had to one another (Fig 1b). These alterations in the gut microbiome were not seen in pediatric patients with newly diagnosed aplastic anemia or with ITP diagnosed > 3 months prior to collection (data not shown). These findings indicate that while there is a dysbiotic gut microbiome in patients with newly diagnosed ITP, there is no dysbiosis present in other pediatric autoimmune cytopenias. Furthermore, the dysbiosis corrects by 3 months post-ITP diagnosis. When we assess specific bacterial families and subgroups we do not find a predominant species that is increased or decreased but rather there are global changes amongst many bacterial species. We did find a trend of lower Alistipes species and increased Eschrechia coli in newly diagnosed ITP patients which is similar to other autoimmune disorders with shifted gut microbiota. Functional pathways analyses showed similar global alterations in vital pathways such as decreased aminoacyl tRNA biosynthesis and homologous recombination. Conclusion: Pediatric patients with newly diagnosed ITP but not other autoimmune cytopenias have gut microbial dysbiosis with perturbations in many bacterial species that may be a cause of, or a result of the underlying mechanism of pathogenesis. Further studies will help determine the role of dysbiosis in the pathobiology of ITP and determine whether intervention alters the duration of disease. Figure 1 Figure 1. Disclosures Lambert: Bayer: Consultancy; Astra Zeneca: Research Funding; Shionogi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; ClinGen, ISTH, ASH, GW University: Honoraria; PDSA: Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Rigel: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sysmex: Research Funding; Principia: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Argenx: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Octapharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Dova: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Background: Success of alternative donor stem cell transplantation (SCT) for acquired and inherited bone marrow failure (BMF) syndromes has previously been limited by significant risks of severe graft versus host disease (GvHD) and graft failure, particularly for patients who lack fully HLA-matched donors. The development of post-transplant cyclophosphamide (ptCy)-based haploidentical donor SCT has mitigated but not eliminated these risks, and the relatively slow engraftment seen with ptCy bone marrow grafts may not be ideal given high rates of infectious disease and bleeding concerns in patients with BMF. Partial T cell depletion of mobilized peripheral stem cell (PSC) grafts can greatly reduce risks of graft versus host disease while facilitating rapid and robust engraftment by providing a high stem cell dose. We previously reported excellent outcomes with minimal GVHD and no graft rejection using peripheral stem cell transplant (PSCT) combined with ex vivo CD3 +/CD19 +depletion and low dose CD3+ T cell addback from matched unrelated donors (MUD) and mismatched unrelated donors (MMUD) in pediatric patients with BMF. Here, we describe the use of selective ex vivoT cell receptor (TCR)αβ +/CD19 +depletion of mobilized PSC from MUD and MMUD for pediatric patients with BMF, which has the advantage of retaining TCRgd +T cells which may help facilitate engraftment and decrease infections. Methods: We report the outcomes of 26 pediatric patients with BMF (excluding MDS-defining clonal evolution) who underwent MUD/MMUD PSCT with TCRαβ +T cell/CD19 +depletion using CliniMACS at The Children's Hospital of Philadelphia from 2017 to 2021. Patients were enrolled on a prospective clinical trial for patients with BMF (NCT03047746, n=21)or on an expanded access study (NCT03145545, n=5). Conditioning regimens consisted of thymoglobulin (9mg/kg), cyclophosphamide (100mg/kg), fludarabine (150mg/m 2), and low dose TBI (200-300 cGy) for patients with acquired BMF disorders, thymoglobulin (9mg/kg), busulfan (PK-adjusted), fludarabine (150mg/m 2), and thiotepa (10mg/kg) for patients with single lineage BMF or thymoglobulin. One patient with Fanconi Anemia received thymoglobulin and fludarabine, with reduced dosing of busulfan and cyclophosphamide. Patients undergoing MSD-BMT for BMF over a similar time period served as a comparison group for engraftment kinetics, rates of GVHD, donor chimerism, immune reconstitution, and overall survival. Results: Subjects included 18 with severe acquired aplastic anemia (SAAA), 4 with SAAA and concurrent paroxysmal nocturnal hemoglobinuria (PNH), 2 patients with acquired BMF not otherwise specified, 1 patient with DBA, and 1 patient with Fanconi Anemia. 11 patients with SAAA underwent SCT as initial therapy while 11 patients had SCT after failing previous medical therapies. Median age at diagnosis was 10.3 years (0.1-20.6) and at transplant 11.1 years (0.9-21). HLA match of unrelated donors was either 10/10 (n=15), or 9/10 (n=11). Median CD34 +and TCR αβ +T cell dose was 12.0x10 6cells/kg (3-22.6) and 0.1x10 5cells/kg (0.0-4.2). Median times to neutrophil and platelet engraftment per CIBMTR criteria were 15 days (10-22) and 15 days (13-19), respectively, both significantly earlier than engraftment following MSD-BMT (Fig 1a). At a median follow-up of 727 days (39-1498), 25 of 26 patients are alive with resolved hematologic disease (Fig 1b). One patient with SAAA+PNH who failed prior IST achieved trilinear engraftment without GVHD, but died on Day+95 due to acute disseminated toxoplasmosis. No patients exhibited immunologic graft rejection. 2/26 patients had grade II acute GVHD that responded to steroids and none developed Grade III-IV acute or chronic extensive GVHD (Fig 1c) CMV viremia/reactivation occurred in 5 subjects, all responding to antiviral pharmacotherapy, and none developed end-organ CMV disease (Fig 1d). One patient developed recipient-derived EBV post-transplant lymphoproliferative disorder requiring multimodal treatment. Only one patient developed BK cystitis. Total peripheral chimerism exceeded 90% in all patients. Immune reconstitution kinetics were similar to that seen in MSD-BMT. Conclusion: MUD/MMUD PSCT with TCRαβ +T cell/CD19 +depletion in patients with BMF enables rapid, durable engraftment with minimal risk of GVHD and immunologic graft rejection. Figure 1 Figure 1. Disclosures Monos: Omixon: Consultancy, Patents & Royalties. Grupp: Jazz Pharmaceuticals: Consultancy, Other: Steering committee, Research Funding; Novartis, Adaptimmune, TCR2, Cellectis, Juno, Vertex, Allogene and Cabaletta: Other: Study steering committees or scientific advisory boards; Novartis, Kite, Vertex, and Servier: Research Funding; Novartis, Roche, GSK, Humanigen, CBMG, Eureka, and Janssen/JnJ: Consultancy.

Cervical carcinoma is associated with high-risk human papillomavirus (HPV) DNA integration and usually occurs after age 21 (peak 45 years), as reflected in screening guidelines. Between 1999 and 2008, cervical carcinoma rate in adolescents aged 15-19 years was 0.15 per 100,000. HPV-negative cervical carcinoma is rare in adolescents. The youngest previously reported case was 15 years old. Treatment options for cervical carcinoma are limited after first-line therapy. Immune checkpoint inhibitors blocking programmed death receptor (PD-1) and its ligand, PD-L1, have shown objective clinical responses and are tolerable in adults with gynecologic cancers. This class of agents is well tolerated in pediatric patients. PD-1/PD-L1 is commonly expressed in gynecologic cancers but its expression may not predict clinical response. We describe an exceptional response to single agent nivolumab postradiation therapy in a 13-year-old adolescent with poorly differentiated cervical carcinoma and widespread metastatic disease.

Unrelated donor (URD) hematopoietic stem cell transplantation (HSCT) is increasingly being utilized to cure non-malignant hematologic diseases (NMHD) in patients who lack HLA matched related donors (MRD). Both graft rejection and graft vs host disease (GVHD) remain major barriers to safe and effective transplant for these patients requiring URDs. Partial T cell depletion combined with peripheral stem cell transplantation (pTCD-PSCT), has the potential advantages of providing a high stem cell dose to facilitate rapid engraftment, maintaining cells that may facilitate engraftment, and decreasing GVHD risk compared to T replete HSCT. Here, we report a single institution, retrospective experience of URD pTCD-PSCT for pediatric patients with NMHD. From 2014 to 2017, 12 pediatric patients with transfusion-dependent NMHD underwent matched (MUD) or mismatched (MMUD) unrelated donor pTCD HSCT in our center using disease-specific conditioning. Donor peripheral stem cells underwent CD3(+) T cell and CD19(+) B cell depletion using CliniMACS, followed by a targeted addback of 1 × 10(5) CD3(+) T cells/kg to the graft prior to infusion. All 12 patients demonstrated rapid trilinear engraftment. At a median follow-up of 740 days (range 279–1466), all patients are alive with over 92% total peripheral blood donor chimerism, and without transfusion dependence or recurrence of their underlying hematologic disease. Immune reconstitution was rapid and comparable to T replete HSCT. No patients developed severe acute GVHD (Grade III-IV) or chronic extensive GVHD, and all patients have discontinued systemic immune suppression. Viral reactivations were common, but no patient developed symptoms of life-threatening infectious disease. Our data indicate that MUD and MMUD pTCD-PSCT are safe and effective approaches that enable rapid engraftment and immune reconstitution, prevent severe GVHD, and expand availability of HSCT to any patients with NMHD who have closely matched unrelated donors.

Objectives To assess initial and long-term outcome of children with persistent/chronic idiopathic thrombocytopenic purpura (ITP) treated with 4 infusions of rituximab and three 4-day cycles of dexamethasone (4R+3Dex) including cohorts with most benefit and/or treatment associated toxicity. Study design All pediatric patients with ITP at Weill-Cornell who received 4R+3Dex were included in this retrospective study. Duration was median time from first rituximab infusion to treatment failure. Patient cohort included 33 children ages 1-18 years with persistent/chronic ITP; 19 were female, 10 of whom were adolescents. Every patient had failed more than 1 and usually several ITP treatments. Results Children were treated with rituximab, 375 mg/m2 weekly for 4 weeks and three 4-day courses of dexamethasone 28 mg/m2 (40 mg max). Average age of nonresponders was 7.75 years, and initial responders averaged 12.7 years (P = .0073); 30% maintained continuing response at 60 months or last check-up. Eight of the 10 patients who underwent remission were female with ITP Conclusions Durable unmaintained ITP remission after 4R+3Dex was seen almost exclusively in female adolescents with

PNH is the most common clonal hematopoietic disorder arising in patients with aAA. PNH is caused by mutations in PIGA, a gene that encodes the catalytic subunit of an enzyme involved in the biosynthesis of GPI anchors, transmembrane glycolipids required for cell surface expression of many proteins. PNH clones likely arise as immune escape mechanisms in aAA by preventing CD1D-restricted T-cell recognition of GPI anchors and GPI-linked autoantigens. Though many patients with aAA treated with IST will develop subclinical PNH clones, only a subset will develop PNH disease, characterized by increased thrombosis, intravascular hemolysis, and potential for severe organ dysfunction. In contrast to IST, allogeneic HSCT for patients with aAA is thought to cure bone marrow aplasia and prevent hematopoietic clonal evolution to PNH. Herein, we present a phenomenon of host-derived PNH disease arising in a patient with aAA many years following MSD-BMT, highlighting the importance of monitoring for this clonal disease in aAA patients with stable mixed donor/recipient chimerism after HSCT. We also provide a literature review for similar occurrences of PNH arising after HSCT.

Inherited platelet disorders (IPD) are increasingly recognized as the cause of clinical bleeding. Advances in genomic technologies have identified a growing number of platelet-associated genes that are currently vetted with phenotypic correlates. These platelet-associated genes are a disparate group, including transcription or related nuclear factors, cytoskeletal proteins, surface receptors, intracellular proteins and granule forming proteins. At present the prevalence of each inherited platelet-associated disorder and the disorders in aggregate are not well defined. We leveraged the recent curating of 123,136 high quality exomes from a cross section of the general population in the form of the genome aggregation database (gnomAD) for analysis. We used the loss-of-function transcript effect estimator (LoFTEE) in conjunction with the gnomAD dataset to study loss of function (LoF) variants in genes of interest. With this set of predicted LoF variants, we generated a LoF frequency for each gene of interest taking into account whether the heterozygote or homozygote state is sufficient and/or necessary for clinical phenotype. These data are analyzed to determine whether each platelet-associated gene is relatively tolerant or intolerant to LoF mutations in the context of their clinical phenotypes. By this analysis, we found approximately 800 novel LoF variants in platelet-related genes in this population of >120,000 individuals. Affected genes known to cause disease phenotype in the heterozygous state (n=33) accounted for 27% of the mutations analyzed. With these data, we calculated the frequency of IPD in the general population secondary to LoF mutations and estimated the relative impact of dominant versus recessive cases of IPD. We demonstrate that the majority of manifest cases of IPD will be due to the dominantly inherited, haploinsufficient IPDs. The transcription factor gene subset (9 of the IPD associated genes) was the most intolerant to LoF variants based on ratio of observed vs. expected number of variants (pLI measurement). Interestingly, the severity of the platelet dysfunction and resultant bleeding from LoF mutations in this subset of genes is not directly related to their intolerance of these mutations. For instance, heterozygous LoF of RUNX1 result in a mild-moderate bleeding disorder; however, a pLI of 0.819 indicates this gene is moderately to very intolerant of LoF variants. These same LoF variants in RUNX1 predispose to myelodysplastic syndromes with a high risk of myeloid leukemia in the form of familial platelet disorder with predisposition to acute myeloid leukemia (FPD/AML), and likely this is driving LoF intolerance. Cytoskeletal protein encoding genes represent another subset of mostly LoF intolerant platelet-related genes. Intracellular protein encoding genes and granule protein genes have varied tolerance and platelet-associated receptor protein genes as a subgroup were most tolerant of haploinsufficiency. There were some genes with similar clinical bleeding phenotypes that had divergent tolerance to LoF. For instance, GP9 and GP1BB both cause Montreal Platelet Syndrome in the haploinsufficient state and had moderate intolerance to LoF mutations (pLI GP9 = 0.804; pLI GP1BB = 0.575). In contrast, while LoF mutations in GP1BA cause the same bleeding phenotype, this gene is much more tolerant to haploinsufficiency (pLI = 0.0002). These data indicate that perhaps there is another unidentified adverse condition associated with GP9 and GP1BB that is driving increased haploinsufficiency intolerance. In summary, we present a comprehensive analysis of known platelet-associated genes, the frequency of LoF mutations in these genes and their relative tolerance of the haploinsufficient state. These data generate an incidence of IPDs of ~0.18% in the general population. Importantly, these data also inform the driving mechanisms of LoF intolerance as there are defective genes resulting in similar bleeding phenotypes, but divergent tolerance to haploinsufficiency, indicating that further investigation is warranted for additional biology. Disclosures Lambert: CSL: Consultancy; Rigel: Consultancy; Sysmex: Consultancy; Amgen: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Summus: Consultancy; Bayer: Membership on an entity's Board of Directors or advisory committees; Shionogi: Consultancy; Educational Concepts in Medicine: Consultancy. Poncz:Incyte Corporation: Consultancy, Research Funding.

Interferon-gamma (IFN-gamma) has potent antiviral activity in neurons which is affected by the production of nitric oxide (NO). This study examines the interactions between cannabinoid receptor-1 (CB(1)), IFNgamma-induced pathways, and inhibition of vesicular stomatitis virus (VSV) replication in neuronal cells. CB(1) is abundantly expressed in neurons of the CNS and the NB41A3 neuroblastoma cell line. CB(1) activation of NB41A3 cells by the synthetic cannabinoid, WIN55,212-2, is associated with an inhibition of Ca(2+) mobilization, leading to diminished nitric oxide synthase (NOS)-1 activity and the production of NO, in vitro. This ultimately results in antagonism of IFN-gamma-mediated antiviral activity and enhanced viral replication. Therefore, activation of cells expressing CB(1) by endogenous (or exogenous) ligands may contribute to decreased inflammation and to increased viral replication in neurons and disease in the CNS.

Since approximately 5–10% of the ~ 50,000 tuberous sclerosis complex (TSC) patients in the US are “MRI-negative,” our goal was to test the hypothesis that they nevertheless exhibit metabolic abnormalities. To test this, we used proton MR spectroscopy to obtain and compare gray and white matter (GM and WM) levels of the neuronal marker, N-acetylaspartate (NAA), the glial marker, myo-inositol (mI), and its associated creatine (Cr), and choline (Cho) between two “MRI-negative” female TSC patients (ages 5 and 43 years) and their matched controls. The NAA, Cr, Cho and mI concentrations, 9.8, 6.3, 1.4, and 5.7 mM, in the pediatric control were similar to those of the patients, whereas the adult patient revealed a 17% WM NAA decrease and 16% WM Cho increase from their published means for healthy adults — both outside their respective 90% prediction intervals. These findings suggest that longer disease duration and/or TSC2 gene mutation may cause axonal dysfunction and demyelination.

Oligodendrocyte-specific protein (OSP)/claudin-11 has been recently implicated in multiple sclerosis pathophysiology. Yet, the pathogenic autoimmunity against OSP has been poorly investigated. We previously showed that OSP-induced experimental autoimmune encephalomyelitis (EAE) and optic neuritis in SJL/J mice are primarily associated with CD4+ T cells reactive against OSP55-80. Dissecting the fine epitope specificity to the level of epitopic residues recognized by OSP-specific encephalitogenic T cells revealed their focused recognition of OSP58M. Accordingly, OSP58M predicted by computer modeling to be a major TCR contact residue shared by the three nonameric core epitopes within OSP55-80, albeit at different MHC-II pockets, was experimentally determined as the primary TCR contact residue crucial for activation and control of encephalitogenic T cells reactive against OSP55-80 or against recombinant OSP. Ala substitution of OSP58M impaired the functional TCR recognition/activation of pathogenic OSP-reactive T cells. Accordingly, the non-stimulatory/non-encephalitogenic pOSP55-58A-66 analogue not only treated EAE induced by pOSP55-80 but also effectively reversed EAE induced by whole OSP. Thus, the selection/activation and control of OSP-pathogenic T cells in H-2(s) mice appeared to be dominated by their predetermined focused recognition of OSP58M. Such a focused recognition by OSP-pathogenic T cells, despite their extensive TCR heterogeneity (Kaushansky, N., Zhong, M. C., Kerlero de Rosbo, N., Hoeftberger, R., Lassmann, H. and Ben-Nun, A. 2006. Epitope specificity of autoreactive T and B cells associated with experimental autoimmune encephalomyelitis and optic neuritis induced by oligodendrocyte-specific protein in SJL/J mice. J. Immunol. 177:7364), may impact profoundly on peripheral self-tolerance to OSP and on altered peptide ligand-mediated immune-specific modulation of the recently described OSP-related autoimmune pathogenesis.

Background: Therapies in ITP primarily increase the platelet count while treatment continues: IVIG, steroids, IV RhIg, romiplostim, eltrombopag and others. Curative effects of treatments other than splenectomy are uncertain; 2 recent studies with rituximab provided disappointing results. ITP in children often spontaneously improves but in children with chronic ITP and adults, this is much less common and management may be difficult. The aim of this study is to explore the efficacy and safety of R+3D. Methods : ITP patients were managed with R+3D if appropriate. Forty-nine adults and 33 children with ITP (newly diagnosed, persistent, and chronic) were treated at Weill Cornell Medical College with the previously described R+3D treatment plan: weekly rituximab infusion 375mg/m2 x 4 weeks + three 4-day cycles of 28mg/m2 (max. 40mg) dexamethasone at 2-week intervals. Patients who came to the platelet disorders center were included even if they received all or part of their treatment elsewhere. Counts were obtained weekly, monthly, and 2-3x monthly thereafter for responders. Response was either partial (PR, plt count > 50,000-100,000) or complete (CR > 100,000). Analysis was descriptive and by Kaplan-Meier. Children and adults are presented separately. Results : The overall estimated response (initial and long-term) in the 49 adults (22 men, 27 women) was associated with greater initial platelet response, female gender, and duration of ITP < 1 year. There was a significant difference in the projected 78% lasting response rate at 5 years in women with ITP of < 1 yr duration. All other groups (men < or > 1 yr duration of ITP and women > 1 yr duration of ITP) had highly inferior results ( Ninety % of the 49 adults (44/49; 11 PRs and 33 CRs) initially responded to R+3Dex at 8 weeks. Children had lower initial responses at 45% (15/33; 2 PRs and 13 CRs). Four adults further improved from PR to CR and 2 children, 1 NR, 1 PR, to CR. Kaplan Meier projected long-term treatment-free response for all adults treated with R+3Dex is 33.5%. Only 1 PR but 22/37 (59.5%) of CRs continue to maintain adequate platelet counts. Parallel projection of long-term response for children is 23.9%. In children, 10/16 (62.5%) responders continue to maintain their response at last follow-up., Female patients maintained a 44% long-term response at 5 years whereas male patients projected a rate of only 19% (p value = 0.009). In children, girls projected a 37% long-term response and boys projected 11% at 5 years. Further divided by the duration of ITP, both adult and pediatric female patients with duration of ITP less than 12 months fared better than females with longer duration of disease (and males of any duration). Conclusions: ITP does not typically display a large gender disparity compared to other autoimmune diseases such as systemic lupus erythematosus), (1.5F:1Mcompared to 9F:1M ). No gender differences were found in responses to first- and second-line treatments in a previous report (Andres et al., 2012). However, gender bias in especially long term response is dramatic and is shown separately in both children and adults; when combined with earlier treatment initiation, the difference in projected long-term, treatment-free remission is remarkable (Table 1). Studies in lymphoma and 2 other indications have also suggested a gender effect in response to rituximab, but attributed it to Pk in elderly women. The influence of gender on autoimmune diseases, ITP in particular, is complicated and warrants further study as to the mechanism of effect. Table 1.Adults (n=49) Female: n=27 // Male: n=22Children (n=33) Female: n=18 // Male: n=15Initial ResponseNR = 5 (10.2%)NR = 18 (54.5%)PR = 11 (22.4%)PR = 2 (6.1%)CR = 33 (67.4%)CR = 13 (39.4%)Female: 26/27 (96.3%) Male: 18/22 (81.8%)Female: 8/18 (44.4%) Male: 7/15 (46.7%)Best ResponseNR = 5 (10.2%)NR = 17 (51.5%)PR = 7 (14.3%)PR = 1 (3.0%)CR = 37 (75.5%)CR = 15 (45.5%)Female: 26/27 (96.3%) Male: 18/22 (81.8%)Female: 9/18 (50%) Male: 7/15 (46.7%)Relapsed21/44 (47.7%)6/16 (37.5%)Female: 10/26 (38.5%) Male: 10/18 (55.5%)Female: 2/9 (22.2%) Male: 4/7 (57.1%)Ongoing23/44 (52.3%)10/16 (62.5%)Female: 16/26 (61.5%) Male: 8/18 (44.4%)Female: 7/9 (77.7%) Male: 3/7 (42.9%) Disclosures Bussel: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; protalex: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; rigel: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cangene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.