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Autosomal recessive CARD9 deficiency predisposes patients to invasive fungal disease. Candida and Trichophyton species are major causes of fungal disease in these patients. Other CARD9-deficient patients display invasive diseases caused by other fungi, such as Exophiala spp. The clinical penetrance of CARD9 deficiency regarding fungal disease is surprisingly not complete until adulthood, though the age remains unclear. Moreover, the immunological features of genetically confirmed yet asymptomatic individuals with CARD9 deficiency have not been reported. Identification of CARD9 mutations by gene panel sequencing and characterization of the cellular phenotype by quantitative PCR, immunoblot, luciferase reporter, and cytometric bead array assays were performed. Gene panel sequencing identified compound heterozygous CARD9 variants, c.1118G>C (p.R373P) and c.586A>G (p.K196E), in a 4-year-old patient with multiple cerebral lesions and systemic lymphadenopathy due to Exophiala dermatitidis. The p.R373P is a known disease-causing variant, whereas the p.K196E is a private variant. Although the patient’s siblings, a 10-year-old brother and an 8-year-old sister, were also compound heterozygous, they have been asymptomatic to date. Normal CARD9 mRNA and protein expression were found in the patient’s CD14+ monocytes. However, these cells exhibited markedly impaired pro-inflammatory cytokine production in response to fungal stimulation. Monocytes from both asymptomatic siblings displayed the same cellular phenotype. CARD9 deficiency should be considered in previously healthy patients with invasive Exophiala dermatitidis disease. Asymptomatic relatives of all ages should be tested for CARD9 deficiency. Detecting cellular defects in asymptomatic individuals is useful for diagnosing CARD9 deficiency.

IRAK4 deficiency is an inborn error of immunity predisposing patients to invasive pyogenic infections. Currently, there is no established simple assay that enables precise characterization of IRAK4 mutant alleles in isolation. Anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis is an autoimmune condition that is characterized by psychiatric symptoms, involuntary movement, seizures, autonomic dysfunction, and central hypoventilation. It typically occurs in adult females associated with tumors. Only a few infantile cases with anti-NMDAR encephalitis have been so far reported. We identified a 10-month-old boy with IRAK4 deficiency presenting with anti-NMDAR encephalitis and human herpes virus 6 (HHV6) reactivation. The diagnosis of IRAK4 deficiency was confirmed by the identification of compound heterozygous mutations c.29_30delAT (p.Y10Cfs*9) and c.35G>C (p.R12P) in the IRAK4 gene, low levels of IRAK4 protein expression in peripheral blood, and defective fibroblastic cell responses to TLR and IL-1 (TIR) agonist. We established a novel NF-κB reporter assay using IRAK4-null HEK293T, which enabled the precise evaluation of IRAK4 mutations. Using this system, we confirmed that both novel mutations identified in the patient are deleterious. Our study provides a new simple and reliable method to analyze IRAK4 mutant alleles. It also suggests the possible link between inborn errors of immunity and early onset anti-NMDAR encephalitis. Electronic supplementary material The online version of this article (10.1007/s10875-020-00885-5) contains supplementary material, which is available to authorized users.

Autosomal recessive (AR) complete signal transducer and activator of transcription 1 (STAT1) deficiency is an extremely rare primary immunodeficiency that causes life-threatening mycobacterial and viral infections. Only seven patients from five unrelated families with this disorder have been so far reported. All causal STAT1 mutations reported are exonic and homozygous. We studied a patient with susceptibility to mycobacteria and virus infections, resulting in identification of AR complete STAT1 deficiency due to compound heterozygous mutations, both located in introns: c.128+2 T>G and c.542-8 A>G. Both mutations were the first intronic STAT1 mutations to cause AR complete STAT1 deficiency. Targeted RNA-seq documented the impairment of STAT1 mRNA expression and contributed to the identification of the intronic mutations. The patient’s cells showed a lack of STAT1 expression and phosphorylation, and severe impairment of the cellular response to IFN-γ and IFN-α. The case reflects the importance of accurate clinical diagnosis and precise evaluation, to include intronic mutations, in the comprehensive genomic study when the patient lacks molecular pathogenesis. In conclusion, AR complete STAT1 deficiency can be caused by compound heterozygous and intronic mutations. Targeted RNA-seq-based systemic gene expression assay may help to increase diagnostic yield in inconclusive cases after comprehensive genomic study.

Background Patients with Mendelian susceptibility to mycobacterial disease (MSMD) experience recurrent and/or persistent infectious diseases associated with poorly virulent mycobacteria. Multifocal osteomyelitis is among the representative manifestations of MSMD. The frequency of multifocal osteomyelitis is especially high in patients with MSMD etiologies that impair cellular response to IFN-γ, such as IFN-γR1, IFN-γR2, or STAT1 deficiency. Objectives This study sought to characterize the mechanism underlying multifocal osteomyelitis in MSMD. Methods GM colonies prepared from bone marrow mononuclear cells from patients with autosomal dominant (AD) IFN-γR1 deficiency, AD STAT1 deficiency, or STAT1 gain of function (GOF) and from healthy controls were differentiated into osteoclasts in the presence or absence of IFN-γ. The inhibitory effect of IFN-γ on osteoclastogenesis was investigated by quantitative PCR, immunoblotting, tartrate-resistant acid phosphatase staining, and pit formation assays. Results Increased osteoclast numbers were identified by examining the histopathology of osteomyelitis in patients with AD IFN-γR1 deficiency or AD STAT1 deficiency. In the presence of receptor activator of nuclear factor kappa-B ligand and M-CSF, GM colonies from patients with AD IFN-γR1 deficiency, AD STAT1 deficiency, or STAT1 GOF differentiated into osteoclasts, similar to GM colonies from healthy volunteers. IFN-γ concentration-dependent inhibition of osteoclast formation was impaired in GM colonies from patients with AD IFN-γR1 deficiency or AD STAT1 deficiency, whereas it was enhanced in GM colonies from patients with STAT1 GOF. Conclusions Osteoclast differentiation is increased in AD IFN-γR1 deficiency and AD STAT1 deficiency due to an impaired response to IFN-γ, leading to excessive osteoclast proliferation and, by inference, increased bone resorption in infected foci, which may underlie multifocal osteomyelitis.

Interleukin-2-inducible T cell kinase (ITK) is an important mediator of T cell receptor signaling. Loss of function mutations in ITK results in hypogammaglobulinemia and CD4+ T cell loss in humans, and the patients often present with EBV-associated B cell lymphoproliferative syndrome. Itk-deficient mice show loss of T cell naivety, impaired cytolytic activity of CD8+ T cells, and defects in CD4+ T cell lineage choice decisions. In mice, Itk mutations were shown to affect Th17-Treg lineage choice in favor of the latter. In this study, we explored whether human ITK reciprocally regulates Th17-Treg balance as its murine ortholog. Whole Exome Sequencing was used to identify the mutation. ITK-deficient peripheral blood lymphocytes were characterized by FACSAria III-based flow cytometric assays with respect to proliferation, apoptosis, cytokine production, and innate lymphoid cell (ILC) frequency. Sorted T cells from healthy donors were exposed to ibrutinib, an irreversible ITK inhibitor, to assess ITK’s contribution to Th17 and Treg cell generation and functions. In this study, we report a child with a novel ITK mutation who showed impaired CD3/CD28 induced proliferation in T cells. ITK-mutant cells were more apoptotic irrespective of TCR activation. More importantly, T cells produced less Th17-associated cytokines IL-17A, IL-22, and GM-CSF. Conversely, Th1-associated IFN-γ production was increased. An irreversible inhibitor of ITK, ibrutinib, blocked ex vivo Th17 generation and IL-17A production, conversely augmented FOXP3 expression only at low doses in Treg cultures. Finally, we analyzed peripheral ILC populations and observed a relative decrease in ILC2 and ILC3 frequency in our ITK-deficient patient. To our knowledge, this is the first report showing that both genetic and chemical inhibition of ITK result in reduced Th17 generation and function in humans. We also report, for the first time, a reduction in ILC2 and ILC3 populations in an ITK-deficient human patient.

Despite the recent development of biological modifiers for inflammatory bowel diseases (IBD), there continues to be considerable interest in fermented medicines because of its negligible adverse effects. We previously showed that the synbiotic Gut Working Tablet (GWT) alleviates experimental colitis. Here we show that GWT is capable of ameliorating jejunoileal mucosal injury, which is frequently seen with IBD. We created experimental jejunoileal mucositis in rats by injection of methotrexate (MTX) which increases intestinal permeability, a hallmark finding of IBD. Administering GWT to MTX-injected rats restored intestinal integrity by reversing villi shortening, crypt loss, and goblet cell depletion in the mucosa. Also GWT reduced activities of myeloperoxidase and lipid peroxidase and increased superoxide dismutase activity, which is critical for maintaining intestinal function. We further found that GWT suppressed mRNA expression of tumor necrosis factor-α(TNF-α) and interleukin-12 (IL-12) in macrophage and reduced TNF-αmRNA expression in specimens with experimental colitis, which is in contrast to VSL#3 that enhanced TNF-αproduction. Together, the current and previous animal studies clearly demonstrate the protective role of GWT in chemically induced enterocolitis. Crohn’s disease, a well-known IBD, can affect any portion of the intestine, and these results suggest that GWT may be useful as a novel therapeutic or maintenance therapy for IBD.

Neutropenia is defined as a decrease in the number of circulating neutrophils in the peripheral blood with absolute neutrophil count less than 1000–1500/μL. Chronic neutropenia in pediatric patients is divided into three groups. Extrinsic factors, such as antibodies, some drugs, and nutritional deficiencies, lead to excessive destruction of neutrophils. Autoimmune neutropenia is a benign form of neutropenia shown in infancy to early childhood. Spontaneous recovery of neutropenia usually occurs within a few months to a few years. Acquired disorders of myeloid and stem cells present hypoplasia of myeloid cells. Congenital neutropenia is intrinsic defects in granulocytes or their progenitors and includes a heterogenous group of disorders. More than ten responsible gene mutations have been identified in congenital neutropenia. Most common congenital neutropenia is due to the gene mutation of neutrophil elastase. The hallmark of profound neutropenia is increased susceptibility to bacterial infections, cutaneous cellulitis, deep tissue abscesses, pneumonia, and septicemia. Almost patients with congenital neutropenia have been responded to administration of G-CSF. However, long-term use of G-CSF has the risk of the development of MDS/AML, suggesting the necessity of the careful follow-up. Hematopoietic stem cell transplantation should be considered for the curable treatment in severe congenital neutropenia.

Severe congenital neutropenia (SCN) is a rare heterogeneous genetic disorder characterized by recurrent bacterial infections from early infancy due to severe chronic neutropenia. Majority of SCN patients have benefitted by the treatment with granulocyte colony-stimulating factor (G-CSF). However, patients on long-term G-CSF therapy have a relative risk of developing myelodysplastic syndrome/acute myeloid leukemia. The only curable treatment for SCN patients is hematopoietic stem cell transplantation (HSCT). Recently, HSCTs with reduced intensity conditioning regimens have been applied to the treatment for SCN patients prior to malignant transformation. However, the optimal conditioning of HSCT for SCN patients has not been established. In this study, we conducted bone marrow cell transplantations (BMT) in 16 patients with SCN using an immunomyelosuppressive conditioning regimen to minimize early and late transplant-related morbidity in Hiroshima University Hospital. A total of 17 BMT procedures were performed in 16 patients with SCN from 2008 to 2019. Five of 16 patients had experienced the engraftment failure of initial HSCT and 4 of them were referred to our hospital for re-transplantation. Fifteen of 16 patients had a heterozygous mutation in the ELANE gene. Bone marrow cells (BM) were obtained from 6 HLA-matched related, 3 HLA-matched unrelated, and 8 HLA-mismatched unrelated (7/8 antigens) donors, respectively. Conditioning regimen consisted of fludarabine, cyclophosphamide, melphalan, total body irradiation (3.6 Gy) with or without antithymocyte globulin. Short-term methotrexate and tacrolimus were administered for the prophylaxis of graft-versus-host disease (GVHD). Engraftment of neutrophils was observed within post-transplant 24 days in all patients. Two patients developed graft failure on day 40 and day 90, respectively, after the temporal engraftment. However, both patients were rescued by second BMT from different HLA-matched unrelated donors receiving the same conditioning regimen. Four patients who received BMT from HLA-matched related donors developed stable mixed chimerism without neutropenia in peripheral blood for 3 to 10 years. Although one patient who received donor lymphocyte infusion due to mixed chimerism developed grade II acute GVHD and limited chronic GVHD, the others did not develop severe GVHD. All patients are alive for 6 months to 11 years after BMT with no signs of severe infections or transplantation-related morbidity. Similar conditioning regimen has been applied to BMT for 35 patients with chronic granulomatous disease (CGD) in our hospital. In that study 4 male adulthood patients with CGD already fathered each child by their wives through spontaneous pregnancy, implying the successful preservation of patients' fertility. Collectively, our results demonstrate that BMT with a sufficient immunosuppressive conditioning regimen may be a feasible and effective treatment for SCN patients, irrespective of initial engraftment failure. The excellent results in our cohort suggest that indications for proceeding to HSCT could be extended to patients without malignant transformation.The further analyses of accumulated cases are necessary to assess the efficacy, safety, and less late adverse effects related to HSCT including fertility. Disclosures No relevant conflicts of interest to declare.

Prophylactic administration of factor VIII products is necessary to prevent bleeding and preserve normal musculoskeletal function in children with severe hemophilia A (HA). Recently, extended half-life recombinant factor VIII (EHL-rFVIII) products have been utilized in HA patients. Therefore, the pharmacokinetics (PK) of EHL-rFVIII in individuals is needed to determine the appropriate administration for personalized prophylaxis according to the age, bleeding phenotype, the presence of arthropathy, and physical activity. The myPKFiTR ver 3.0 has been developed as the device to estimate the personalized dosing with a 2 sample PK based on the population PK (Bayesian) tool. In this study we report our single-center experience to study PK profiles and to individualize dose and dosing interval based on myPKFiTR. Eight patients with severe HA aged from 10 to 20 years were enrolled in this study for personalized prophylaxis. The half-life of EHL-rFVIII was approximately 15 to 18 hours in all patients studied. The clearance of FVIII was inversely correlated with the half-life of EHL-rFVIII. The EHL-rFVIII products have been basically administered twice a week. The trough levels of FVIII were more than 3% in all patients. The prophylactic regimen in adolescents was individually determined according to the personal simulation of PK study and to patients' life style and physical activities. Adolescent patients actively participated in sports, such as track and field, basketball, and football after school. The FVIII level after school was easily estimated by the use of myPKFiTR according to the dose and duration of replacement. The doses of EHL-rFVIII products were individually determined to have more than 10 to 30% of FVIII level at the time of sports activity. During personalized prophylaxis (6 to 18 months), all of patients studied have been no bleeds during sports as well as no spontaneous bleeds. Additionally, myPKFiTR has the capability of presenting the real-time FVIII level on the screen of smartphone after the replacement of EHL-rFVIII based on the individual PK. Some patients have referred their own FVIII level before the beginning of sports through their smartphone and then have decided the necessity of the replacement. These experiences suggest the enhancement of treatment concordance in the supportive relationship between patients and medical staff. Thus, the use of myPKFiTR may be essential for the optimization of prophylactic administration of EHL-rFVIII and for the medical adherence and concordance in each individual. Disclosures No relevant conflicts of interest to declare.

CMCD is a rare congenital disorder characterized by persistent or recurrent skin, nail, and mucosal membrane infections caused by Candida albicans. Heterozygous GOF STAT1 mutations have been shown to confer AD CMCD as a result of impaired dephosphorylation of STAT1. We aimed to identify and characterize STAT1 mutations in CMCD patients and to develop a simple diagnostic assay of CMCD. Genetic analysis of STAT1 was performed in patients and their relatives. The mutations identified were characterized by immunoblot and reporter assay using transient gene expression experiments. Patients' leukocytes are investigated by flow cytometry and immunoblot. Six GOF mutations were identified, three of which are reported for the first time, that affect the CCD and DBD of STAT1 in two sporadic and four multiplex cases in 10 CMCD patients from Japan. Two of the 10 patients presented with clinical symptoms atypical to CMCD, including other fungal and viral infections, and three patients developed bronchiectasis. Immunoblot analyses of patients' leukocytes showed abnormally high levels of pSTAT1 following IFN-γ stimulation. Based on this finding, we performed a flow cytometry-based functional analysis of STAT1 GOF alleles using IFN-γ stimulation and the tyrosine kinase inhibitor, staurosporine. The higher levels of pSTAT1 observed in primary CD14+ cells from patients compared with control cells persisted and were amplified by the presence of staurosporine. We developed a flow cytometry-based STAT1 functional screening method that would greatly facilitate the diagnosis of CMCD patients with GOF STAT1 mutations.

A 2-year-old boy presented with a 21-hydroxylase deficiency, associated with advanced-stage neuroblastoma primarily occurring in the left adrenal gland. He required intensive chemotherapy with polypharmacy, followed by cord blood stem cell transplantation to treat the neuroblastoma. The precise adjustment of cortisol levels was crucial in this patient to prevent adrenal crisis. We administered hydrocortisone by continuous infusion while monitoring blood cortisol levels. As there are no published reports on the target cortisol levels for children, we used two control infants with advanced-stage neuroblastoma, also undergoing chemotherapy and cord blood stem cell transplantation, to guide the continuous hydrocortisone therapy. The daily dose of hydrocortisone during chemotherapy required about threefold the normal treatment to avoid adrenal insufficiency. Continuous hydrocortisone therapy is feasible for preventing adrenal crisis and this report may provide an effective management for hydrocortisone replacement in 21-hydroxylase-deficient patients undergoing chemotherapy and hematopoietic stem cell transplantation.

X-linked ectodermal dysplasia with immunodeficiency (XL-ED-ID) is caused by hypomorphic mutations in NEMO, which encodes nuclear factor-kappaB (NF-κB) essential modulator. We identified a novel mutation, 769-1 G>C, at the splicing acceptor site of exon 7 in NEMO in a Japanese patient with XL-ED-ID. Although various abnormally spliced NEMO messenger RNAs (mRNAs) were observed, a small amount of wild-type (WT) mRNA was also identified. Decreased NEMO protein expression was detected in various lineages of leukocytes. Although one abnormally spliced NEMO protein showed residual NF-κB transcription activity, it did not seem to exert a dominant-negative effect against WT-NEMO activity. CD4(+) T cell proliferation was impaired in response to measles and mumps, but not rubella. These results were consistent with the clinical and laboratory findings of the patient, suggesting the functional importance of NEMO against specific viral infections. The 769-1 G>C mutation is responsible for decreased WT-NEMO protein expression, resulting in the development of XL-ED-ID.

The regular prophylactic replacement therapy with clotting factors has significantly reduced the arthropathy in patients with hemophilia. Early detection of the signs of joint damage is important to prevent the progressive joint damage. For the early detection, we have introduced the regular ultrasound (US) joint examination in patients with hemophilia from 2014 based on the hemophilia early arthropathy detection with ultrasound (Head-US) scanning protocol. US examination has been performed at the time of annual comprehensive clinic in Hiroshima University Hemophilia Center. Forty-five patients aged 1 to 33 years were enrolled and more than 280 joints were evaluated in this study. US joint examination was performed within 30 minutes without the sedation in all aged children. Abnormal findings, such as cartilage changes, subchondral bone erosion, synovial hypertrophy and/or joint effusion were observed in 8 out of 45 patients. The replacement therapy with clotting factors of all 8 patients aged 13 to 33 years was an insufficient prophylaxis during early childhood. The residual 37 patients aged less than 15 years have relatively followed the prophylactic treatment. The prophylactic regimen has been individually determined based on the trough levels of factor IX concentration in plasma for hemophilia B and pharmacokinetics of factor VIII using MyPKFitR for hemophilia A patients, respectively. The result suggests the importance of the careful prophylactic replacement therapy considering the factor concentration in plasma. Four patients aged 4, 5, 18, and 19 years, respectively, suffered from painful and/or swollen joint episodes from 2014 to 2018. Urgent US examination without the use of any sedatives revealed ankle, knee, or hip joint bleeding in each case. According to the replacement therapy for breakthrough bleeding, hemoarthropathy was accessed by consecutive examination of US. These results suggest that US joint examination could be necessary for the early detection of joint damage and for appropriate treatment of hemoarthropathy. Collectively, utilization of US examination would be essential for the management through the life in patients with hemophilia. Disclosures No relevant conflicts of interest to declare.

CD4+ 25+ regulatory T cells (Tregs) play a role in controlling the development and progression of autoimmunity. The transcription factors Foxp3 and NFATC2 (NFAT1) play key roles in regulating the development and function of Tregs. The present study examined the involvement of Tregs in the pathophysiology of autoimmune neutropenia in children. Tregs were analysed by flow cytometry, based on the expressions of CD4, CD25, and intracellular Foxp3. The expressions of FOXP3 and NFATC2 mRNA in the CD4+ 25+ cells were determined by quantitative real-time polymerase chain reaction. The percentage of CD4+ 25(high) Tregs in patients with autoimmune neutropenia was significantly lower than that in age-matched healthy subjects. The intracellular expression of Foxp3 of CD4+ 25+ cells in patients similarly decreased in comparison to that in healthy subjects. The expression of FOXP3 and NFATC2 mRNA of CD4+ 25+ cells in patients also significantly decreased in comparison to that in healthy subjects. These results suggest that the deficiency of Tregs might thus play an important role in the immunopathophysiology of autoimmune neutropenia in children.

Aims: Metyrapone causes a decrease in the serum cortisol level without affecting ACTH production in ectopic tumors. We report a case who presented with Cushing’s syndrome due to an ectopic ACTH-producing thymic carcinoid. In the present case, it was demonstrated that metyrapone administration resulted in a significant decrease in the plasma ACTH and serum cortisol levels. We hypothesized that the steroid hormone may promote proopiomelanocortin (POMC) gene expression in the carcinoid cells. Methods: An 11-year-old boy presented with Cushing’s syndrome. Prior to the detection of a thymic tumor, metyrapone was administered to ameliorate the symptoms of Cushing’s syndrome. Interestingly, plasma ACTH as well as serum cortisol levels immediately decreased after metyrapone administration. The levels of cortisol and ACTH were observed to be normal after complete surgical resection of the tumor. Biological characterization of the tumor cells was by in vitro analysis. Results: Thein vitro culture of the tumor cells showed an increased expression of POMC in the presence of cortisol. A CpG methylation assay showed that the demethylation of the POMC promoter was induced by a steroid hormone. Conclusion: These findings suggest that the ectopic ACTH-producing tumor may partly be regulated by the elevated levels of cortisol.

The production of factor VIII (FVIII) inhibitory antibodies is a serious problem in patients with hemophilia A. Immune tolerance induction (ITI) is the only strategy proven to eradicate persistent inhibitors and has been shown to be successful in 70 % of patients with hemophilia A. However, a minority of hemophilia patients present life-long inhibitors. To eliminate such inhibitors, we designed an intravenous immunoglobulin (IVIG) strategy in combination with high dose recombinant FVIII for ITI in hemophilia A children with inhibitors. Four previously untreated patients produced inhibitors within 16 exposures to FVIII. The peak inhibitor titers in these patients ranged from 3 to 14 BU/mL. The patients received ITI combined with IVIG within 1.5 months after the inhibitors were detected. All patients showed a negative titer for inhibitors by 28 days, with no anamnestic responses. The recovery of FVIII in the plasma concentration was normalized within three months after initiation of ITI. An additional course of IVIG administration led to induction of complete tolerance by 20 months after initiation of ITI therapy in all patients. ITI treatment with high-dose FVIII combined with IVIG may be effective for the early elimination of inhibitors.

Primary adrenal lymphoma is uncommon, and the majority cases of this disorder are found in elderly individuals. We describe a 17-year-old boy with persistent fever, hemophagocytic lymphohistiocytosis, and a bilateral tumor of the adrenal glands. The disease was progressive and did not respond to treatment such as immunosuppression therapy or plasma exchange. Postmortem analysis revealed nasal-type natural killer cell lymphoma in association with Epstein-Barr virus infection. To our knowledge, this case is the first of primary adrenal lymphoma with the natural killer cell phenotype to be reported. The characterization of this unusual case should be included in the differential diagnosis of adrenal gland tumors.

Severe congenital neutropenia (SCN) is a rare heterogeneous genetic disorder characterized by severe chronic neutropenia, with absolute neutrophil counts below 0.5×109/L, and by recurrent bacterial infections from early infancy. Granulocyte colony-stimulating factor (G-CSF) is widely used for the treatment of neutropenia in patients with SCN. However, the long-term G-CSF therapy has a relative risk of developing myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). The only curative treatment available for SCN patients is hematopoietic stem cell transplantation (HSCT). Recently, HSCTs with reduced intensity conditioning (RIC) regimens have been applied to the treatment of SCN patients without malignant transformation who have become G-CSF refractory. However, the optimal conditions of HSCT for SCN patients have not been established. In this study, we conducted bone marrow cell transplantations (BMT) in ten patients with SCN using an immunosuppressive conditioning regimen to minimize early and late transplant-related morbidity in Hiroshima University Hospital. Ten patients with a total of 11 HSCT procedures in our institution (performed from 2007 to 2015) were enrolled in this study. Four of the ten patients had experienced engraftment failure of the initial HSCT and three of them were referred to our hospital for re-transplantation. Heterozygous mutation inthe ELANE gene was identified in nine of ten patients. These nine patients received BMT less than 10 years of age. All ten patients had recurrently experienced moderate to severe bacterial or fungal infection before HSCT and received temporal or regular administration of G-CSF. Bone marrow cells (BM) were obtained from five HLA-matched related (MRD), three HLA-matched unrelated (MUD), and three HLA-mismatched unrelated (7/8) donors (MMUD), respectively. The conditioning regimen basically consisted of fludarabine (100 to 125 mg/m2), cyclophosphamide (100 to 150 mg/kg), melphalan (70 to 90 mg/m2), total body irradiation (3 to 3.6 Gy), and/or anti-thymocyte globulin (10 to 12 mg/kg). Short-term methotrexate and tacrolimus were administered for the prophylaxis of graft-versus-host disease (GVHD). Engraftment of neutrophils was successfully observed within 24 days of post-transplantation in all patients. All patients achieved complete chimerism at the time of engraftment. Two patients who underwent BMT from MRD and one patient who underwent BMT from MUD showed the gradual decrease of donor-derived cells. Donor lymphocyte infusion treatment successfully achieved the complete chimerism or stable mixed chimerism in these 3 patients. Although 3 patients experienced the acute GVHD (Grade I-II), the addition of glucocorticoids to tacrolimus prevented the extension of acute GVHD. Only one patient developed mild chronic GVHD presenting limited type of skin involvement. All patients are alive for 9 months to 9 years after HSCT with no signs of severe infections or transplantation-related morbidity. Our results demonstrate that BMT together with a sufficient immunosuppressive conditioning regimen may be a feasible and effective treatment for SCN patients, irrespective of initial engraftment failure. Although our results through the small number of cohort is limited to conclude, the BMT with the optimal donors may lead to the increased opportunity for lower risk of SCN patients especially at younger age as a curative treatment. The further analyses of accumulated cases are necessary to assess the efficacy, safety, and less late adverse effects related to HSCT including fertility. Disclosures No relevant conflicts of interest to declare.

The t(7;11)(p15;p15) translocation has been reported as a rare and recurrent chromosomal abnormality in acute myeloid leukemia (AML) patients. The NUP98-HOXA9 fusion gene with t(7;11)(p15;p15) was identified and revealed to be essential for leukemogenesis and myeloproliferative disease. To date, t(7;11)(p15;p15) with NUP98-HOXA11 fusion has been reported only in one case of ph-negative chronic myeloid leukemia (CML). Here, we report a case of a 3-year-old girl with juvenile myelomonocytic leukemia (JMML) carrying t(7;11)(p15;p15) abnormality with NUP98-HOXA11 fusion. AML chemotherapy followed by bone marrow transplantation (BMT) was found to be effective in treating this disorder, and she remains in complete remission for 3 years after BMT. We suggest the possibility that AML chemotherapy might be effective for treating JMML with t(7;11)(p15;p15) abnormality and NUP98-HOXA11 fusion.

The derivation of induced pluripotent stem (iPS) cells from individuals of genetic disorders offers new opportunities for basic research into these diseases and the development of therapeutic compounds. Severe congenital neutropenia (SCN) is a serious disorder characterized by severe neutropenia at birth. SCN is associated with heterozygous mutations in the neutrophil elastase [elastase, neutrophil-expressed (ELANE)] gene, but the mechanisms that disrupt neutrophil development have not yet been clarified because of the current lack of an appropriate disease model. Here, we generated iPS cells from an individual with SCN (SCN-iPS cells). Granulopoiesis from SCN-iPS cells revealed neutrophil maturation arrest and little sensitivity to granulocyte-colony stimulating factor, reflecting a disease status of SCN. Molecular analysis of the granulopoiesis from the SCN-iPS cells vs. control iPS cells showed reduced expression of genes related to the wingless-type mmtv integration site family, member 3a (Wnt3a)/β-catenin pathway [e.g., lymphoid enhancer-binding factor 1], whereas Wnt3a administration induced elevation lymphoid enhancer-binding factor 1-expression and the maturation of SCN-iPS cell-derived neutrophils. These results indicate that SCN-iPS cells provide a useful disease model for SCN, and the activation of the Wnt3a/β-catenin pathway may offer a novel therapy for SCN with ELANE mutation.

The ELANE is known as the responsible gene for both cyclic neutropenia (CyN) and severe congenital neutropenia (SCN). However, relations between mutations in the ELANE gene and abnormal myelopoiesis in the different phenotype of these diseases still remain unclear. It has been reported that induced pluripotent stem cell (iPSC) from an individual patient with SCN (SCN-iPSC) demonstrated maturation arrest of myeloid progenitor cells and poor response to granulocyte-colony stimulating factor as similarly observed in patient's bone marrow cells. Thus, the study on myelopoiesis using disease specific iPSC seems to provide disease pathogenesis as a novel in vitro experimental model. In this study, we established iPSC line from an individual patient with CyN (CyN-iPSC) with heterozygous mutation in ELANE gene (Exon5, R191Q point mutation). Then we compared myelopoiesis among healthy Control-iPS (253G1), SCN-iPS (Exon5, C194X point mutation) , and CyN-iPSC. Undifferentiated colonies derived from CyN-iPSC were staind with pluripotency markers (OCT3/4 and NANOG). CyN-iPSC retained a normal karyotype and ELANE locus mutation of the original samples. In vitro myelopoiesis was examined by using a serum- and feeder-free monolayer hematopoietic culture system. iPSC colonies were cultured on growth factor-reduced Matrigel-coated cell culture dishes in modified Tenneille Serum Replacer 1 (mTeSR™1) medium (StemCell Technologies, Inc.), containing BSA, rh bFGF, rh TGFβ, Lithium Chloride, Pipecolic acid, GABA. Medium was replaced every four days. Then medium was changed to StemPro®-34 SMF Complete Medium plus nutrient supplement (Life technologies Corp.). The iPSC were cultured with BMP4 (80 ng/mL) for four days, and then replaced with VEGF165 (80 ng/mL), bFGF (25.7 ng/mL), and SCF (100 ng/mL) on Day 4. On Day 6, cytokines were replaced with a combination of SCF (50 ng/mL), IL-3 (50 ng/mL), and G-CSF (50 ng/mL). Medium was replaced every 3 - 4 days. No significant difference in the ratio of proliferating CD33+ cells were noted between CyN-iPSCs and Control-iPSCs. CyN-iPSCs showed less capability in the proliferation and maturation for CD15+ cells on days 20 to 40 than Control-iPSCs. The decreased number of CD15+ cells derived from CyN-iPSc implies the defect in mature neutrophil survival. In contrast, CD15+ / CD33+ cells derived from SCN-iPSCs were hardly observed in this culture condition, suggesting the defects of proliferation and maturation in SCN-iPSCs. We next examined the colony formation of CD34+ cells derived from CyN-iPSCs, Control-iPSCs, and SCN-iPSCs. CD34+ cells were obtainded at the day 12 of primary culture of iPSCs and purified by cell sorting using FACS-Aria®. No significant differences in the number of G-colony and GM-colony between CD34+ cells from CyN-iPSCs and Control-iPSCs. In contrast, CD34+ cells from SCN-iPSCs gave rise to the significantly decreased number of G-colony and GM-colony. The observations of myeloid proliferation/maturation and colony formation of CD34+ cells were almost compatible with those obtained from bone marrow cells in patients with SCN and CyN. Furthermore, neutrophils differentiated from CyN-iPSCs showed the excessive cell death, whereas SCN-iPSCs presented the defective myelopoiesis. These results suggest that the analyses using CyN-iPSCs and SCN-iPSCs may be useful tool for investigating the relation of gene mutation and pathophysiology in both diseases. Disclosures No relevant conflicts of interest to declare.

Mendelian susceptibility to mycobacterial diseases (MSMD) is a rare congenital phagocytic disorder characterized by susceptibility to clinical diseases caused by weakly virulent mycobacteria, such as Mycobacterium bovis Bacille Calmette-Guérin (BCG) and non-tuberculous mycobacteria. In individuals with MSMD, mutations have been found in several genes that all encode proteins in the type 1 cytokine pathway that is essential for killing or controlling mycobacteria. The various gene mutations, such as IL12B, IL12RB1, IFNGR1 IFNGR2, and STAT1, have been identified for the responsible genes in patients with MSMD. STAT1 mediates most of the biological functions of both type I IFN (IFN-α and β) and type II IFN (IFN-γ ) and is important in immune response to viruses and other intracellular pathogens. The loss-of-function formed IFN-γ receptor 1 and STAT1 deficiency presents MSMD. Osteomyelitis is a bone infection by bacteria and other microorganism, and is one of clinical features presented in more than 80% of patients with MSMD. The histopathology of biopsied specimen shows noncaseating granuloma with abundant number of osteoclasts. Osteoclasts, which play a critical role in pathological bone loss, are multinucleated cells of hematopoietic origin that are formed from monocyte/macrophage lineage precursor cells. Osteoclastogenesis is an intricate process that involves many stages, such as differentiation to tartrate-resistant acid phosphatase (TRAP)-positive cells, fusion to form multinucleated cells, activation to resorb bone, and spontaneous apoptosis. IFN-γ is known to strongly suppress osteoclast formation in mice. However, the mechanisms underlying the effects of IFN-γ osteoclast formation and function still remain unclear in human. In this study, we examined the effect of IFN-γ on the formation and function of osteoclasts derived from patients with AD IFNGR1 or STAT1 deficiency. Osteoclast precursor cells derived from the bone marrow mononuclear cells were enriched by incubation with SCF, IL-3 and GM-CSF followed by the culture of M-CSF and RANKL-containing media with or without IFN-γ. In the absence of IFN-γ, no significant difference in in vitro osteoclastogenesis observed by TRAP staining was noted between patients and healthy subjects. The addition of IFN-γ in the culture dose-dependently suppressed osteoclastogenesis. Ten IU/ml of IFN-γ sufficiently inhibited osteoclast formation in normal subjects, while more than 50 IU/ml of IFN-γ was required to suppress osteoclastogenesis in patients. Osteoclast formation of patients with IFN-γ-STAT1 signaling defect was less susceptible to IFN-γ than those of healthy subjects, suggesting the association with the abundant osteoclast involvement in patients' bony inflammation. Next, bone resorption assay, pit-formation was microscopically measured to evaluate the osteoclast activity. Bone resorption by osteoclasts was inhibited by the addition of IFN-γ. However, the suppressive activity of IFN-γ for pit formation was significantly lower in patients with IFN-γ-STAT1 signaling defect than that in healthy subjects, resulting in the enhanced activity of patients' osteoclasts. The down-regulation of nuclear factor of activated T cells (NFATc1) mRNA expression induced by IFN-γ is essential for the osteoclast formation and its function. Osteoclasts derived from patients showed the significant impairment of down-regulation of NFATc1 mRNA expression in the stimulation with IFN-γ. These results suggest that the qualitative and quantitative augmentations of osteoclasts may be associated with the development of chronic and multifocal osteomyelitis in patients with MSMD through the impairment of IFN-γ-STAT1 signaling. Disclosures No relevant conflicts of interest to declare.

We report a patient with neonatal Coxsackie B2 meningo-encephalitis in whom magnetic resonance imaging (MRI) could be performed serially from the early stage of the disease. The patient was a 12-day-old girl born at a gestational age of 37 weeks. She was hospitalized due to poor suckling. During her hospital stay, she developed clonic seizures in the right upper and lower limbs. Coxsackie B2 virus was detected, and a diagnosis of viral encephalitis was made. The first diffusion-weighted images (DWI) showed an abnormal high-intensity area restricted to the corpus callosum and posterior limb of the internal capsule 8 h after onset of seizures. Repeated MRI revealed damage to the white matter, which finally changed into diffuse excessive necrosis followed by cystic leukomalacia. Early DWI is valuable for the early detection and diagnosis of neonatal meningo-encephalitis. This is the first report of the detailed neuroradiological course of neonatal Coxsackie B2 meningo-encephalitis.

Early thrombocytopenia is a common hematological abnormality in sick neonates. Here, we examined the relationship between early thrombocytopenia in neonates and parameters associated with thrombopoiesis to identify predictive factors at birth. Two hundred and forty-four neonates admitted to the neonatal intensive care unit were divided into thrombocytopenic (n = 55, 23%) and non-thrombocytopenic (n = 189, 77%) groups based on platelet counts, which were monitored within 72 h of birth. Immature platelet fraction (IPF) and platelet count at birth were determined simultaneously soon after phlebotomy with an automated hematology analyzer. Megakaryocytes and their precursors positive for CD41 in peripheral blood were examined at birth by flow cytometry. The thrombocytopenic group showed significantly higher IPF percentage and lower percentage of CD41+ mononuclear cells (MNCs) than did the non-thrombocytopenic group (P0.01). Moreover, the percentage of CD41+ MNCs significantly differentiated neonates with platelet counts150 x 10(3)/microL at birth and nadir platelet count150 x 10(3)/microL over the clinical course from neonates without thrombocytopenia. These observations suggest that the percentage of CD41+ MNCs at birth and IPF percentage are useful predictors of early thrombocytopenia in the majority of sick neonates.

Chronic mucocutaneous candidiasis (CMCD) is a rare congenital disorder characterized by persistent or recurrent skin, nails and mucosal membranes infections caused by Candida albicans. Several studies suggest that impairment of development in Th17 linage and/or IL-17 signaling could be responsible for development of CMCD and seven responsible genes, CARD9, STAT3, IL12B, IL12RB1, IL17RA, IL17F, and AIRE have been identified. Recently, heterozygous mutations in coiled-coil domain (CCD) and DNA-binding domain (DBD) of STAT1 are identified in approximately 40% of patients with CMCD. Signal transducer and activation of transcription 1 (STAT1) is a DNA-binding factor which regulates specific gene transcription. STAT1 mutations identified in patients with CMCD are gain-of-function (GOF), gain-of-gamma-activated factor (GAF) DNA binding and gain-of-gamma-activated sequence (GAS) transcription activity in response to IFN-γ, IFN-α and IL-27. Based on the results of transient gene experiments, impairment in dephosphorylation of STAT1 has been considered to be a molecular pathogenesis underlying the increased phosphorylation of STAT1 at Tyr701 (pSTAT1). In this paper, we aimed to identify and characterize STAT1 mutations in CMCD patients, and to develop a simple diagnostic assay of CMCD. Five sporadic and five familial cases of CMCD, from a total of 15 patients from 10 kindreds in Japan, are investigated. Six heterozygous missense mutations, including three novel mutations, in CCD and DBD of STAT1 were identified in two sporadic and four familial cases in 10 patients with CMCD. Thus, STAT1 mutations were commonly identified in Japanese patients with CMCD. We investigated functional significance of these mutations by transient gene expression experiments using U3C STAT1 null fibrosarcoma cells. Similar to the previous studies, all mutant proteins showed increased pSTAT1 in response to IFN-α and IFN-γ. Increased GAF-DNA binding and GAS transcription activity were observed in mutant expressed cells. Thus, these mutations are GOF mutations against GAF mediated transcription activity. Next, we studied dephosphorylation of STAT1 using peripheral blood mononuclear cell (PBMCs) from the patients. As the STAT1 GOF mutations are thought to be associated with impairment in dephosphorylation of STAT1, we used staurosporine, the tyrosine kinase inhibitor which inhibits JAK-STAT signaling upstream of STAT1, to clarify the difference between STAT1 WT alleles and STAT1 GOF alleles. If the STAT1 dephosphorylation normally occurs in the nucleus, pSTAT1 should promptly decrease following staurosporine treatment. PBMCs from fourteen healthy individuals and ten patients with CMCD carrying GOF STAT1 mutations were incubated with staurosporine followed by IFN-γ stimulation and analyzed by flow cytometry. Some overlap was observed, but MFI values for pSTAT1 in response to IFN-γ were significantly higher in CD14+ cells from the patients than in those from the controls PBMCs from the patients. This excess phosphorylation persisted after 15 minutes of treatment with staurosporine. Moreover, in these conditions, there was no overlap in MFI of pSTAT1 between the patients and healthy controls. These findings suggest that excess pSTAT1 is caused by an impairment of dephosphorylation and this flow cytometry-based technique is likely to be useful for the rapid assessment of STAT1 function in CMCD patients. Disclosures No relevant conflicts of interest to declare.

Severe congenital neutropenia (SCN) is characterized by severe chronic neutropenia with a absolute neutrophil count of less than 0.5×109/L, maturation arrest of myeloid precursors at the promyelocyts/myelocyte stage, and the recurrent bacterial infections from early infancy. The induction of G-CSF therapy dramatically improved the prognosis of most patients with SCN. Analysis on patients on the Severe Chronic Neutropenia International Registry has demonstrated that the patients with SCN on long-term G-CSF treatment are at a risk of developing myelodysplastic syndrome or acute myeloid leukemia (MDS/AML). Recently, hematopoietic stem cell transplantation (HSCT) has been applied for a curative treatment in SCN patients without malignant transformation. However, the optimal conditions of HSCT for SCN have not been established. The failure of engraftment has been reported in some of SCN patients receiving HSCT with reduced intensity conditioning (RIC) or using cord blood as a donor source. Here, we tried to undergo retransplantation of bone marrow cells for 4 SCN patients with ELANE mutation who referred to our hospital because of the engraftment failure of initial HSCT. The summary of initial HSCT is shown in Table. The source of stem cells and the conditioning regimen were heterogenous in four patients. In this study we used bone marrow cells as stem cell source for all patients to transfuse the sufficiently total nucleated cells. Bone marrow cells were obtained from an HLA-matched related, an HLA-matched unrelated, and two HLA-mismatched unrelated (7/8) donors, respectively. Conditioning regimen consisted of fludarabine (125 mg/m2), cyclophosphamide (140 mg/kg), anti-thymocyte globulin (ATG, 10-12 mg/kg), melpharan (90 mg/m2), and TLI (3.6 Gy). Short-term methotrexate and tacrolimus were administered for the prophylaxis of graft versus host disease (GVHD). Engraftment of neutrophils was observed within post-transplant 21 days in all patients. Three of 4 patients have achieved complete engraftment without any signs of GVHD during their course. One patient required additional donor-lymphocyte infusion to sustain the stable engraftment. All patients are alive for 1 to 5 years after HSCT with no signs of infections or transplantation-related morbidity. Furthermore, we observed successful BMT using similar conditioning regimen in other 4 SCN patients without prior transplantation. In our cohort, sufficient administration of ATG is thought to be critical for preventing both engraftment failure and acute GVHD. These results suggest that bone marrow transplantation using fludarabine-based conditioning regimen with a sufficient dose of ATG may be a feasible and effective treatment for SCN patients irrespective of initial engraftment failure. Indications for proceeding to HSCT have been under discussion in patients with SCN. Accumulation of cases and further analysis are necessary to assess the efficacy and safety of this regimen including late complication related to HSCT. Table. HSCT characterictecs at the initial transplantation Case 1 2 3 4 Etiology ELANE G185R ELANE V72M ELANE 4501-4503 del ELANE V72M Age at the time of HSCT 10 months 2 years 3 years 9 months 15 months Stem cell source cord blood bone marrow cord blood bone marrow HLA disparity mismatched unrelated donor (5/6) matched unrelated donor (6/6) matched related donor (6/6) mismatched unrelated donor (6/8) Transfused NCC (108/kg) 0.7 1.9 0.49 1 Transfused CD34 cells (106/kg) 0.699 1.9 0.07 1.07 Conditioning regimen BU 16, CY 120, Flu 120 CY 140, Flu 125, L-PAM 90 TBI 3, ATG 2.5 CY 100, Flu 120 TBI 3, ALG 40 Flu 125, L-PAM 140 TBI 2, ATG 5 Posttransplant day of rejection 72 days 55 days after 4 times of DLI 14 days one year after mixed chimerism DLI (times) 0 4 3 0 Busulfan (BU, mg/kg), Cyclophophamide (CY, mg/kg), Fludarabine (Flu, mg/m2), Melpharan (L-PAM, mg/m2), Total body irradiation (TBI, Gy), Anti-thymoglobulin (ATG, mg/kg), Anti-lymphoglobulin (ALG, mg/kg), donor lymphocyte infusion (DLI) Disclosures No relevant conflicts of interest to declare.

Abstract 4831 Chronic mucocutaneous candidiasis (CMCD) is a rare congenital disorder characterized by persistent or recurrent skin, nails and mucosal membranes infections caused by Candida albicans. Several studies suggest that impairment of development in Th17 lineage and/or IL-17 signaling could be responsible for development of CMCD and seven responsible genes, CARD9, STAT3, IL12B, IL12RB1, IL17RA, IL17F, and AIRE have been identified. Recently, heterozygous mutations in coiled-coil domain (CCD) and DNA-binding domain (DBD) of STAT1 are identified in approximately 40% of patients with CMCD. Signal transducer and activation of transcription 1 (STAT1) is a DNA-binding factor which regulates specific gene transcription. IFN-γ stimulation results in phosphorylation of STAT1 at Tyr701 (pSTAT1) to induce the homodimerization, a gamma-activated factor (GAF), through the conformational change and the nuclear import. The GAF binds to the gamma-activated sequence (GAS) to induce the transcriptional activities. STAT1 mutations identified in patients with CMCD are gain-of-phosphorylation, gain-of-GAF DNA binding and gain-of-GAS transcription activity in response to IFN-γ, IFN-α and IL-27. Based on the results of transient gene experiments, impairment in dephosphorylation of STAT1 has been considered to be a molecular pathogenesis underlying the increased pSTAT1. Here we report six heterozygous missense mutations in CCD and DBD of STAT1, including two novel heterozygous STAT1 mutation, 1060C>A (L354M) and 986C>T (P329L), in two sporadic and four multiplex cases with CMCD in Japan. We investigated functional significance of these mutations by transient gene expression experiments using U3C STAT1 null fibrosarcoma cells. Similar to the previous studies, all mutant proteins showed increased pSTAT1 in response to IFN-α and IFN-γ. Increased GAF-DNA binding and GAS transcription activity were observed in mutant expressed cells. Thus, these mutations are gain-of-function mutations against GAF mediated transcription activity. We then studied dephosphorylation of STAT1 using peripheral blood mononuclear cells (PBMCs) from the patients. The PBMCs were incubated with Staurosporine, tyrosine kinase inhibitor, followed by IFN-γ stimulation and analyzed by flowcytometry and immunoblot. In both experiments, PBMCs from the patients showed increased pSTAT1 after IFN-γ stimulation. Furthermore, we observed persistent pSTAT1 after Staurosporine treatment. These findings suggest that excess pSTAT1 is caused by an impairment of dephosphorylation. The flowcytometry analysis showed no overlap in mean flow intensity of pSTAT1 between 6 patients and 11 healthy controls after Staurosporine treatment. Therefore, this method can be useful for rapid test of STAT1 function in patients with CMCD. Disclosures: No relevant conflicts of interest to declare.

Abstract 730 Severe congenital neutropenia (SCN) is a rare disorder characterized by severe neutropenia present at birth, an arrest of neutrophilic differentiation at the promyelocyte or myelocyte stage, and a propensity to develop acute myeloid leukemia and myelodysplasia. Mutations of the ELA2 gene encoding neutrophil elastase have been identified in majority cases of SCN. To date, more than 50 distinct mutations of the ELA2 gene have been reported in patients with SCN. However, the mechanisms by which these mutations disrupt neutrophil development are unclear. To understand the roles of ELA2 mutation in SCN, we established three human induced pluripotent stem (iPS) cell clones (SPN0101, SPN0102 and SPN0103) from bone marrow stromal cells of a patient having heterozygous mutation in ELA2 gene by transfection with retrovirus vector whichexpressed human OCT3/4, SOX2, KLF4, and c-MYC (SCN-iPS cells). The silencing of exogenous genes and the capability to differentiate into three germ lines by teratoma formation were confirmed in the three SCN-iPS cell clones. We first examined the hematopoietic differentiation of SCN-iPS and control iPS cells which were generated from healthy donors by the same method to SCN-iPS cells, using coculture system with murine embryonic aorta-gonad-mesonephros region-drived stromal cell line (AGM-3 cells) (Ma F. et al., PNAS, 2009). The cocultured cells were harvested at day 12 and analyzed for the hematopoietic colony formation by 10,000 CD34+ cells which were separated using magnetic beads system. There was no difference between SCN-iPS and control iPS cells in the number and size of erythroid and mixed-lineage colonies (erythroid colonies; 11.5±3.7 in SCN-iPS cells and 10.3±4.5 in control and mixed-lineage colonies; 25.5±8.0 in SCN-iPS cells and 18.7±4.2 in control). However, the number of myeloid colonies derived from SCN-iPS cells was significantly smaller than that in control (48.0±21.3 in SCN-iPS cells and 206.7±37.6 in control, p We next examined the neutrophil development in more detail in suspension culture. 10,000 CD34+ cells derived from SCN-iPS and control iPS cells were cocultured with AGM-3 stromal cells in the presence of various cytokines including 10 ng/mL of G-CSF, but not erythropoietin to exclude the growth of erythroid cells. The cells from control iPS cell increased 23.3-fold for 2 weeks, and most of the cultured cells were neutrophils. By contrast, the cells from SCN-iPS cells gradually decreased, and contained few neutrophils, but mainly monocytic cells. These results indicated thatthe development of neutrophils was selectively impaired in hematopoiesis derived from SCN-iPS cells, although other lineages of cells, such as erythroid and monocytic cells, were not affected, and that the stimulation of high concentration of G-CSF compensated the impaired neutrophil development to some extent in SCN-iPS cells. The present study demonstrated that hematopoiesis derived from SCN-iPS cells reflected the pathophysiological status of SCN patients including neutrocytopenia and its improvement by G-CSF treatment, and established SCN-iPS cells could contribute to understand the pathophysiology and leukemogenesis in SCN. Further researches are under investigation to clarify the relationship between ELA2 mutation and clinical status in SCN including leukemogenesis. Disclosures: No relevant conflicts of interest to declare.

Abstract 1732 X-linked ectodermal dysplasia with immunodeficiency (XL-EDA-ID) is caused by hypomorphic mutations in the nuclear factor κB (NF-κB) essential modulator (NEMO) gene, also called IKBKG, and these mutations impair but do not abolish NF-κB signaling, resulting in distinct clinical and immunologic phenotypes. We presented the patient with XL-EDA-ID showing a novel splice-site mutation. In this report we precisely study the involvement of this mutation in the molecular pathogenesis of XL-EDA-ID. The patient is 12 years old Japanese boy. He had conical-shaped teeth and hypodontia. He had suffered from recurrent bacterial infections and these pathogenic bacteria were mostly streptococcus pneumonia. The laboratory examination revealed that the number of white blood cell counts, the classification of lymphocytes, and the serum immunoglobulin levels were within normal range. However the specific antibodies against measles and streptococcus pneumonia were negative in spite of these infections. We identified a novel hemizygous mutation, 769-1 G>C, at the splicing acceptor site of exon 7 in the IKBKG gene. In order to clarify the effect of this mutation on mRNA, we performed a cloning analysis. Although various abnormal spliced NEMO (mutant NEMO) mRNAs were observed, a small amount of wild-type NEMO (WT NEMO) mRNA was also identified. The rate of WT and mutant was variable on the time of blood collection. Further we performed FACS and immunoblot analysis in order to evaluate the effect of this mutation at protein level. Two major bands which were presumed to be derived from WT and one mutant were detected in immunoblots using EBV transfected B cells, however the expression levels of these bands were markedly decreased compared to those of the healthy controls. The NEMO protein expression was confirmed to be decreased in various lineages of leukocytes. We generated WT and two representative mutant NEMO constructs and measured their NF-κB transcriptional activity using reporter assay. One mutant NEMO abolished NF-κB transcriptional activity, the other showed weak activity. However, neither of mutant NENO showed a dominant-negative effect against WT NEMO activity. CD14+ cells from the patient produced a lower level of TNF-α in response to IFN-γ stimulation, on the other hand CD3+ cells produced very little IFN-γ in response to IL-12. CD4+ T-cell proliferation was impaired in response to measles and mumps, but not rubella. The hemizygous 769-1 G>C mutation was shown to cause the decreased expression of WT NEMO protein, resulting in the decrease in NF-κB activation and the development of XL-EDA-ID. Disclosures: No relevant conflicts of interest to declare.

Abstract 3339 Poster Board III-227 Chronic granulomatous disease (CGD) is the most common inherited disorders of phagocytic function caused by abnormal nicotinamide adenine dinucleotide phosphate (NADPH) oxidase which produces reactive oxygen species. Defects in four of the NADPH components are responsible for CGD: gp91phox, p47phox, p67phox and p22phox. Due to the defects in production of superoxide, patients are highly susceptible to catalase-positive infections including fungi, as well as developing granuloma and autoimmune complications. To date, hematopoietic stem cell transplantation (HSCT) is the only curative treatment for patients with CGD. The HSCT for CGD patients using myeloablative conditioning regimen consisting of busulfan and cyclophosphamide (CY) has been proven to cure the disease. However, HSCT with this myeloablative conditioning regimen has considerable risk of transplantation-related morbidity (TRM) and mortality in patients with life-threatening infection and long-term complications including graft-versus host disease (GVHD), pulmonary late effects, and gonadal failure. To avoid the risk of TRM and long-term complications, we underwent bone marrow transplantation (BMT) with reduced-intensity conditioning (RIC) for 12 patients with CGD from 2002 to 2009 in Hiroshima University. Seven patients were transplanted from related donors and five patients from unrelated donors. Conditioning regimens consisted of fludarabine, CY and TBI (3 Gy) with or without antithymoglobulin or melphalan (L-PAM). Three of twelve patients who had severe life-threatening infections such as multiple brain abscesses, multiple liver abscesses or multiple pulmonary abscesses with high level of CRP (>15 mg/dl) underwent BMT. One of three patients who had suffered from severe fungal infection in both lungs was died from pulmonary hemorrhage due to the engraftment syndrome on day 45 after BMT. No TRM during the conditioning and early period after BMT was observed in the remaining 11 patients, irrespective of the presence of active and intractable infections/inflammation. The patients alive were eliminated from infections and/or inflammation with complete chimerism. Four patients undergoing BMT using fludarabine-based regimen without L-PAM required donor lymphocyte infusion to achieve complete donor chimerism. The addition of L-PAM to fludarabine-based RIC rapidly induced the complete engraftment of donor cells without any toxicity in 7 patients. The frequency and severity of acute or chronic GVHD were not significant and sufficiently tolerable. Karnofsky performance scale of all patients alive has been 100% after BMT. Furthermore, apparent endocrinological problems including gonadal function were not observed during the limited period after BMT. These results suggest HSCT using RIC with L-PAM is effective therapy for CGD patients for successful complete engraftment and minimal toxicity. Disclosures No relevant conflicts of interest to declare.

Abstract 2665 Poster Board II-641 X-linked ectodermal dysplasia with immunodeficiency (XL-EDA-ID) is an X-linked recessive disease characterized by hypohidrosis, missing or malformed teeth, coarse hair, dry skin and immunodeficiency. This disorder is caused by hypomorphic mutations in IKBKG (the inhibitory κB kinase γ gene), which encodes NF-κB essential modulator (NEMO). NEMO is an essential subunit of the IκB kinase (IKK) complex which plays an important role in NF-αB activation. The boy aged 12 years has presented mild mental retardation, conical-shaped teeth and hypodontia. He had suffered from recurrent bacterial infections, e.g. 3 episodes of bacterial meningitis, cellulitis, left knee arthritis and osteomyelitis. The majority of pathogenic bacteria were Streptococcus pneumonia and Staphylococcus aureus. Complete blood counts and classification of white blood cells were within normal range. The lymphocyte subpopulation, serum complement levels, and the function of neutrophils were also normal. The NK cell activity was significantly low despite of the presence of normal number of CD16- and CD56-positive cells. Serum immunoglobulin levels were IgG 966 mg/dl, IgA 292 mg/dl, IgM 76 mg/dl, respectively. The concentration of IgG2 was relatively low (248 mg/dl, 25% of IgG). No specific antibodies against measles and Streptococcus pneumonia were developed in spite of the history of infections. We identified a novel hemizygous mutation, IVS6 -1 G>C, at the splicing acceptor site of exon 7 in the IKBKG gene. Flow cytometric analysis of NEMO protein showed the significantly low, but not deficient expression in T cell, B cell, NK cell and monocytes in the peripheral blood of the patient. In addition, the number of the CD19+CD27+ memory B cells was significantly reduced. The INF-κ production in response to IL-12 stimulation was impaired in CD3-positive cells from the patient, and the TNF-α production in response to IFN-α stimulation was also impaired in CD14-positive cells. These results suggest the involvement of NEMO in the function of T cells, B cells, NK cells, and monocytes. To further clarify the effect of this splice site mutation, we performed RT-PCR of patient's cDNA using primers that span the sequences around exon 7 of IKBKG. The PCR product was cloned into p-GEMT-easy cloning vector, and sequenced 24 individual clones. Notably, the sequence analysis of 24 clones demonstrated that 7 clones were derived from normal splicing and the other 17 clones from various abnormal splicing. The NEMO mRNA derived from abnormal splicing was presumed to produce non-functional NEMO protein based on the large conformational change. Taken together, these results suggest that immunological impairment found in this patient may be qualitative abnormality of NEMO protein in lymphocytes and monocytes derived from the splicing mutation of the gene. Disclosures: No relevant conflicts of interest to declare.

Neonate alloimmune neutropenia (NAIN) is caused by the transplacental transfer of maternal alloantibodies directed against antigens on the infant’s neutrophils. To date, there are scant studies about its clinical characteristics and characterization of antineutrophil antibodies though some case reports are found. In this study we analyzed 11 cases with NAIN from January 2005 to December 2007. The diagnosis of NAIN was confirmed by the transient neutropenia less than 500/μl of absolute neutrophil count (ANC), the detection of maternal antineutrophil antibody, the incompatibility of neutrophil antigens between parents, and their mothers without autoimmune diseases. Antineutrophil antibodies were detected by granulocyte indirect immunofluorescence test using flow cytometry. To quantify the strength of the antibodies, the ratio of the mean fluorescence channel of each sample to that of control serum was expressed as relative fluorescence intensity according to the method reported previously (Blood99: 3468, 2002). The median age at diagnosis in NAIN patients was 8 days after birth ranged from 0 to 30 days. The average of ANC at the presentation was 170/μl ranged from 0 to 500/μl. All antineutrophil antibodies detected in sera of both neonates and their mothers were against HNA-1 antigens. The alloantibody against HNA-1a was found in 2 cases, that against HNA-1b was in 6 cases, and that against FcγR IIIb was found in 3 cases. The fact that the frequencies of homozygote of HNA-1a and HNA-1b in Japanese population were approximately 50% and 12%, respectively may reflect the frequency of alloantibody specificity in NAIN. During the neutropenic period, 7 cases with NAIN showed mild to moderate infections associated with neutropenia, such as pyrexia and pyodermia. In contrast, 4 of 11 cases with NAIN did not have any infectious episodes in their clinical course. In all patients, the spontaneous recovery of neutropenia with the disappearance of alloantibody was observed within 6 months (median 85 days ranged from 3 weeks to 6 months). The duration until spontaneous resolution of neutropenia was dependent on the strength of alloantibody found in sera of the mothers and neonates. Two patients with significantly high strength of alloantibodies had the relatively long duration to restore the neutropenia(4 months and 6 months). In conclusion, the specificity of antineutrophil antibodies in patients with alloimmune neutropenia is dependent on the frequencies of neutrophil antigens in Japanese population. The quantification of alloantibodies in neonates and their mothers may be useful in considering the clinical course of neutropenia in neonates.

Graft-versus-host disease (GVHD) is a significant complication in hematopoietic stem cell transplantation (SCT). On the other hand, graft-versus-tumor (GVT) effect has been known to be concerned with the prevention of relapses in various hematological or non-hematological malignant disorders. The regulation of GVHD without suppressing GVT effect is a pivotal role in the success of hematopoietic SCT. Recently CD4 + CD25 + regulatory T cells have been recognized to regulate the maintenance of self-tolerance, and associate with several autoimmune diseases. In transplant immunity, CD4 + CD25 + regulatory T cells have been reported to regulate GVHD without suppressing GVT effect in several animal studies. Natural killer T (NKT) like cells also have been recognized to associated with the maintenance of self-tolerance by inducing CD4 + CD25 + regulatory T cells through the production of IL-2. In this study, we examined the roles of CD4 + CD25 + regulatory T cells and NKT like cells in cases underwent hematopoietic SCT. Blood samples from patients underwent SCT in our institution during past 3 years (7 months through 26 years of age, n =19) were obtained every two weeks until day 90 after SCT. Primary disorders of patients were non-malignant hematological disease such as aplastic anemia (n=3), chronic granulomatous disease (n=8) and malignant disease such as leukemia (n=5), and solid tumors (n=3). Pre-conditioning regimens used in this study were myeloablative regimens in 10 cases and reduced intensity regimens in 9 cases, respectively. The frequencies of CD4 + CD25 + regulatory T cells were assessed by the expression of CD4 and CD25, and those of NKT like cells were assessed by the expression of CD3, CD16, and CD56 using flow cytometry. The mRNA expression of FOXP3 in purified CD4 + CD25 + regulatory T cells were determined by quantitative real-time PCR method. In 13 patients who had none or Grade 1 acute GVHD, the frequency of CD4 + CD25 + regulatory T cells in CD4 + T cells was increased up to 25–90% at early period after SCT and normalized below 20% after day 45 post SCT. On the other hand, four of 6 patients who had acute GVHD (more than Grade 2) showed that the frequency of CD4 + CD25 + regulatory T cells in CD4 + T cells persisted below 20%. In other one patient, the development of acute GVHD (Grade 2) was associated with decreasing the frequency of CD4 + CD25 + regulatory T cells (30 to 10%) and the recovery of GVHD was found with increasing CD4 + CD25 + regulatory T cells (10 to 30%). The mean frequency of CD4 + CD25 + regulatory T cells in CD4 + T cells on day 15 after SCT was 44% in patients without GVHD and 21% in patients with GVHD (p=0.07). No difference in the expressions of FOXP3 mRNA in purified CD4 + CD25 + regulatory T cells was noted between patients with GVHD and those without GVHD. The reconstitution pattern of CD3 + CD16 + CD56 + NKT like cells after SCT was not associated with the development of GVHD. These results suggest that the development of acute GVHD may be strongly associated with the reconstitution of donor derived CD4 + CD25 + regulatory T cells in CD4 + T cells. The measurement of CD4 + CD25 + regulatory T cells in CD4 + T cells might lead to the early diagnosis and the prevention of acute GVHD. (Future studies will be needed to examine the association between the frequency of regulatory T cells and GVT effect.)

Abstract 3213 Severe congenital neutropenia (SCN) includes a variety of hematologic disorders characterized by severe neutropenia, with absolute neutrophil counts (ANC) below 0.5 × 109/L, and associated with severe systemic bacterial infections from early infancy. Mutations in ELANE, HAX1, G6PC3, WAS and GFI1 have been so far identified in patients with SCN. The Severe Chronic Neutropenia International Registry (SCNIR) has collected data to monitor the clinical course, treatments, and disease outcomes for SCN patients. In this study, we analyzed the clinical and genetic characteristics of patients with SCN based on the Japanese cohort study collecting the data on chronic neutropenia using a standardized case report form. This study is approved by the ethics committees of the Hiroshima University School of Medicine. Forty-six patients with SCN in Japan were enrolled in this study. Mean present age of patients was 13.6 years old ranged from 1 to 39. The Mean age at diagnosis was 4.6 months ranged from 0 to 24 months. Approximately 90% of patients were diagnosed before 12 months. Twenty-three patients were female. As initial clinical presentation, subcutaneous abscess and cutaneous cellulitis were dominated (37%), followed by unknown fever (17%), stomatitis (13%) and lymphadenitis (13%). On the other hand, infections which patients had experienced after diagnosis were bacterial pneumonia (50%), cutaneous infections (50%) and oral infections such as stomatitis (48%) and gingivitis (48 %). Total 8 patients (17%) including 4 patients with mutations in HAX1 suffered from psychomotor retardation that was higher than that in general Japanese population (about 1%), suggesting that psychomotor retardation may be one of considerable complications in patients with SCN. Thirty-three out of 46 patients with SCN were preformed gene analysis and 29 patients (approximately 88 %) were identified mutations; 25 patients had heterozygous mutations in ELANE, 4 patients had homozygous mutations in HAX1. The proportion of mutations in ELANE (76%) was higher and that in HAX1 (12%) was relatively lower in Japan compared with those in Western counties. Thirty-six patients (78%) were treated with G-CSF, including regular use in 26 patients (56%) and on demand use during infections in 10 patients (22%). In 26 patients treated with regular G-CSF therapy, the mean cumulative duration was 6.6 years, ranged from 0.4 to 19. Two patients showed no response to G-CSF therapy. Four patients among total 46 patients developed MDS/AML and 3 patients were alive after receiving hematopoietic stem cell transplantation (HSCT). Total 12 out of 46 patients (26%) with SCN were underwent HSCT before malignant transformations due to recurrent infections and/or the long-term use of G-CSF. Reduced intensity conditionings (RIC) were used in part of patients. All patients were alive after HSCT. In Japan, high proportion of patients was treated with HSCT using RIC before malignant transformations. The accumulation of cases is necessary to establish suitable conditioning regimen and appropriate indication of HSCT in patients with SCN. Disclosures: No relevant conflicts of interest to declare.