High-dose Cepharanthin for pediatric chronic immune thrombocytopenia in Japan.

BACKGROUND: A nationwide, multicenter and observational study was retrospectively conducted to evaluate the clinical utility of Cepharanthin (CEP) for pediatric patients with chronic immune thrombocytopenia (ITP). METHODS: Clinical and laboratory data for 46 Japanese patients aged <16 years who were diagnosed as having chronic ITP in 14 hospitals during 2001-2011, and were treated with CEP for >12 months, were analyzed. RESULTS: Median daily CEP dose was 1 mg/kg (range, 0.12-2 mg/kg). Median platelet count prior to CEP was 20.5 × 109 /L (IQR, 8.3-53.0 × 109 /L), and then significantly increased to 58.5 × 109 /L (IQR, 22.8-115.0 × 109 /L) and 69.0 × 109 /L (IQR, 23.0-134.0 × 109 /L) at 12 and 24 months of treatment, respectively. No life-threatening bleeds or moderate-severe adverse events were reported. Of 38 patients who received both corticosteroids (CS) and CEP, 17 patients (45%) were weaned from CS, and 15 patients (39%) attained the reduced dose of CS. The duration from the start of CEP to the stopping of CS was a median of 413 days (range, 49-1734 days) in patients who were weaned from CS. CONCLUSIONS: CEP alone or combined with CS was useful for the management of pediatric chronic ITPs.

Immunoregulatory function of neonatal nucleated red blood cells in humans.

We found that human cord blood nucleated red blood cells (NRBCs) have a regulatory function in the innate immune reaction. These cells suppressed the production of inflammatory cytokines including TNF-α and IL-1ß from monocytes in response to lipopolysaccharide (LPS). The NRBCs exerted their regulatory function even without cell-to-cell contact with the monocytes. However, IL-10 production from the monocytes by LPS stimulation in the presence of NRBCs was higher than that from LPS-stimulated monocytes cultured in the absence of NRBCs. Addition of an anti-IL-10 receptor blocking antibody restored the inflammatory cytokine production from the monocytes, suggesting that the functional change of the monocytes caused by the interaction with NRBCs was mediated by the increased IL-10 production. A whole-genome microarray analysis revealed that the monocytes expressed increased amounts of IL-10 superfamily genes after interacting with NRBCs. IL-19, which is a member of the IL-10 superfamily, enhanced IL-10 production from the monocytes, which suggested a cooperative role of the IL-10 superfamily in the suppression of inflammatory cytokine production from monocytes. Arginase, which was reported to play an important role in the suppressive function of NRBCs in mice monocytes, was found to have no significant role in human monocytes. The NRBCs seem to have a regulatory role through the induction of IL-10/IL-19 production by monocytes to suppress a vigorous innate immune reaction, which can be harmful to fetuses.

Invasive Bacterial Infection in Patients with Interleukin-1 Receptor-associated Kinase 4 Deficiency: Case Report.

Interleukin-1 receptor-associated kinase 4 (IRAK4) deficiency (OMIM #607676) is a rare primary immunodeficiency of innate immune defect. We identified 10 patients from 6 families with IRAK4 deficiency in Japan, and analyzed the clinical characteristics of this disease. Nine patients had homozygous c.123_124insA mutation, and 1 patient had c.123_124insA and another nonsense mutation (547C>T). Umbilical cord separation occurred on the 14th day after birth or thereafter. Two patients had no severe infections owing to the prophylactic antibiotic treatment. Severe invasive bacterial infections occurred before the age of 3 in the other 8 patients. Among them, 7 patients had pneumococcal meningitis. Five patients died of invasive bacterial infection during infancy, although intravenous antibiotic treatment was started within 24 hours after onset in 4 patients among them. Analysis of cerebrospinal fluid of the patients who had fatal meningitis revealed very low glucose levels with only mild pleocytosis. The clinical courses of invasive bacterial infections were often rapidly progressive despite the early, appropriate antibiotic treatment in IRAK4 deficiency patients. The early diagnosis and appropriate prophylaxis of invasive bacterial infections are necessary for the patients.

Age-specific onset and distribution of the natural anticoagulant deficiency in pediatric thromboembolism.

BACKGROUND: The early diagnosis of inherited thrombophilia in children is challenging because of the rarity and hemostatic maturation. METHODS: We explored protein C (PC), protein S (PS), and antithrombin (AT) deficiencies in 306 thromboembolic patients aged ≤20 y using the screening of plasma activity and genetic analysis. RESULTS: Reduced activities were determined in 122 patients (40%). Low PC patients were most frequently found in the lowest age group (0-2 y, 45%), while low PS or low AT patients were found in the highest age group (16-20 y; PS: 30% and AT: 20%). Genetic study was completed in 62 patients having no other causes of thromboembolism. Mutations were determined in 18 patients (8 PC, 8 PS, and 2 AT genes). Six of eight patients with PC gene mutation were found in age 0-2 y (75%), while six of eight patients with PS gene mutation were in 7-20 y. Two AT gene-mutated patients were older than 4 y. Four PC-deficient and two PS-deficient patients carried compound heterozygous mutations. All but one PC gene-mutated patient suffered from intracranial thromboembolism, while PS/AT gene-mutated patients mostly developed extracranial venous thromboembolism. CONCLUSION: Stroke in low PC infants and deep vein thrombosis in low PS/AT school age children could be targeted for genetic screening of pediatric thrombophilias.

Wiskott-Aldrich syndrome in a girl caused by heterozygous WASP mutation and extremely skewed X-chromosome inactivation: a novel association with maternal uniparental isodisomy 6.

Wiskott-Aldrich syndrome (WAS) is an X-linked disease characterized by microthrombocytopenia, eczema and immune deficiency, caused primarily by mutations in the WASP (Wiskott-Aldrich syndrome protein) gene. Female carriers are usually asymptomatic because of the preferential activation of the normal, nonmutated X-chromosome in their hematopoietic cells. We report our observations of a female child with WAS, who displayed symptoms of congenital thrombocytopenia. DNA sequencing analysis of the WASP gene revealed a heterozygous nonsense mutation in exon 10. The expressions of WASP and normal WASP mRNA were defective. We found preferential inactivation of the X-chromosome on which wild-type WASP was located. Single-nucleotide polymorphism microarray testing and the analysis of the polymorphic variable number of tandem repeat regions revealed maternal uniparental isodisomy of chromosome 6 (UPD6). Our results underscore the importance of WASP evaluation in females with congenital thrombocytopenia and suggest that UPD6 might be related to the pathophysiology of nonrandom X-chromosome inactivation.

Early progression of atherosclerosis in children with chronic infantile neurological cutaneous and articular syndrome.

OBJECTIVE: Chronic inflammation plays a key role in the development of atherosclerosis. Early progression of atherosclerosis has been reported in patients with RA. Cryopyrin-associated periodic syndromes (CAPS) are autosomal dominant autoinflammatory disorders caused by heterozygous NLRP3 gene mutations. Chronic infantile neurological cutaneous and articular (CINCA) syndrome is the most severe form of CAPS and patients display early onset of rash, fever, uveitis and joint manifestations. However, there has been no previous report on atherosclerosis in patients with CAPS. The objective of this study is to assess the development of atherosclerosis in patients with CINCA syndrome. METHODS: Intima-media thickness (IMT) of the carotid arteries, stiffness parameter ß, ankle brachial index (ABI) and pressure wave velocity (PWV) were evaluated by ultrasonography in 3 patients with CINCA syndrome [mean age 9.0 years (S.D. 5.3)] and 19 age-matched healthy controls [9.3 years (S.D. 4.3)]. RESULTS: The levels of carotid IMT, stiffness parameter ß and PWV in CINCA syndrome patients were significantly higher than those in healthy controls [0.51 mm (S.D. 0.05) vs 0.44 (0.04), P = 0.0021; 6.1 (S.D. 1.7) vs 3.9 (1.0), P = 0.0018; 1203 cm/s (S.D. 328) vs 855 (114), P = 0.017, respectively]. CONCLUSION: Patients with CINCA syndrome showed signs of atherosclerosis from their early childhood. The results of this study emphasize the importance of chronic inflammation in the development of atherosclerosis. Further analysis on atherosclerosis in young patients with CINCA syndrome may provide more insights into the pathogenesis of cardiovascular disease.

Severe acute rhabdomyolysis induced by multi-agent chemotherapy for alveolar rhabdomyosarcoma in a 15-year-old female: a case report.

This is the first paper to report the association of cancer chemotherapy with rhabdomyolysis in children. A previously healthy, 15-year-old Japanese female was diagnosed as having alveolar rhabdomyosarcoma. She received the first cycle of multi-agent chemotherapy without any adverse effects. However, she developed severe acute rhabdomyolysis shortly after the second cycle of multi-agent chemotherapy, which consisted of etoposide, ifosfamide, actinomycin-D and vincristine. Her condition deteriorated rapidly and she was treated with mechanical ventilation and fluid replacement. After further evaluation, anticancer drugs were thought to be responsible for the rhabdomyolysis.

Treatment choice of immunotherapy or further chemotherapy for Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis.

BACKGROUND: Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis (EBV-HLH) leads to an aggressive and often fatal course without appropriate treatment. Etoposide therapy is crucial for the better prognosis, although it remains unknown what patients need cytotoxic agents. Since we have complied with step-up strategy in a tertiary center, treatment outcomes were studied to search predictors for disease course. METHODS: The study enrolled 22 EBV-HLH patients treated between 1999 and 2010 in Kyushu University. Immunotherapy, chemotherapy and stem cell transplantation (SCT) proceeded in stages unless patients attained a consecutive >21 days-afebrile remission. Clinical and laboratory data and outcomes were retrospectively analyzed. RESULTS: Median age of 9 males and 13 females was 5 years (range: 9 months-41 years). Sixteen patients (73%) presented at age <15 years. Two patients remitted spontaneously, 12 attained remissions after immunotherapy, 5 after chemotherapy, and 1 after successful SCT. The remaining two patients died after chemotherapy and SCT, respectively. Median EBV load was 1 × 10(5) copies/ml of peripheral blood (range: 200-5 × 10(7)). T-cells were exclusively targeted (94%; 15/16 examined) often with EBV/T-cell receptor clonality. EBV status indicated 19 primary infections and 3 reactivations. Either death occurred in EBV-reactivated patients who underwent chemotherapy ± SCT. Age at primary infection in pediatric patients increased in the last 5 years. Patients having prolonged fever (P = 0.017) or high soluble CD25 levels (P = 0.017) at diagnosis were at higher risk for requiring chemotherapy assessed by multivariate analyses. CONCLUSIONS: No cytotoxic agents were needed for >60% of EBV-HLH patients. Early immunotherapy may modulate T-cell activation and reduce the chance of unnecessary chemotherapy.

Histone 3 lysine 9 (H3K9) methyltransferase recruitment to the interleukin-2 (IL-2) promoter is a mechanism of suppression of IL-2 transcription by the transforming growth factor-ß-Smad pathway.

Suppression of IL-2 ßproduction from T cells is an important process for the immune regulation by TGF-ß. However, the mechanism by which this suppression occurs remains to be established. Here, we demonstrate that Smad2 and Smad3, two major TGF-ß-downstream transcription factors, are redundantly essential for TGF-ß-mediated suppression of IL-2 production in CD4(+) T cells using Smad2- and Smad3-deficient T cells. Both Smad2 and Smad3 were recruited into the proximal region of the IL-2 promoter in response to TGF-ß. We then investigated the histone methylation status of the IL-2 promoter. Although both histone H3 lysine 9 (H3K9) and H3K27 trimethylation have been implicated in gene silencing, only H3K9 trimethylation was increased in the proximal region of the IL-2 promoter in a Smad2/3-dependent manner, whereas H3K27 trimethylation was not. The H3K9 methyltransferases Setdb1 and Suv39h1 bound to Smad3 and suppressed IL-2 promoter activity in collaboration with Smad3. Overexpression of Suv39h1 in 68-41 T cells strongly inhibited IL-2 production in response to T cell receptor stimulation irrespective of the presence or absence of TGF-ß, whereas Setdb1 overexpression only slightly suppressed IL-2 production. Silencing of Suv39h1 by shRNA reverted the suppressive effect of TGF-ß on IL-2 production. Furthermore, TGF-ß induced Suv39h1 recruitment to the proximal region of the IL-2 promoter in wild type primary T cells; however, this was not observed in Smad2(-/-)Smad3(+/-) T cells. Thus, we propose that Smads recruit H3K9 methyltransferases Suv39h1 to the IL-2 promoter, thereby inducing suppressive histone methylation and inhibiting T cell receptor-mediated IL-2 transcription.

Smad2 and Smad3 are redundantly essential for the TGF-beta-mediated regulation of regulatory T plasticity and Th1 development.

Although it has been well established that TGF-beta plays a pivotal role in immune regulation, the roles of its downstream transcription factors, Smad2 and Smad3, have not been fully clarified. Specifically, the function of Smad2 in the immune system has not been investigated because of the embryonic lethality of Smad2-deficient mice. In this study, we generated T cell-specific Smad2 conditional knockout (KO) mice and unexpectedly found that Smad2 and Smad3 were redundantly essential for TGF-beta-mediated induction of Foxp3-expressing regulatory T cells and suppression of IFN-gamma production in CD4(+) T cells. Consistent with these observations, Smad2/Smad3-double KO mice, but not single KO mice, developed fatal inflammatory diseases with higher IFN-gamma production and reduced Foxp3 expression in CD4(+) T cells at the periphery. Although it has been suggested that Foxp3 induction might underlie TGF-beta-mediated immunosuppression, TGF-beta still can suppress Th1 cell development in Foxp3-deficient T cells, suggesting that the Smad2/3 pathway inhibits Th1 cell development with Foxp3-independent mechanisms. We also found that Th17 cell development was reduced in Smad-deficient CD4(+) T cells because of higher production of Th17-inhibitory cytokines from these T cells. However, TGF-beta-mediated induction of RORgamma t, a master regulator of Th17 cell, was independent of both Smad2 and Smad3, suggesting that TGF-beta regulates Th17 development through Smad2/3-dependent and -independent mechanisms.

Loss of suppressor of cytokine signaling 1 in helper T cells leads to defective Th17 differentiation by enhancing antagonistic effects of IFN-gamma on STAT3 and Smads.

Suppressor of cytokine signaling 1 (SOCS1) is an important negative regulator for cytokines; however, the role of SOCS1 in Th17 differentiation has not been clarified. We generated T cell-specific SOCS1-deficient mice and found that these mice were extremely resistant to a Th17-dependent autoimmune disease model, experimental autoimmune encephalomyelitis. SOCS1-deficient naive CD4(+) T cells were predominantly differentiated into Th1 and poorly into Th17 in vitro. These phenotypes were canceled in IFN-gamma(-/-) background, suggesting that a large amount of IFN-gamma in SOCS1-deficient T cells suppressed Th17 differentiation. IL-6 plus TGF-beta enhanced retinoic acid receptor-related orphan receptor (ROR)-gammat expression and suppressed IFN-gamma production in wild-type T cells, whereas these effects were severely impaired in SOCS1-deficient T cells. These phenotypes can be partly explained by STAT3 suppression by enhanced SOCS3 induction through hyper-STAT1 activation in SOCS1-deficient T cells. In addition, SOCS1-deficient T cells were much less sensitive to TGF-beta. Suppression of Th1 differentiation by TGF-beta was impaired in SOCS1-deficient T cells. TGF-beta-mediated Smad transcriptional activity was severely inhibited in SOCS1-deficient cells in the presence of IFN-gamma. Such impairment of TGF-beta functions were not observed in SOCS3-overexpressed cells, indicating that suppression of Smads was independent of SOCS3. Therefore, SOCS1 is necessary for Th17 differentiation by suppressing antagonistic effect of IFN-gamma on both STAT3 and Smads. Induction of SOCS3 can partly explain IFN-gamma-mediated STAT3 suppression, while other mechanism(s) will be involved in IFN-gamma-mediated Smad suppression. SOCS1-deficient T cells will be very useful to investigate the molecular mechanism for the STAT1-mediated suppression of Th17 development.

Selective expansion of foxp3-positive regulatory T cells and immunosuppression by suppressors of cytokine signaling 3-deficient dendritic cells.

Dendritic cells (DCs) induce immunity and immunological tolerance as APCs. It has been shown that DCs secreting IL-10 induce IL-10(+) Tr1-type regulatory T (Treg) cells, whereas Foxp3-positive Treg cells are expanded from naive CD4(+) T cells by coculturing with mature DCs. However, the regulatory mechanism of expansion of Foxp3(+) Treg cells by DCs has not been clarified. In this study, we demonstrated that suppressors of cytokine signaling (SOCS)-3-deficient DCs have a strong potential as Foxp3(+) T cell-inducing tolerogenic DCs. SOCS3(-/-) DCs expressed lower levels of class II MHC, CD40, CD86, and IL-12 than wild-type (WT)-DCs both in vitro and in vivo, and showed constitutive activation of STAT3. Foxp3(-) effector T cells were predominantly expanded by the priming with WT-DCs, whereas Foxp3(+) Treg cells were selectively expanded by SOCS3(-/-) DCs. Adoptive transfer of SOCS3(-/-) DCs reduced the severity of experimental autoimmune encephalomyelitis. Foxp3(+) T cell expansion was blocked by anti-TGF-beta Ab, and SOCS3(-/-) DCs produced higher levels of TGF-beta than WT-DCs, suggesting that TGF-beta plays an essential role in the expansion of Foxp3(+) Treg cells. These results indicate an important role of SOCS3 in determining on immunity or tolerance by DCs.

[Alveolar rhabdomyosarcoma of unknown origin mimicking acute leukemia at the initial presentation].

A 14-year-old boy presented with a short history of general fatigue. Laboratory examination of the peripheral blood revealed white blood cells 11,300/microl, hemoglobin 10.4 g/dl, platelets 45,000/microl, fibrinogen < 50 mg/dl, fibrin/fibrinogen degradation products 536 microg/ml and lactate dehydrogenase 1,684 U/l. A bone marrow aspirate contained 89.6% of undifferentiated tumor cells. A hematological malignancy was suspected and the patient was treated with idarubicin and cytarabine. However, further examination revealed that tumor cells were positive for CD56 and lacked lineage markers of lymphoid or myeloid cells. They were positive for PAS, HHF35 and desmin, and negative for MPO. Reverse transcriptase polymerase chain reaction demonstrated PAX3/FKHR fusion transcripts, confirming the diagnosis of alveolar rhabdomyosarcoma. Radiological examination revealed only one enlarged lymph node being 1.5 cm in diameter at the paraaortic region in the abdomen, and failed to find a primary tumor. After three courses of chemotherapy containing etoposide, cyclophosphamide, pirarubicin, cisplatin and vincristine, tumor cells were eradicated from the bone marrow. The patient received an allogeneic bone marrow transplantation eight months after diagnosis, although he died of hepatic veno-occlusive disease on day 21. Alveolar rhabdomyosarcoma often develops in older children and younger adults, and its bone marrow infiltration may mimic acute leukemia.