Ryuta Nishikomori

Pyrrolidine dithiocarbamate, a potent inhibitor of nuclear factor κB (NF-κB) activation, prevents apoptosis in human promyelocytic leukemia HL-60 cells and thymocytes

We examined the effect of pyrrolidine dithiocarbamate (PDTC), which potently blocks the activation of nuclear factor kappa B (NF-kappa B), on the induction of apoptosis by a variety of agents. Treatment of a human promyelocytic leukemia cell line, HL-60, with 10 micrograms/mL etoposide or 2 microM 1-beta-D-arabinofuranosylcytosine induced NF-kappa B activation within 1 hr and subsequently caused apoptosis within 3-4 hr. The simultaneous addition of 50-500 microM PDTC with these agents blocked NF-kappa B activation and completely abrogated both morphologically apoptotic changes and internucleosomal DNA fragmentation for up to 6 hr. However, PDTC failed to inhibit the endonuclease activity contained in the whole cell lysates. The inhibitory effect of PDTC was also observed in etoposide- and dexamethasone-induced apoptosis in human thymocytes at a concentration of 1-10 microM. Since PDTC has both antioxidant and metal-ion chelating activities, we tested the effects of N-acetyl-L-cysteine (NAC) (antioxidant) or o-phenanthroline (OP) (metal-ion chelator) on the induction of apoptosis. Pretreatment of HL-60 cells or thymocytes with 100-500 microM OP for 2 hr, but not 10-60 mM NAC, suppressed subsequent occurrence of apoptosis induced by etoposide. These results suggest that the activation of NF-kappa B plays an important role in the apoptotic process of human hematopoietic cells.

High Incidence of NLRP3 Somatic Mosaicism in Patients With Chronic Infantile Neurologic, Cutaneous, Articular Syndrome: Results of an International Multicenter Collaborative Study

OBJECTIVE Chronic infantile neurologic, cutaneous, articular (CINCA) syndrome, also known as neonatal-onset multisystem inflammatory disease (NOMID), is a dominantly inherited systemic autoinflammatory disease. Although heterozygous germline gain-of-function NLRP3 mutations are a known cause of this disease, conventional genetic analyses fail to detect disease-causing mutations in ∼40% of patients. Since somatic NLRP3 mosaicism has been detected in several mutation-negative NOMID/CINCA syndrome patients, we undertook this study to determine the precise contribution of somatic NLRP3 mosaicism to the etiology of NOMID/CINCA syndrome. METHODS An international case-control study was performed to detect somatic NLRP3 mosaicism in NOMID/CINCA syndrome patients who had shown no mutation during conventional sequencing. Subcloning and sequencing of NLRP3 was performed in these mutation-negative NOMID/CINCA syndrome patients and their healthy relatives. Clinical features were analyzed to identify potential genotype-phenotype associations. RESULTS Somatic NLRP3 mosaicism was identified in 18 of the 26 patients (69.2%). Estimates of the level of mosaicism ranged from 4.2% to 35.8% (mean ± SD 12.1 ± 7.9%). Mosaicism was not detected in any of the 19 healthy relatives (18 of 26 patients versus 0 of 19 relatives; P < 0.0001). In vitro functional assays indicated that the detected somatic NLRP3 mutations had disease-causing functional effects. No differences in NLRP3 mosaicism were detected between different cell lineages. Among nondescript clinical features, a lower incidence of mental retardation was noted in patients with somatic mosaicism. Genotype-matched comparison confirmed that patients with somatic NLRP3 mosaicism presented with milder neurologic symptoms. CONCLUSION Somatic NLRP3 mutations were identified in 69.2% of patients with mutation-negative NOMID/CINCA syndrome. This indicates that somatic NLRP3 mosaicism is a major cause of NOMID/CINCA syndrome.

STAT4 serine phosphorylation is critical for IL-12-induced IFN-γ production but not for cell proliferation

T helper 1 (TH1) differentiation and IFN-γ production are crucial in cell-mediated immune responses. IL-12 is an important regulator of this process and mediates its effects through signal transducer and activator of transcription 4 (STAT4). IFN-γ production is also regulated by the p38 mitogen-activated kinase pathway, although the mechanisms are ill-defined. We show here that GADD45-β and GADD45-γ can induce STAT4 S721 phosphorylation via the MKK6/p38 pathway. Thus, STAT4 could be a target that accounts for the defects in cell-mediated immunity associated with perturbations in the p38 pathway. To investigate the biological significance of STAT4 S721 phosphorylation, we reconstituted primary spleen cells from STAT4-deficient mice with wild-type and mutated STAT4, by using a retroviral gene transduction. We demonstrated that expression of wild-type STAT4, but not the S721A mutant, restored normal TH1 differentiation and IFN-γ synthesis. The inability of STAT4 S721 to restore IFN-γ production was not caused by decreased IL-12R expression because the STAT4 S721 mutant also failed to restore IFN-γ production in STAT4-deficient IL-12Rβ2 transgenic cells. Importantly, STAT4 S721A-transduced cells showed normal proliferative response to IL-12, illustrating that serine phosphorylation is not required for IL-12-induced proliferation. Additionally, the results imply the existence of STAT4 serine phosphorylation-dependent and -independent target genes. We conclude that phosphorylation of STAT4 on both tyrosine and serine residues is important in promoting normal TH1 differentiation and IFN-γ secretion.

Activated STAT4 Has an Essential Role in Th1 Differentiation and Proliferation That Is Independent of Its Role in the Maintenance of IL-12Rβ2 Chain Expression and Signaling

In this study we demonstrated that CD4+ T cells from STAT4−/− mice exhibit reduced IL-12R expression and poor IL-12R signaling function. This raised the question of whether activated STAT4 participates in Th1 cell development mainly through its effects on IL-12 signaling. In a first approach to this question we determined the capacity of CD4+ T cells from STAT4−/− bearing an IL-12Rβ2 chain transgene (and thus capable of normal IL-12R expression and signaling) to undergo Th1 differentiation when stimulated by Con A and APCs. We found that such cells were still unable to exhibit IL-12-mediated IFN-γ production. In a second approach to this question, we created Th2 cell lines (D10 cells) transfected with STAT4-expressing plasmids with various tyrosine→phenylalanine mutations and CD4+ T cell lines from IL-12β2−/− mice infected with retroviruses expressing similarly STAT4 mutations that nevertheless express surface IL-12Rβ2 chains. We then showed that constructs that were unable to support STAT4 tyrosine phosphorylation (in D10 cells) as a result of mutation were also incapable of supporting IL-12-induced IFN-γ production (in IL-12Rβ2−/− cells). Thus, by two complementary approaches we demonstrated that activated STAT4 has an essential downstream role in Th1 cell differentiation that is independent of its role in the support of IL-12Rβ2 chain signaling. This implies that STAT4 is an essential element in the early events of Th1 differentiation.

Obesity and the prevalence of allergic diseases in schoolchildren

Although the association between obesity and bronchial asthma (BA) has been gaining more attention, few studies have been conducted concerning the relationship between obesity and other allergic diseases. The objective of this study was to determine whether and how childhood obesity is associated with allergic diseases other than BA, such as atopic dermatitis (AD), allergic rhinitis (AR), allergic conjunctivitis (AC), and either AR or AC (AR/AC). A questionnaire was administered to the parents of 50,086 Japanese schoolchildren. Associations between childhood obesity and the various allergic diseases were evaluated by univariate and multivariate logistic models. Significant associations were found between higher body mass index (BMI) and AD (p = 0.03), and lower BMI and AC (p < 0.0001), and AR/AC (p < 0.0001). There was a significantly higher prevalence of BA in girls with obesity (p = 0.009) than in those without obesity. Significantly lower prevalence of AC (p = 0.01) and AR/AC (p = 0.002) among children with obesity, and AR (p = 0.04) and AR/AC (p = 0.0004) among boys with obesity were observed than those without obesity. Those who were obese and had AD were significantly more likely to have severe symptoms (p = 0.01). Overall, childhood obesity has positive associations with BA prevalence and AD severity, whereas it has negative associations with AR and AC prevalence, especially among boys. Changes in the immunologic balance accompanied by obesity might have different effects on each type of allergic disease. Exploring the mechanisms by which childhood obesity affects allergic status should lead to new management options for childhood allergy.

FR901228 induces tumor regression associated with induction of Fas ligand and activation of Fas signaling in human osteosarcoma cells.

We investigated the antitumor effects of FR901228, a HDAC inhibitor, on human osteosarcoma cells, in vitro and in vivo to explore its possible utility in the treatment of pediatric bone cancers. FR901228 caused marked growth inhibition with a 50% inhibitory concentration of 1.2–7.3 nM and induction of apoptosis in all eight osteosarcoma cell lines tested. These effects of FR901228 were also observed in vivo xenograft models on BALB/c nude mice, and treatment with 5.6 mg/kg/day resulting in a >70% reduction in the mean final tumor volume compared with the mean initial tumor volume. TUNEL assays demonstrated extensive apoptosis in tumor sections of mice treated with FR901228. Induction of apoptosis was preceded by increased expression of Fas ligand (FasL) mRNA, resulting in expression of membrane-bound FasL, which was followed by sequential activation of caspase-8 and -3. The level of apoptosis induction was reduced using a neutralizing anti-FasL antibody and overexpression of either the dominant-negative FADD or the viral FLICE inhibitory protein. Furthermore, treatment with a suboptimal dose of FR901228 greatly sensitized osteosarcoma cells to agonistic anti-Fas antibody-mediated apoptosis. These findings suggest that FR901228 is a highly promising antitumor agent against osteosarcoma, inducing apoptosis by the activation of the Fas/FasL system.

Breastfeeding and the prevalence of allergic diseases in schoolchildren: Does reverse causation matter?

Kusunoki T, Morimoto T, Nishikomori R, Yasumi T, Heike T, Mukaida K, Fujii T, Nakahata T. Breastfeeding and the prevalence of allergic diseases in schoolchildren: Does reverse causation matter?
Pediatr Allergy Immunol 2010: 21: 60–66.
© 2010 John Wiley & Sons A/S

Complex I (reduced nicotinamide-adenine dinucleotide-coenzyme Q reductase) deficiency in two patients with probable Leigh syndrome

Two infants who had clinical and radiographic findings consistent with Leigh syndrome were found to have deficiency of complex I (reduced nicotinamide-adenine dinucleotide--coenzyme Q reductase) activity. Significant abnormalities were found on computed tomographic scans and magnetic resonance images of the brain. Lactate and pyruvate concentrations in blood and cerebrospinal fluid were elevated, and muscle biopsy specimens showed abnormal mitochondria. These data indicate that Leigh syndrome, as well as MELAS syndrome (mitochondrial encephalopathy, myopathy, lactic acidosis, and stroke-like episodes) may result from complex I deficiency.

Reduced numbers and proapoptotic features of mucosal-associated invariant T cells as a characteristic finding in patients with inflammatory bowel disease

Background:Mucosal-associated invariant T (MAIT) cells are innate-like T cells involved in the homeostasis of mucosal immunity; however, their role in inflammatory bowel disease (IBD) is unclear. Methods:Flow cytometry was used to enumerate peripheral blood MAIT cells in 88 patients with ulcerative colitis (UC), 68 with Crohns disease (CD), and in 57 healthy controls. Immunohistochemistry identified MAIT cells in intestinal tissue samples from patients with UC (n = 5) and CD (n = 10), and in control colon (n = 5) and small intestine (n = 9) samples. In addition, expression of activated caspases by MAIT cells in the peripheral blood of 14 patients with UC and 15 patients with CD, and 16 healthy controls was examined. Results:Peripheral blood analysis revealed that patients with IBD had significantly fewer MAIT cells than healthy controls (P < 0.0001). The number of MAIT cells in the inflamed intestinal mucosae of patients with UC and CD was also lower than that in control mucosae (P = 0.0079 and 0.041, respectively). The number of activated caspase-expressing MAIT cells in the peripheral blood of patients with UC and CD was higher than that in healthy controls (P = 0.0061 and 0.0075, respectively), suggesting that the reduced MAIT cell numbers in IBD are associated with an increased level of apoptosis among these cells. Conclusions:The number of MAIT cells in the peripheral blood and inflamed mucosae of patients with UC and CD was lower than that in non-IBD controls. Also, MAIT cells from patients with IBD exhibited proapoptotic features. These data suggest the pathological involvement and the potential for therapeutic manipulation of these cells in patients with IBD.

BALB/c Mice Bearing a Transgenic IL-12 Receptor β2 Gene Exhibit a Nonhealing Phenotype to Leishmania major Infection Despite Intact IL-12 Signaling

In BALB/c mice infected with Leishmania major, early secretion of IL-4 leads to a Th2-type response and nonhealing. We explored the role of IL-4-induced down-regulation of the IL-12Rβ2 chain in the establishment of this Th2 response. First, we showed that the draining lymph nodes of resistant C57BL/6 mice infected with L. major were enriched in CD4+/IL-12Rβ2 chain+ cells producing IFN-γ. Next, we demonstrated that BALB/c background mice bearing an IL-12Rβ2-chain transgene manifested a nonhealing phenotype similar to wild-type littermates despite the persistence of their ability to undergo STAT4 activation. Finally, we found that such transgenic mice display more severe infection than wild-type littermates when treated with IL-12 7 days after infection, and under this condition, the mice display increased Leishmania Ag-induced IL-4 secretion. These studies indicate that although CD4+/IL-12Rβ2 chain+ T cells are important components of the Th1 response, maintenance of IL-12Rβ2 chain expression is not sufficient to change a Th2 response to a Th1 response in vivo and thus to allow BALB/c mice to heal L. major infection.