Knowledge and Compliance with Malaria National Treatment Guidelines among Primary Health Care Workers in a Rural Area in Northern Nigeria.

BACKGROUND: Knowledge and compliance with malaria treatment guidelines are among the major issues affecting treatment outcome for malaria in Nigeria. Primary health care (PHC) facilities are the first point of contact with the national health system for patients with malaria and other diseases. OBJECTIVE: This study assessed the knowledge and compliance with malaria National Treatment Guidelines (NTG) among PHC workers in Lere local government area of Kaduna State, North western Nigeria. METHODS: This was a descriptive cross-sectional study conducted among 42 community health workers. The total population of all eligible participants was used for subject selection. Data were analysed with SPSS IBM version 25.0 and STATA/SE 12. The level of statistical significance p-value was set at p<0.05. RESULTS: The mean age of the respondents was 38.02±9.23 years. Majority of the respondents were males (25; 59.5%) and community health extension workers (CHEWs) (24; 57.1%). Almost one-third (28.6%) of the PHC workers had poor knowledge of the recommendations of the NTG for malaria, while 14.3% had poor compliance with the NTG. Bivariate analysis showed a significant relationship between older age and good knowledge of the NTG ( χ2 =0.03, p=0.04). Multivariate analysis further revealed that the odds for poor knowledge of NTG was 40% higher among CHEWs compared to other health workers (AOR=1.40, 95% CI=0.25-7.93). The odds for good knowledge was lower by 55% among those who had practiced for <10 years compared to >10 years (OR=0.45, 95% CI=0.06-3.32). CONCLUSION: Poor knowledge and compliance to malaria NTG were commoner among lower cadre (CHEWs) staff with relatively fewer years in PHC practice. There is a need for training, retraining and equitable distribution of the NTG to ensure access and also improve knowledge and utilisation of the NTG for malaria by rural PHC workers.

CONTEXTE: La connaissance et le respect des directives de traitement du paludisme sont parmi les principaux problèmes qui affectent les résultats du traitement du paludisme au Nigéria. Les établissements de soins de santé primaires (SSP) sont le premier point de contact avec le système national de santé pour les patients atteints de paludisme et d'autres maladies. OBJECTIF DE L'ÉTUDE: Cette étude a évalué les connaissances et le respect des directives nationales de traitement du paludisme (NTG) parmi les travailleurs des SSP dans la zone de gouvernement local de Lere de l'État de Kaduna, dans le nord-ouest du Nigeria. MÉTHODES: Il s'agit d'une étude descriptive transversale menée auprès de 42 agents de santé communautaires. La population totale de tous les participants éligibles a été utilisée pour la sélection des sujets. Les données ont été analysées avec SPSS IBM version 25.0 et STATA/SE 12. Le niveau de signification statistique a été fixé à p<0,05. RÉSULTATS: L'âge moyen des personnes interrogées était de 38,02±9,23 ans. La majorité des personnes interrogées étaient des hommes (25 ; 59,5%) et des agents de vulgarisation en santé communautaire (24 ; 57,1%). Près d'un tiers (28,6 %) des agents de santé publique connaissaient mal les recommandations de la NTG pour le paludisme, tandis que 14,3 % ne respectaient pas la NTG. L'analyse bivariée a montré une relation significative entre l'âge avancé et la bonne connaissance du NTG ( χ2=0,03, p=0,04). L'analyse multivariée a également révélé que la probabilité d'une mauvaise connaissance de la NTG était 40% plus élevée chez les CHEW que chez les autres agents de santé (AOR=1,40, 95% CI=0,25-7,93). La probabilité d'une bonne connaissance était inférieure de 55% chez ceux qui avaient pratiqué pendant <10 ans par rapport à >10 ans (OR=0,45, 95% CI=0,06-3,32). CONCLUSION: Le manque de connaissances et d'observance des NTG sur le paludisme était plus fréquent parmi le personnel des cadres inférieurs (CHEWs) ayant relativement moins d'années de pratique dans les soins de santé primaires. Il est nécessaire de former, de recycler et de distribuer équitablement les NTG pour garantir l'accès et améliorer la connaissance et l'utilisation des NTG pour le paludisme par les travailleurs des SSP en milieu rural. Mots clés: Connaissance, Observance, Personnel de soins de santé primaires, Directives, Paludisme.

Relationship between the housing coldness/warmth evaluation by CASBEE Housing Health Checklist and psychological distress based on TMM Community-Based Cohort Study: a cross-sectional analysis.

OBJECTIVES: Previous studies have reported the relationship between housing environment and health, although due to cost and effort, it was difficult to conduct housing condition surveys on a large scale. The CASBEE Housing Health Checklist (the Checklist) made it possible to easily evaluate the housing condition from the resident's perspective. This study examined the relationship between housing coldness/warmth evaluation using the Checklist and psychological distress in a large-scale general Japanese population. STUDY DESIGN: A cross-sectional study. METHODS: We analysed data from 29,380 people aged ≥20 years who lived in Miyagi Prefecture, Japan. As an assessment of housing coldness/warmth, we used the Checklist. We classified participants' total scores on the Checklist related to coldness/warmth into quartiles. The Kessler 6 scale was used as an indicator of psychological distress. Multivariable logistic regression models were used to estimate the adjusted odds ratio (OR) and 95% confidence intervals (CIs). Adjusted OR and P-values for linear trends were calculated using the quartiles of the Checklists' score. RESULTS: Among participants in Q1 (i.e., poorer subjective house condition), the percentage of people with psychological distress was high. Compared to the highest quartile, Q1 showed poorer evaluation of housing coldness/warmth, and higher OR for psychological distress. The OR (95% CI) of psychological distress for Q3, Q2, and Q1 compared with Q4 were 1.93 (1.74-2.14), 2.82 (2.55-3.12), and 5.78 (5.25-6.35), respectively. CONCLUSIONS: Housing coldness/warmth evaluation was significantly related to psychological distress. This finding suggests that maintaining a comfortable thermal environment at home could be important for residents' mental health.

Toll-like receptor 3 in nasal CD103+ dendritic cells is involved in immunoglobulin A production.

Intranasal inoculation with influenza hemagglutinin subunit with polyinosine-polycytidylic (polyI:C), a synthetic analog for double-stranded RNA, enhances production of vaccine-specific immunoglobulin (Ig) A, which is superior to IgG in prophylactic immunity. The mechanism whereby polyI:C skews to IgA production in the nasal-associated lymph tissue (NALT) was investigated in mouse models. Nasally instilled polyI:C was endocytosed into CD103+ dendritic cells (DCs) and induced T-cell activation, including interferon (IFN)-γ production. According to knockout mouse studies, polyI:C activated the Toll-like receptor 3 signal via the adapter TICAM-1 (also called TRIF), that mainly caused T-cell-dependent IgA production. Nasal CD103+ DCs activated transforming growth factor-ß signaling and activation-induced cytidine deaminase upon polyI:C stimulation. IgA rather than IgG production was impaired in Batf3-/- mice, where CD103+ DCs are defective. Genomic recombination occurred in IgA-producing cells in association with polyI:C-stimulated DCs and nasal microenvironment. PolyI:C induced B-cell-activating factor expression and weakly triggered T-cell-independent IgA production. PolyI:C simultaneously activated mitochondrial antiviral signaling and then type I IFN receptor pathways, which only minimally participated in IgA production. Taken together, CD103+ DCs in NALT are indispensable for the adjuvant activity of polyI:C in enhancing vaccine-specific IgA induction and protective immunity against influenza viruses.

Novel biallelic mutations in the PNPT1 gene encoding a mitochondrial-RNA-import protein PNPase cause delayed myelination.

Recent studies suggest that impaired transcription or mitochondrial translation of small RNAs can cause abnormal myelination. A polynucleotide phosphorylase (PNPase) encoded by PNPT1 facilitates the import of small RNAs into mitochondria. PNPT1 mutations have been reported in patients with neurodevelopmental diseases with mitochondrial dysfunction. We report here 2 siblings with PNPT1 mutations who presented delayed myelination as well as mitochondrial dysfunction. We identified compound heterozygous mutations (c.227G>A; p.Gly76Asp and c.574C>T; p.Arg192*) in PNPT1 by quartet whole-exome sequencing. Analyses of skin fibroblasts from the patient showed that PNPase expression was markedly decreased and that import of the small RNA RNaseP into mitochondria was impaired. Exogenous expression of wild-type PNPT1, but not mutants, rescued ATP production in patient skin fibroblasts, suggesting the pathogenicity of the identified mutations. Our cases expand the phenotypic spectrum of PNPT1 mutations that can cause delayed myelination.

Earlier BMI rebound and lower pre-rebound BMI as risk of obesity among Japanese preschool children.

OBJECTIVES: Longitudinal growth data of children were analyzed to clarify the relationship between the timing of body mass index (BMI) rebound and obesity risk in later ages. SUBJECTS/METHODS: Of 54 558 children born between April 2004 and March 2005 and longitudinally measured in April and October every year in the preschool period, 15 255 children were analyzed wherein no longitudinal measurement is missing after 1 year of age. BMI rebound age was determined as the age with smallest BMI value across longitudinal individual data after 1 year of age. Rebound age was compared between overweight and non-overweight groups. The subjects were divided into groups based on the timing of rebound. The sex- and age-adjusted mean of the BMI, height and weight s.d. scores for age group, along with 6 months weight and height gain, were compared among groups using analysis of covariance. RESULTS: Among those who were overweight at 66-71 months of age, BMI rebound age obtained at approximately 3 years of age was compared with the non-overweight group, whose BMI rebound age was utmost 66 months or later (P<0.001). The comparison among BMI age group showed that earlier BMI rebound results in larger BMI (P<0.001) and larger weight and height gain after the rebound (P<0.001). Among the group with BMI rebound earlier than 30 months of age, low BMI was observed (P<0.001). Slight elevation of height and weight gain was observed before the BMI rebound among groups with rebound age earlier than 60 months of age (P<0.001). CONCLUSION: Earlier BMI rebound timing with pre-rebound low BMI leads to greater childhood obesity risk; hence, early detection and prevention is necessary for such cases.

Longitudinal changes in body mass index of children affected by the Great East Japan Earthquake.

BACKGROUND: The evacuation and disruption in housing caused by the 2011 Great East Japan Earthquake and following nuclear radiation may have influenced child health in many respects. However, studies regarding longitudinal childhood growth are limited. Therefore, in this study we aimed to explore the influence of the earthquake on longitudinal changes in body mass index in preschool children. METHODS: Participants were children from nursery schools who cooperated with the study in the Iwate, Miyagi and Fukushima prefectures. The exposed group consisted of children who experienced the earthquake during their preschool-age period (4-5 years old). The historical control group included children who were born 2 years earlier than the exposed children in the same prefectures. Trajectories regarding body mass index and prevalence of overweight/obesity were compared between the two groups using multilevel analysis. Differences in the changes in BMI between before and after the earthquake, and proportion of overweight/obesity was compared between the two groups. We also conducted subgroup analysis by defining children with specific personal disaster experiences within the exposed group. RESULTS: A total of 9722 children were included in the study. Children in the exposed group had higher body mass indices and a higher proportion of overweight after the earthquake than the control group. These differences were more obvious when confined to exposed children with specific personal disaster experiences. CONCLUSIONS: Children's growth and development-related health issues such as increased BMI after natural disasters should evoke great attention.

Homozygous c.14576G>A variant of RNF213 predicts early-onset and severe form of moyamoya disease.

OBJECTIVE: RNF213 was recently reported as a susceptibility gene for moyamoya disease (MMD). Our aim was to clarify the correlation between the RNF213 genotype and MMD phenotype. METHODS: The entire coding region of the RNF213 gene was sequenced in 204 patients with MMD, and corresponding variants were checked in 62 pairs of parents, 13 mothers and 4 fathers of the patients, and 283 normal controls. Clinical information was collected. Genotype-phenotype correlations were statistically analyzed. RESULTS: The c.14576G>A variant was identified in 95.1% of patients with familial MMD, 79.2% of patients with sporadic MMD, and 1.8% of controls, thus confirming its association with MMD, with an odds ratio of 259 and p < 0.001 for either heterozygotes or homozygotes. Homozygous c.14576G>A was observed in 15 patients but not in the controls and unaffected parents. The incidence rate for homozygotes was calculated to be >78%. Homozygotes had a significantly earlier age at onset compared with heterozygotes or wild types (median age at onset 3, 7, and 8 years, respectively). Of homozygotes, 60% were diagnosed with MMD before age 4, and all had infarctions as the first symptom. Infarctions at initial presentation and involvement of posterior cerebral arteries, both known as poor prognostic factors for MMD, were of significantly higher frequency in homozygotes than in heterozygotes and wild types. Variants other than c.14576G>A were not associated with clinical phenotypes. CONCLUSIONS: The homozygous c.14576G>A variant in RNF213 could be a good DNA biomarker for predicting the severe type of MMD, for which early medical/surgical intervention is recommended, and may provide a better monitoring and prevention strategy.

Normal hepatic hemodynamics during early postoperative period in recipients with adult live donor liver transplantation.

To recognize "normal" hepatic hemodynamics after live donor liver transplantation (LDLT), we analyzed Doppler parameters on recipients with a right liver graft and donors after extended left hepatectomy. Theoretically these values should be the same. From April 2000 to October 2004, 20 LDLTs were performed using a right liver graft. The 10 recipients without postoperative complications and their donors were included in this study. Portal venous velocity (PVV; cm/s), hepatic arterial peak systolic velocity (cm/s), and hepatic venous peak velocity (HVPV; cm/s) were measured during the first 2 weeks. In donors PVV and HVPV after LDLT were significantly higher after than before left hepatectomy: 19.2 +/- 4.2 vs. 31.5 +/- 13.0 cm/s (P = .013) and 23.0 +/- 7.2 vs. 41.8 +/- 10.3 cm/s respectively (P = .010). However, there were mild degrees of increased PVV and HVPV. In recipients, a markedly increased PVV (106.3 +/- 45.2 cm/s on day 1) was significantly higher than that in donors on each postoperative day. The hepatic arterial resistive index in recipients was also significantly higher than that in donors on each postoperative day, for example, 0.72 +/- 0.11 vs 0.62 +/- 0.04 on day 1 (P = .0326). In conclusion, we have shown "abnormal" hepatic hemodynamics in even those recipients without complications during the early postoperative period after LDLT.

Mild glycine encephalopathy (NKH) in a large kindred due to a silent exonic GLDC splice mutation.

BACKGROUND: Classic neonatal-onset glycine encephalopathy (GE) is devastating and life threatening. Milder, later onset variants have been reported but were usually sporadic and incompletely defined. OBJECTIVE: To determine the clinical and biochemical phenotype and molecular basis of mild GE in nine children from a consanguineous Israeli Bedouin kindred. METHODS: Genomic DNA was screened for GLDC, AMT, and GCSH gene mutations. GLDC expression in lymphoblasts was studied by Northern blot and reverse transcriptase PCR analysis. RESULTS: Clinical features included hypotonia, abnormal movements, convulsions, and moderate mental retardation with relative sparing of gross motor function, activities of daily living skills, and receptive language. Aggression and irritability were prominent. CSF-to-plasma glycine ratio was mildly to moderately elevated. All nine patients were homozygous and their parents heterozygous for a novel, translationally silent GLDC exon 22 transversion c.2607C>A. Lymphoblast GLDC mRNA levels were considerably reduced. Three aberrantly spliced cDNA species were identified: exon 22 and exon 22 to 23 skipping, and insertion of an 87-base pair cryptic exon. Homozygosity for c.2607C>A was also identified in an unrelated but haplotypically identical patient with an unusually favorable outcome despite severe neonatal-onset GE. Mutation analysis enabled prenatal diagnosis of three unaffected and one affected pregnancies. CONCLUSIONS: The mutation in this kindred led to missplicing and reduced GLDC (glycine decarboxylase) expression. The 4 to 6% of normally spliced GLDC mRNA in the patients may account for their relatively favorable clinical outcome compared with patients with classic glycine encephalopathy.

Glycine decarboxylase mutations: a distinctive phenotype of nonketotic hyperglycinemia in adults.

Three unrelated adult patients with mild hyperglycinemia, infantile hypotonia, mental retardation, behavioral hyperirritability, and aggressive outbursts were screened for glycine decarboxylase (GLDC) mutations; two novel missense mutations (A389V and R739H) were found. Both mutations had a 6 to 8% of normal GLDC activities when expressed in COS7 cells.

Association of the hCLCA1 gene with childhood and adult asthma.

Asthma is caused by bronchial inflammation. This inflammation involves mucus overproduction and hypersecretion. Recently, a mouse model of asthma showed that gob-5 is involved in the pathogenesis of asthma. The gob-5 gene is involved in mucus secretion and its expression is upregulated upon antigen attack in sensitized mice. The observation suggests that human homologue of gob-5, hCLCA1 (human calcium-dependent chloride channel-1), may be involved in human disease. We screened for single-nucleotide polymorphisms (SNPs) in hCLCA1 in the Japanese population. We identified eight SNPs, and performed association studies using 384 child patients with asthma, 480 adult patients with asthma, and 672 controls. In haplotype analysis, we found a different haplotype distribution pattern between controls and childhood asthma (P<0.0001) and between controls and adult asthma (P=0.0031). We identified a high-risk haplotype (CATCAAGT haplotype; P=0.0014) and a low-risk haplotype (TGCCAAGT haplotype; P=0.00010) in cases of childhood asthma. In diplotype analysis, patients who had the CATCAAGT haplotype showed a higher risk for childhood asthma than those who did not (P=0.0011). Individuals who had the TGCCAAGT haplotype showed a lower risk for childhood asthma than those who did not (P<0.0001). Our data suggested that variation of the hCLCA1 gene affects patients' susceptibility for asthma.

Long-term correction of hyperphenylalaninemia by AAV-mediated gene transfer leads to behavioral recovery in phenylketonuria mice.

Classical phenylketonuria (PKU) is a metabolic disorder caused by a deficiency of the hepatic enzyme phenylalanine hydroxylase (PAH). If untreated, accumulation of phenylalanine will damage the developing brain of affected individuals, leading to severe mental retardation. Here, we show that a liver-directed PAH gene transfer brought about long-term correction of hyperphenylalaninemia and behavioral improvement in a mouse model of PKU. A recombinant adeno-associated virus (AAV) vector carrying the murine PAH cDNA was constructed and administered to PAH-deficient mice (strain PAH(enu2)) via the portal vein. Within 2 weeks of treatment, the hyperphenylalaninemic phenotype improved and completely normalized in the animals treated with higher vector doses. The therapeutic effect persisted for 40 weeks in male mice, while serum phenylalanine concentrations in female animals gradually returned to pretreatment levels. Notably, this long-term correction of hyperphenylalaninemia was associated with a reversal of hypoactivity observed in PAH(enu2) mice. While locomotory activity over 24 h and exploratory behavior were significantly decreased in untreated PAH(enu2) mice compared with the age-matched controls, these indices were completely normalized in 12-month-old male PKU mice with lowered serum phenylalanine. These results demonstrate that AAV-mediated liver transduction ameliorated the PKU phenotype, including central nervous system dysfunctions.

Structure of human holocarboxylase synthetase gene and mutation spectrum of holocarboxylase synthetase deficiency.

Holocarboxylase synthetase (HLCS) is an enzyme that catalyzes the incorporation of biotin into apo-carboxylases, and its deficiency causes biotin-responsive multiple carboxylase deficiency. The reported sequences of cDNA for human HLCS from liver, lymphocyte, and KG-1 myeloid cell lines differ at their 5' regions. To elucidate variations of the human HLCS mRNA and longer 5' cDNA ends, we performed screening of the human liver cDNA library and rapid amplification of the cDNA ends (RACE). Our results suggest the existence of three types of HLCS mRNA that start at different exons. The first type starts at exon 1, and the second type starts at exon 3, and both are found in various human tissues. The third type, corresponding to the cDNA from the KG-1 cell, starts at exon 2 of the HLCS gene. Various splicing patterns from exons 3-6 were also observed. None of the variations of cDNA found created a new initiation codon. Mutation screening from exons 6-14, therefore, was sufficient to detect amino acid changes in HLCS in patients. Our direct sequencing strategy for screening mutations in the HLCS gene revealed mutations in five Japanese patients and seven non-Japanese patients. Our analyses involving 12 Japanese and 13 non-Japanese patients and studies by others indicate that (1) there is no panethnically prevalent mutation; (2) the Arg508Trp, Gly581Ser, and Val550Met mutations are found in both Japanese and non-Japanese populations; (3) the IVS10+5G-->A mutation is predominant and probably a founder mutation in European patients; (4) the 655-656insA, Leu237Pro, and 780delG mutations are unique in Japanese patients; (5) the spectrum of the mutations in the HLCS gene may vary substantially among different ethnic groups.

GJB2 (connexin 26) mutations and childhood deafness in Thailand.

HYPOTHESIS: The purpose of this study was to elucidate whether GJB2 mutations are responsible for childhood deafness in Southeast Asia. BACKGROUND: GJB2 mutations are responsible for a large part of childhood deafness in many countries. In Whites, there is a common mutation (35delG) that accounts for about 70 to 80% of the GJB2 mutations. Previously, we and others reported a common GJB2 mutation (235delC) in Japanese patients with prelingual deafness. The association of the 235delC mutation with a single haplotype suggested a founder effect of the mutation. METHODS: We analyzed the GJB2 gene in 17 deaf patients from 12 unrelated families in Thailand. Genomic DNA was extracted from peripheral lymphocytes of each patient and the entire coding region of the GJB2 gene was sequenced. RESULTS: GJB2 mutations were found in 4 patients in 3 families. Patient 1 was a homozygote of 235delC. Patient 2 was a compound heterozygote of 235delC and W24X (71G --> A). Patient 3A and 3B (in 1 family) were heterozygotes of a novel mutation M34L (100A --> T). CONCLUSION: The 235delC mutation may be widely distributed in Asian countries outside of Japan.

A novel mutation in glial fibrillary acidic protein gene in a patient with Alexander disease.

Alexander disease is a rare, progressive, leukoencephalopathy whose hallmark is the widespread accumulation of Rosenthal fibers. The most common form affects infants and young children, and is characterized by progressive failure of central myelination, usually leading to death before adulthood. Definitive diagnosis of Alexander disease has required biopsy or autopsy to demonstrate the presence of Rosenthal fibers. However, missense mutations in the coding region of the glial fibrillary acidic protein (GFAP) gene have recently been associated with a high percentage of pathologically proven cases. Here we report that a 10-year-old Japanese patient who showed clinical signs of Alexander disease is heterozygous for a C to T transition in which predicts a novel A244V amino acid substitution in the conserved 2A alpha-helix domain of GFAP. The nucleotide change was not found in 65 normal individuals (130 alleles). These results provide further support for a causative role for GFAP mutations in Alexander disease, and suggest DNA sequencing as an alternative diagnostic to biopsy.

Structure and expression of the glycine cleavage system in rat central nervous system.

The glycine cleavage system (GCS) is a mitochondrial multienzyme system consisting of four individual proteins, three specific components (P-, T-, and H-proteins) and one house-keeping enzyme, dihydrolipoamide dehydrogenase. Inherited deficiency of the GCS causes nonketotic hyperglycinemia (NKH), an inborn error of glycine metabolism. NKH is characterized by massive accumulation of glycine in serum and cerebrospinal fluids and severe neuronal dysfunction in neonates. To elucidate the neuropathogenesis of NKH, we cloned cDNAs encoding three specific components of the GCS and studied the gene expression in rat central nervous system. P-, T-, and H-protein cDNAs encoded 1024, 403, and 170 amino acids, respectively. In situ hybridization analysis revealed that P-protein mRNA was expressed mainly in glial-like cells, including Bergmann glias in the cerebellum, while T- and H-protein mRNAs were detected in both glial-like cells and neurons. T- and H-protein mRNAs, but not P-protein mRNA, were expressed in the spinal cord. Primary astrocyte cultures established from cerebral cortex had higher GCS activities than hepatocytes whereas those from spinal cord expressed only H-protein mRNA and had no enzymatic activity. An important role of glycine as inhibitory neurotransmitter has been established in the brainstem and spinal cord and another role of glycine as an excitation modulator of N-methyl-D-aspartate receptor is suggested in the hippocampus, cerebral cortex, olfactory bulbus, and cerebellum. Our results suggest that the GCS plays a major role in the forebrain and cerebellum rather than in the spinal cord, and that N-methyl-D-aspartate receptor may participate in neuropathogenesis of NKH.

Complete suppression of insulitis and diabetes in NOD mice lacking interferon regulatory factor-1.

Interferon regulatory factor-1 (IRF-1), a transcriptional factor, regulates type I interferon and interferon-induced genes. It was reported that IRF-1 regulates important molecules required for inflammation and immune reactions. To investigate the role of IRF-1 in the development of autoimmune diabetes, we established IRF-1 deficient (IRF-1(-/-)) non-obese diabetic (NOD) mice. IRF-1-deficient C57BL/6J mice were out-crossed to NOD mice, and F1 were backcrossed to NOD mice. At the N8 generation, the heterozygote for IRF-1 mutation was intercrossed and N8F1 was obtained. Out of three NOD genotypes, IRF-1(+/+) and IRF-1(+/-) developed spontaneous diabetes with an incidence of 47% (9/19) and 50% (10/20) by 30 weeks of age, respectively; whereas IRF-1(-/-) did not develop diabetes (0/18, P< 0.01 vs. (+/+) and (+/-)). Histologically, IRF-1(+/+) and IRF-1(+/-) had various degrees of insulitis, but IRF-1(-/-) had no insulitis. In comparison with IRF-1(+/+), the percentage of CD4(+) and Mac-1(+) splenic cells significantly increased, whereas CD3(+), CD8(+) and B220(+) cells decreased in IRF-1(-/-). Furthermore, spleen cell proliferation in response to Con A or murine GAD65 peptide, a major autoantigen of the pancreatic beta-cell, significantly increased, and the IFN-gamma/IL-10 ratio in the culture supernatant significantly decreased in IRF-1(-/-), suggesting Th2 deviation in cytokine balance. These results indicate that IRF-1 plays a key role in developing insulitis and diabetes in NOD mice.

Chromosomal localization, structure, single-nucleotide polymorphisms, and expression of the human H-protein gene of the glycine cleavage system (GCSH), a candidate gene for nonketotic hyperglycinemia.

Nonketotic hyperglycinemia (NKH) is an inborn error of metabolism caused by deficiency in the glycine cleavage system (GCS); this system consists of four individual constituents, P-, T-, H-, and L-proteins. Several mutations have been identified in P- and T-protein genes, but not in the H-protein gene (GCSH), despite the presence of case reports of H-protein deficiency. To facilitate the mutational and functional analyses of GCSH, we isolated and characterized a human p1-derived artificial chromosome (PAC) clone encoding GCSH. GCSH spanned 13.5kb and consisted of five exons. Using the PAC clone as a probe, we mapped GCSH to chromosome 16q24 by fluorescence in situ hybridization. The transcription initiation site was determined by the oligonucleotide-cap method, and potential binding sites for several transcriptional factors were found in the 5' upstream region. Direct sequencing analysis revealed five single-nucleotide polymorphisms. The expression profiles of P-, T-, and H-protein mRNAs were studied by dot-blot analysis, using total RNA from various human tissues. GCSH was expressed in all 29 tissues examined, while T-protein mRNA was detected in 27 of the 29 tissues. In contrast, the P-protein gene was expressed in a limited number of tissues, such as liver, kidney, brain, pituitary gland, and thyroid gland, suggesting distinct transcriptional regulation of each GCS constituent.