Increased production of nitric oxide by phagocytic stimulated neutrophils in patients with chronic granulomatous disease.

Chronic granulomatous disease (CGD) is an inherited disorder in which phagocytic leukocytes fail to generate superoxide (O(2)(-)) and antimicrobial oxidants. Patients with CGD develop recurrent and often life-threatening infections due to catalase-positive microorganisms. We attempted to measure the production of nitric oxide and reactive oxygen species in polymorphonuclear neutrophils (PMNs) from patients with CGD using a flow cytometry technique. Venous blood was obtained from 5 male patients with X-linked CGD and from 10 healthy volunteers. The nitric oxide production by PMNs after phagocytosis was measured using diaminofluorescein-2 diacetate, a fluorescent indicator of intracellular nitric oxide production. After erythrocytes were hypotonically lysed, the cell pellet was applied to a cytofluorometer. Although the production of hydrogen peroxide by PMNs from patients with CGD was almost absent, nitric oxide production was detected at nearly half the level as was seen in the PMNs from healthy volunteers (CGD vs. healthy volunteers=140.1±28.6 vs. 256.4±10.3, mean fluorescence intensity; P<0.01). In conclusion, we demonstrated that human PMNs produces nitric oxide after phagocytic stimulation. Also nitric oxide production after phagocytic stimulation by PMNs of patients with CGD is observed although its amount is lower than that observed on healthy control. Despite the fact that CGD patients cannot produce H(2)O(2) which is essential for intracellular bacteriocidal process after phagocytosis, our data suggested that the phylactic effect in PMNs induced by nitric oxide could be at least partially related to the survival of patients with CGD.

GSTT1 gene abnormality in minimal change nephrotic syndrome with elevated serum immunoglobulin E.

INTRODUCTION: Imbalance between T-helper 1 (Th1) and 2 (Th2) lymphocytes and effects of reactive oxygen species (ROS) upon glomerular capillary walls have been implicated in minimal change nephrotic syndrome (MCNS). METHODS: By polymerase chain reaction and comparative genomic hybridization, we evaluated mutations of the GSTT1 gene (GSTT1), a member of the glutathione S-transferase (GST) supergene family associated with both protection of cells from ROS and control of allergic reactions and serum immunoglobulin (Ig) E. RESULTS: Among 15 children with MCNS, IgE elevation (over 2,000 IU/l) and GSTT1 deletion was found in 2 who showed severe allergic symptoms. Serum ROS concentrations in these 2 patients were significantly higher than in healthy controls or other MCNS patients. In addition, a Th2 shift caused by increased serum interleukin (IL)- 4 was observed. CONCLUSION: These results suggest presence of a GSTT1 abnormality in some children with MCNS having marked serum IgE elevations and various allergic complications. Defective ROS degradation and Th1/Th2 imbalance caused by GSTT1 abnormality could initiate proteinuria leading to MCNS.

Production of nitric oxide is lower in Shiga toxin-stimulated neutrophils of infants compared to those of children or adults.

Hemolytic uremic syndrome (HUS) in infants is mainly caused by the Shiga toxin (Stx), which is produced by pathogenic Escherichia coli O157:H7. Infants are prone to develop HUS in comparison to older children and adults, but its underlying mechanism remains unknown. Recent observations suggest that reactive oxygen species (ROS) and reactive nitrogen species (RNS) including nitric oxide (NO) may be involved in the pathogenesis of HUS. We therefore measured NO production by neutrophils prepared from infants (6-27 months old), children (5.3-11 years old) or adults (25-47 years old). The NO production was measured by a flow cytometric analysis with a fluorescent indicator (expressed as mean fluorescence intensity), and mRNA expression of inducible NO synthase (iNOS) was analyzed by reverse transcription-polymerase chain reaction (RT-PCR). The amount of NO produced was significantly lower in Stx-stimulated neutrophils prepared from infants (45.8 ± 23.3) than that in those from children (120.5 ± 81.5) or adults (127.7 ± 45.8) (n = 10 each group, P < 0.05). The expression level of iNOS mRNA was lower in Stx-stimulated neutrophils of the infants than the level in those of children or adults. In conclusion, Stx increased NO production in neutrophils probably via iNOS. Importantly, the degree of the Stx-mediated increase in NO production was lower in neutrophils of infants compared to those of children or adults, which may explain the higher incidence of HUS in infants. These results suggest that NO may contribute to the cellular defense mechanisms against Stx.

Diagnosis of autoimmune neutropenia by neutrophil-bound IgG and IgM antibodies.

Autoimmune neutropenia (AIN) in infancy is caused by antineutrophil (granulocyte-specific) autoantibodies. These antibodies are rarely found in circulation because their serum levels are extremely low. We hypothesized that a direct granulocyte immunofluorescence test (D-GIFT) that enables us to detect neutrophil-bound autoantibodies consisting of both immunoglobulin (Ig) G and IgM has better diagnostic value than the detection of circulating autoantibodies. Whole blood (100 µL) was obtained from 50 infants with AIN, 12 infants with transient neutropenia, and 37 control infants. D-GIFT was performed using both fluorescein isothiocyanate-conjugated antihuman IgG Fc portion monoclonal antibodies and fluorescein isothiocyanate antihuman IgM monoclonal antibodies. Results were assessed as relative fluorescence intensity (RFI). The RFIs of antineutrophil IgG-bound and antineutrophil IgM-bound cells in patients with AIN were significantly higher than those in patients with transient neutropenia and in controls. Positive results, as assessed by RFI scores of more than 1.81 in either antineutrophil IgG-bound or antineutrophil IgM-bound cells, showed the sensitivity and specificity of D-GIFT, and the areas under the receiver operating characteristic curve (0.98, 0.98, and 0.997, respectively) in the diagnosis of AIN. D-GIFT detecting both neutrophil-bound IgG autoantibodies and IgM autoantibodies has discriminatory power for identifying patients with AIN and, therefore, can be a useful diagnostic test.

Upregulation of hepatic 11ß-hydroxysteroid dehydrogenase-1 expression in calcium-deficient rats.

BACKGROUND: Many epidemiologic studies have reported a link between calcium (Ca) deficiency and metabolic syndrome. In this study, we examine Ca deficiency in rats and whether changes in glucocorticoid metabolism are induced. METHODS: Twelve-week-old female Wistar rats were weaned onto a very-low-Ca diet (low-Ca group) or a control diet (control group) for 2 weeks. Quantitative real-time PCR was used to assess mRNA for 11ß-hydroxysteroid dehydrogenase-1 (11ß-HSD1), 11ß-HSD2, phosphoenolpyruvate carboxykinase, peroxisome proliferator-activated receptor-α, and glucocorticoid receptor in the liver. Concentrations of adiponectin, leptin, corticosterone, intact parathyroid hormone, asymmetrical dimethylarginine and insulin in fasting serum were determined using a rat-specific enzyme-linked immunosorbent assay. Glucose concentrations were measured using a glucose oxidase system. Serum ionized Ca levels were measured with an automatic ion-selective electrode analyzer. Serum nitrite/nitrate levels were measured using a colorimetric assay kit. RESULTS: After 2 weeks, no differences in serum glucose, corticosterone or insulin levels were observed. The low-Ca group rats showed higher homeostasis model assessment insulin resistance, lower adiponectin and higher intact parathyroid hormone levels. Serum nitrite/nitrate and asymmetrical dimethylarginine were significantly higher in the low-Ca group than in the control group. The expression of hepatic 11ß-HSD1 mRNA was upregulated, while hepatic phosphoenolpyruvate carboxykinase expression was downregulated in the low-Ca group. Glucocorticoid receptor, peroxisome proliferator-activated receptor-α and 11ß-HSD2 expression levels showed a similar tendency. CONCLUSION: A low-Ca diet alters glucocorticoid metabolism, which leads to hepatic upregulation of 11ß-HSD1, and is possibly a key mechanism inducing the metabolic complications of Ca deficiency.

Increased nitric oxide production by T- and B-cells in idiopathic nephrotic syndrome.

The pathogenesis of idiopathic nephrotic syndrome (INS) remains unclear. To study the role of nitric oxide (NO) in INS, we measured intracellular NO produced by T- and B-cells using a novel fluorescent indicator. Twelve children with INS (mean age 7.3 years; group A-1: in relapse, group A-2: in remission) were enrolled in the study together with 16 children with other renal diseases (9.5 years; group B) and 42 healthy control children (7.7 years; group C). The amount of NO produced by CD3+ cells (T-cells) and CD19+ cells (B-cells) and of plasma NO(x) was measured by flow cytometry and colorimetry, respectively. The average amount of NO produced by CD3+ and CD19+ cells in group A-1 subjects was significantly higher than that produced by these cells in group A-2 and B patients and the healthy controls (group C), respectively (P < 0.01), and it decreased after the patients achieved remission (P < 0.01). Plasma NO(x) levels in group A-1 patients was also highest among the different groups (P < 0.01). There were no significant differences in intracellular NO and plasma NO(x) among group A-2, B, and C subjects (P > 0.05). A significant correlation between plasma NO(x) and urinary protein excretion was found only in group A patients and not in group B patients. We conclude that an aberrant immune system may exist not only in T-cells but also in B-cells, and NO may play some role in INS.

Increased nitric oxide production by neutrophils in early stage of Kawasaki disease.

Recent observations suggested that nitric oxide (NO) has a role in triggering the early endothelial dysfunction in Kawasaki disease (KD). We investigated the amount of NO in conjunction with reactive oxygen species (ROS) produced by neutrophils in children with acute KD by a newly developed flow cytometric analysis. Forty children with acute KD (n = 14), non-KD febrile disease (n = 14), and afebrile control (n = 12) were enrolled (age, 3 to 88 months). Neutrophils in KD produced significantly higher amount of NO compared to others (p < 0.05). With regard to ROS, significant increase was not only found in KD but also in non-KD febrile children (p < 0.05 and p < 0.01, respectively). In KD patients, the amount of NO produced by neutrophils decreased after immunoglobulin (IVIG) treatment, while there was no significant change in ROS production. The amount of NO in KD patients also correlated well with the days from the onset. One patient who developed coronary arterial lesion showed the highest value of NO. In conclusion, neutrophils in acute KD generate both NO and ROS considerably, while NO production is exclusive in the early stage of KD before IVIG treatment. Abnormal immune system in KD might be characterized by an overproduction of NO, whereas the role of NO in endothelial damage remains to be elucidated.

A calcium-deficient diet in pregnant, nursing rats induces hypomethylation of specific cytosines in the 11ß-hydroxysteroid dehydrogenase-1 promoter in pup liver.

Prenatal undernutrition affects offspring phenotype via changes in the epigenetic regulation of specific genes. We hypothesized that pregnant females that were fed a calcium (Ca)-deficient diet would have offspring with altered hepatic glucocorticoid-related gene expression and altered epigenetic gene regulation. Female Wistar rats ate either a Ca-deficient or control diet from 3 weeks before conception to 21 days after parturition. Pups were allowed to nurse from their original mothers and then euthanized on day 21. Methylation of individual cytosine-guanine dinucleotides in the phosphoenolpyruvate carboxykinase (Pck1), peroxisome proliferator-activated receptor α (Ppara), glucocorticoid receptor (Nr3c1), 11ß-hydroxysteroid dehydrogenase-1 (Hsd11b1), and 11ß-hydroxysteroid dehydrogenase-2 (Hsd11b2) promoters was measured in liver tissue using pyrosequencing. For each gene, quantitative real-time polymerase chain reaction was used to assess mRNA levels in liver tissue. Overall Hsd11b1 methylation was lower in the Ca-deficient group than in the control group; however, overall methylation of each other gene did not differ between groups. Serum corticosterone levels in male pups from Ca-deficient dams were higher than those in control pups. Expression of Pck1 and Nr3c1 was lower in the Ca-deficient group than in the control group. A Ca-deficient diet for a dam during gestation and early nursing may alter glucocorticoid metabolism and lead to higher intracellular glucocorticoid concentrations in the hepatic cells of her offspring; moreover, this abnormal glucocorticoid metabolism may induce the metabolic complications that are associated with Ca deficiency. These findings indicated that prenatal nutrition affected glucocorticoid metabolism in offspring in part by affecting the epigenome of offspring.

MEFV Variants in Patients with PFAPA Syndrome in Japan.

BACKGROUND: The pathogenesis of PFAPA (periodic fever, aphthous stomatitis, pharyngitis, adenitis) syndrome is unknown as yet. In order to understand whether genes implicated in other auto-inflammatory diseases might be involved in the pathogenesis of PFAPA, all variants in the genes causing familial Mediterranean fever (FMF), tumor necrosis factor (TNF) receptor-associated periodic syndrome (TRAPS), and Hyper IgD syndrome were analyzed in children with PFAPA. PATIENTS AND METHODS: All variants in MEFV, TNFRSF1A, and MVK were analyzed in 20 patients with PFAPA. PFAPA were diagnosed by previous published criteria. The findings of all analyses in PFAPA patients were compared with those of unaffected normal subjects (n=62). RESULTS: In the 13 children of 20 with PFAPA, the heterozygous variants of MEFV (5 patients: E148Q-L110P, 2 patients: E148Q, 1 patient: E148Q-L110P/E148Q, 1 patient: E148Q-P369S-R408Q-E84K, 1 patient: E148Q-L110P-P369S-A408G, 1 patient: R202Q, 1 patient: P115R) were found. No variants belonging to TNFRSF1A or MVK were detected in children with PFAPA. The frequency of the E148Q-L110P variants in children with PFAPA was significantly higher than that observed in unaffected normal subjects (7/20 versus 8/62). The duration of the episodes of illness in PFAPA children with MEFV variants was shorter than that of patients without variants. CONCLUSION: Genes involved in the development and progression of MEFV may affect the incidence and the phenotype of PFAPA in children.

Down-regulation of hepatic phosphoenolpyruvate carboxykinase expression in magnesium-deficient rats.

Many epidemiological studies have reported the link between magnesium deficiency and metabolic syndrome. We examined whether magnesium deficiency in rats induces changes in glucocorticoid metabolism. Twelve-week-old, female Wistar rats were weaned onto a very low-magnesium diet or a control diet for two weeks. Quantitative real-time PCR was used to assess mRNA for 11ß hydroxysteroid dehydrogenase-1 (11ß-HSD1), 11ß-HSD2, phosphoenolpyruvate carboxykinase (PEPCK), peroxisome proliferator-activated receptor α (PPARα), and glucocorticoid receptor in the liver. Concentrations of adiponectin, leptin, corticosterone, insulin and asymmetric dimethylarginine (ADMA) in fasting serum were determined using a rat-specific enzyme-linked immunosorbent assay. After two weeks, no differences in serum glucose, leptin, corticosterone, or adiponectin levels were observed between the groups. Magnesium-deficient rats showed higher HOMA-IR, insulin, ionized calcium, ADMA levels and diastolic blood pressure. There were no significant differences in hepatic mRNA expression levels of GR, 11ß-HSD1, 11ß-HSD2, or PPARα between the groups. We observed lower expression of hepatic PEPCK mRNA, in the magnesium-deficient rats, thus suggesting a possible compensatory mechanism to diminish glycogenesis. A low-magnesium diet alters glucocorticoid metabolism, which leads to endothelial damage. Higher ADMA induces hypertension and insulin resistance. Hyperinsulinemia induces hepatic down-regulation of PEPCK, and is possibly a key mechanism inducing the metabolic complications of magnesium deficiency.

Magnesium deficiency in pregnant rats alters methylation of specific cytosines in the hepatic hydroxysteroid dehydrogenase-2 promoter of the offspring.

Prenatal under-nutrition involves changes in the epigenetic regulation of specific genes. Maternal magnesium (Mg) deficiency affects maternal glucocorticoid metabolism, but the mechanisms underlying changes in glucocorticoid homeostasis of offspring are not well understood. In this study, we investigated the effects of feeding pregnant rats a Mg-deficient diet (0.003% magnesium) on the methylation of cytosine-guanine (CpG) dinucleotides in hepatic glucocorticoid genes of neonatal offspring, compared with controls (0.082% magnesium). Methylation of CpG dinucleotides in the peroxisome proliferator-activated receptor α (Ppara), glucocorticoid receptor (Nr3c1) and 11ß-hydroxysteroid dehydrogenase-2 (Hsd11b2) promoters in the liver were measured by pyrosequencing. Quantitative real-time PCR was used to assess hepatic mRNA expression of each gene. Mean methylation of the Hsd11b2 promoter in the Mg-deficient offspring (33.2%) was higher than in controls (10.4%). This was due to a specific increase at CpG dinucleotides 1 (20.0% vs. control 10.1%), 2 (58.8% vs. 17.0%), 3 (29.7% vs. 6.2%) and 4 (38.7% vs. 8.8%) (p < 0.05). Ppara and Nr3c1 methylation status and expression did not differ between the groups. No significant difference was noted between male and female pups, which were equally represented. Therefore, a Mg-deficient diet alters glucocorticoid metabolism, predicting higher hepatic intracellular glucocorticoid concentrations, and is possibly a key mechanism that induces the metabolic complications of Mg deficiency.