Shoji Tsuji

Role of reactive oxygen species in neutrophil apoptosis following ingestion of heat‐killed Staphylococcus aureus

Neutrophils, short‐lived leucocytes that die by apoptosis, play an important role in the first stage of defense against bacterial infections. It has been reported that phagocytosis of intact bacteria or Candida albicans can accelerate neutrophil apoptosis. However, the mechanism of phagocytosis‐mediated neutrophil apoptosis is not well characterized. In this study, we evaluated whether ingestion of heat‐killed Staphylococcus aureus (S. aureus) enhances neutrophil apoptosis and whether this type of apoptosis is mediated by oxidative stress by using antioxidants and polymorphonuclear leucocytes (PMNs) from patients with chronic granulomatous disease (CGD). Co‐culture of PMNs with varying doses of S. aureus resulted in accelerated PMN death in a dose‐ and time‐dependent manner. Increased PMN apoptosis was observed by both Annexin V and PI staining. Similar results were observed in PMNs of CGD patients. Dimethyl sulphoxide (DMSO, an OH• scavenger) did not significantly inhibit either S. aureus‐ingested PMN apoptosis or spontaneous PMN apoptosis. On the other hand glutathione (GSH, an H2O2 scavenger) significantly inhibited both types of apoptosis.

Increased nitric oxide production by neutrophils from patients with chronic granulomatous disease on trimethoprim–sulfamethoxazole

Chronic granulomatous disease (CGD) is an inherited disease characterized by severe and recurrent bacterial and fungal infections. Phagocytic cells of CGD patients are unable to produce superoxide anion, and their efficiency in bacterial killing is significantly impaired. In these patients, the prophylactic and therapeutic validity of a long-term use of trimethoprim-sulfamethoxazole (TMP-SMX) has been well established. However a role of nitric oxide (NO) produced by phagocytic cells from CGD patients is unknown, and the mechanism of TMP-SMX in CGD is unclear. We have directly measured NO production in whole human blood by using 4,5-diaminofluorescein as a novel fluorescent indicator for intracellular NO. Intracellular NO production of gated neutrophils increased time dependently when stimulated by lipopolysaccharide (LPS) and calcium ionophore. Although all polymorphonuclear leukocyte (PMN) specimens from patients with CGD failed to generate hydrogen peroxide, NO production by CGD PMNs was significantly increased compared with that of control PMNs (p<0.05). TMP-SMX with LPS significantly increased compared with LPS-stimulated samples at clinical (n=5, p<0.05) and 10-fold clinical concentrations (n=5, p<0.01). TMP-SMX with LPS in CGD PMNs significantly increased the production of NO in comparison with the LPS stimulation at 10-fold clinical concentrations (n=5, p<0.05). In conclusion, our data indicate the possibility that NO production by neutrophils from patients with CGD treated with TMP-SMX has a role of bactericidal activity instead of O(2)(-) in host defense mechanism.

Dizygotic twin sisters with myelokathexis: Mechanism of its neutropenia

Dizygotic twin sisters were first found to have neutropenia at 1 year of age when evaluated for recurrent pulmonary infections. Since then they have remained neutropenic (0.05∼0.5 × 109/l). Despite of their neutropenia, myeloid hyperplasia was evident on a marrow smear examination, and a number of cells were hypersegmented with fine interlobular bridging with chromatin strands and cytoplasmic vacuolation. Electron microscopy showed apoptotic cells with condensed nuclei and apoptotic bodies in the cytoplasm. Although life span, hydrogen peroxide production, phagocytosis, spreading, and chemotaxis of peripheral neutrophils were normal, the survival of bone marrow neutrophils in both infants was markedly decreased when compared with that of normal bone marrow neutrophils. During the bone marrow culture apoptotic neutrophils were observed at an earlier stage in both patients than in normal controls, biochemically and morphologically. Morphology of bone marrow neutrophils in both patients resembled that of cultured control bone marrow neutrophils. Peripheral neutropenia and appearance of characteristic neutrophils in the bone marrow in myelokathexis are considered to be an expression of apoptosis of bone marrow neutrophils. Am. J. Hematol. 62:106–111, 1999.

A young child with pseudohypoaldosteronism type II by a mutation of Cullin 3

BackgroundPseudohypoaldosteronism type II (PHA II), also referred to as Gordon syndrome, is a rare renal tubular disease that is inherited in an autosomal manner. Though mutations in WNK1 and WNK4 partially account for this disorder, in 2012, 2 research groups showed that KLHL3 and CUL3 were the causative genes for PHA II. Here, we firstly report on the Japanese child of PHA II caused by a mutation of CUL 3.Case presentationThe patient was a 3-year-old Japanese girl having healthy unrelated parents. She was initially observed to have hyperkalemia, hyperchloremia, metabolic acidosis, and hypertension. A close investigation led to the diagnosis of PHA II, upon which abnormal findings of laboratory examinations and hypertension were immediately normalized by administering thiazides. Genetic analysis of WNK1 and WNK4 revealed no mutations. However, analysis of the CUL3 gene of the patient showed abnormal splicing caused by the modification of exon 9. The patient is currently 17 years old and does not exhibit hypertension or any abnormal findings on laboratory examination.ConclusionsIn this patient, CUL3 was found to play a fundamental role in the regulation of blood pressure, potassium levels, and acid–base balance.

B-type natriuretic peptide for assessment of haemodynamically significant patent ductus arteriosus in premature infants.

Haemodynamically significant patent ductus arteriosus (hsPDA) is frequently observed in premature infants. This study was conducted to explore whether the blood BNP can be a valuable biomarker to assess the necessity of treatment for hsPDA in premature infants.

Differential diagnosis and clinical course of autoimmune neutropenia in infancy: comparison with congenital neutropenia

Aim: Autoimmune neutropenia in infancy (AIN) is caused by granulocyte‐specific autoantibodies. Clinical presentation and diagnosis have not been well studied, resulting in cumbersome diagnostic investigations and unnecessary treatment such as granulocyte colony‐stimulating factor (G‐CSF) therapy. Methods: Clinical, laboratory and immunological data of 18 infants with AIN were evaluated. Granulocyte‐specific autoantibodies were detected by the direct granulocyte immuno‐fluorescence test (D‐GIFT), indirect granulocyte immunofluorescence test (I‐GIFT) and immuno‐blotting. Results: The average age of onset and resolution of neutropenia in AIN was 7.4 ± 3.4 mo (mean ± SD) and 20.4 ± 4.9 mo, respectively. Sixteen of the 18 patients presented with mild infectious symptoms; the other 2 patients were detected by chance and presented with no infectious symptoms. D‐GIFT was positive in all patients, and I‐GIFT was positive in 17 of these 18 patients. Most patients showed preferential binding to neutrophils from NA(1 + 2−)‐phenotyped donors by I‐GIFT and immunoblotting. An antibiotic (sulfamethoxazole‐trimethoprim) was given to 15 patients for prophylaxis. G‐CSF was given to only one infectious patient.

Pathogenesis of childhood idiopathic nephrotic syndrome: a paradigm shift from T-cells to podocytes

BackgroundNephrotic syndrome is the most common cause of kidney disease in children, but its pathogenesis remains unclear. This article reviews the novel aspects of the mechanisms underlying massive proteinuria in minimal-change disease, which is the most common form of childhood nephrotic syndrome.Data sourcesThis article integrates the findings of a PubMed database search for English language articles published in the past 40 years (from September 1974 to February 2014) using the key words “pathogenesis”, “minimal change nephrotic syndrome” or “idiopathic nephrotic syndrome”.ResultsUnknown humoral factors associated with T-cell dysfunction have been thought to play an important role in the pathogenesis of minimal-change disease. However, recent findings are changing this paradigm, i.e., visceral glomerular epithelial cells (podocytes) may be involved via expression of molecules such as CD80 and angiopoietin-like 4.ConclusionsRecent evidence suggests that minimalchange disease results from interactions between humoral factors and dysfunctional podocytes. In addition to immunosuppressant drugs that target lymphocytes, a biological agent such as an antibody against the abnormal molecule(s) expressed by podocytes may provide novel drug treatment for minimal-change disease.

Urinary Biomarkers for Screening for Renal Scarring in Children with Febrile Urinary Tract Infection: Pilot Study

PURPOSE Recurrent febrile urinary tract infections during infancy cause renal scarring, which is characterized by progressive focal interstitial fibrosis and may lead to renal failure. Renal scarring can be diagnosed through scintigraphy, although it seems impractical to perform renal scintigraphy for all infants with febrile urinary tract infections. Therefore, it is important to search for a biomarker to identify the presence of renal scarring. We hypothesized that urinary biomarkers of nephropathy may increase in infants with renal scarring following febrile urinary tract infections. MATERIALS AND METHODS A total of 49 infants who underwent renal scintigraphy for febrile urinary tract infections were enrolled in the study. Several measurements were performed using urine samples, including total proteins, beta2-microglobulins, N-acetyl-β-D-glucosaminidase, neutrophil gelatinase associated lipocalin, liver-type fatty acid binding protein and angiotensinogen. Values were corrected by creatinine and compared between patients with and without renal scarring. RESULTS Among urinary biomarkers only angiotensinogen in patients with scarring (median 14.6 μg/gm creatinine) demonstrated significantly higher levels than in patients without scarring (3.6 μg/gm creatinine, p <0.001). CONCLUSIONS Urinary angiotensinogen may be useful for diagnosing the presence of renal scarring.

Close association between proteinuria and regulatory T cells in patients with idiopathic nephrotic syndrome

BackgroundIdiopathic nephrotic syndrome (INS) has been considered to be a T cell disorder. Supporting this hypothesis is the reported occurrence of remission following measles infection, which suppresses T cell function. In contrast, there has been no case report suggesting an association between influenza B virus infection and the remission of INS.Case-Diagnosis/TreatmentWe report the case of a 5-year-old boy with INS who achieved remission without steroid treatment in response to influenza B virus infection. Although he relapsed soon after remission, he was successfully treated with prednisolone. Both the induction of remission and the response to prednisolone were associated with an increase in the number of circulating regulatory T cells (Tregs), assessed as CD4+CD25+Foxp3+ cells. These results suggest that both influenza B virus infection and steroid administration increased the number of circulating Tregs, thus leading to the remission of INS.ConclusionsIn summary, our case indicates an important role for Tregs in the development of the proteinuria associated with INS and sheds light on its pathogenesis. Further studies are warranted.

Change in urinary 8-hydroxydeoxyguanosine in idiopathic nephrotic syndrome

Sirs, We read with great interest the article entitled “Antioxidant status of children with idiopathic nephrotic syndrome” by Mishra et al. which was recently published in Pediatric Nephrology [1]. Oxidative stress (OS), defined as a disturbance in the reactive oxygen species (ROS) and antioxidant balance, can result either from low levels of antioxidants and/or from an increased production of ROS. It has been postulated that childhood idiopathic nephrotic syndrome (INS) is associated with OS due to increased levels of ROS and decreased levels of antioxidants [2], although results to the contradictory have been published [3]. Mishra et al. [1] report evidence of OS and impaired antioxidant defense during acute INS. 8-Hydroxydeoxyguanosine (8-OHdG), which originates from damaged DNA repaired by non-specific endonucleases and specific glycosylases and is eliminated into the urine, is currently accepted as a sensitive biomarker for oxidative DNA damage, as OS leads to the damage of not only lipids and proteins, but also nucleic acids [4]. In order to investigate oxidative cellular damage in INS, we are currently performing serial measurements of urinary 8-OHdG using a novel analyzer (model No. ICR001; Techno-Medica, Yokohama, Japan). Here, we report changes in urinary 8-OHdG during five nephrotic relapses in four patients with INS (ages 3, 4, 15, and 20 years, respectively) and three patients with poststreptococcal acute glomerulonephritis (PSAGN; ages 4, 7, and 16 years, respectively). As shown in Fig. 1, the urinary levels of 8-OHdG (ng/mL) corrected by creatinine (ng/mg Cr) in patients with INS were significantly higher in patients with nephrotic relapse (active phase) than in those in remission (convalescent phase; p=0.04, Wilcoxon signed-rank test); in contrast, there was no significant change between the active and convalescent phase in those patients with PSAGN (p=0.59). Thus, our finding of oxidative DNA damage assessed by urinary 8-OHdG is in agreement with the observation by Mishra et al. [1] that OS in INS is characterized by increased levels of ROS with decreased levels of antioxidant in the active phase and by normalized ROS in the