Yoshimitsu Gotoh

Characterization of the cytokine network at a single cell level in mice with collagen-induced arthritis using a dual color ELISPOT assay.

Collagen-induced arthritis (CIA) in mice has been classified as a Thl-mediated disease. However, most evidence for this has been obtained by indirect experiments; for example, the administration of neutralizing anti-interferon-gamma (IFN-gamma) antibody reduced the severity of arthritis. To obtain direct evidence about the cytokine balance in CIA mice, we analyzed the cytokine-secreting cell in CIA mice at the single cell level using a dual color enzyme-linked immunospot (ELISPOT) assay, which enabled us to analyze interleukin-2 (IL-2)-secreting cells or IL-4-secreting cells or both simultaneously. Furthermore, to characterize the cytokine network in the pathogenesis of CIA, the frequency of the cells secreting IL-12, which induced the development of naive Th cells into Th1 cells, was analyzed. The results show that in the prearthritic phase, the number of IL-12-secreting cells in spleen and peritoneal exuded cells is increased, and Th1 cells in lymph node and spleen are dominant. In contrast, after the onset of clinical arthritis, the number of IL-12-secreting cells in spleen, lymph node, and peritoneal exuded cells is decreased, and there is a shift from a Thl-dominant to a Th2-dominant state in lymph node and spleen. The results indicated that the pathogenesis of CIA is associated with a disruption in the normal ratio of Th1/Th2 at cell level.

Age-dependent decrease in serum transforming growth factor (TGF)-beta 1 in healthy Japanese individuals; population study of serum TGF-beta 1 level in Japanese

Transforming growth factor-beta1 (TGF-β1), a multi-functional cytokine, is involved in regulating a variety of cellular activities and the serum/plasma TGF-β1 level is altered with various diseases. However, most published reports have described adult patients, and so we investigated the clinical significance of serum TGF-β1 level in pediatric patients. The diagnostic application of the measurement of serum TGF-β1 level depends critically on the control value, however, there is no information on the control value of serum TGF-β1 for children. In the present study, we determined the serum TGF-β1 level of healthy Japanese children as a control value with enzyme-linked immunosorbent assay (ELISA). The serum TGF-β1 level of children (0–14 years old) was significantly higher than that of adults (over 15 years old) (p < 0.01). Thus, it is recommended that when the serum TGF-β1 levels of patients are evaluated, they should be compared with those of age-matched controls.

Determination of age-related changes in human soluble interleukin 2 receptor in body fluids of normal subjects as a control value against disease states

A highly sensitive enzyme-linked immunosorbent assay (ELISA) system was developed for human soluble interleukin-2 receptor (sIL-2R) with an ELISA-amplification system (ELAST((R))). The sensitivity of the new method was 20-fold higher than that without the amplification. Thus very low concentrations of sIL-2R in urine can be detected. With this method, serum and urine concentrations of sIL-2R were analyzed for healthy Japanese subjects with age 1-67 years. Mean sIL-2R concentrations in both serum and urine of children were significantly higher than those of adults. However, the concentrations of children showed a progressive decline to those of adults by the age of 15 years. There was no difference in the values between males and females. The results provide a control value of sIL-2R against those in disease states such as nephrotic syndrome. Since the trends in serum and urine were found to be similar, urinary sIL-2R measurement may provide sufficient information, without measuring the blood concentration.

A case of atypical hemolytic uremic syndrome due to anti-factor H antibody in a patient presenting with a factor XII deficiency identified two novel mutations.

A 9-year-old boy with pallor and macrohematuria showed hemolytic anemia, thrombocytopenia and renal failure. There was no history of diarrhea and the stool culture was negative. A diagnosis of atypical hemolytic uremic syndrome (HUS) was confirmed; however, the cause of the prolonged activated partial thromboplastin time (APTT) was unknown. Plasma exchange and hemodialysis were performed because of progressive hemolytic anemia and renal dysfunction. Fresh frozen plasma was administered frequently to correct the prolonged APTT after hemolysis was controlled and C3 levels had recovered. Factor H (FH) and factor I (IF) levels were normal and we did not detect mutations of FH, IF and membrane cofactor protein. Further investigation revealed the presence of anti-FH antibody in the patient’s plasma and a deficiency of coagulation factor XII. Analysis of the patient’s coagulation system displayed <3% functional activity of factor XII, whereas levels of other coagulation factors were within the normal range. Two novel mutations (W222G and R447S) were identified upon analysis of the factor XII gene in this patient. Moreover, further investigation revealed that compound heterozygous mutations were present in two of the patient’s three siblings, while the third sibling only had a mutation at W222G. The patient was treated for atypical HUS; however, no treatment was required for factor XII deficiency as he did not display a hemorrhagic tendency. We report here a rare case of atypical HUS due to anti-FH antibody presenting with a coagulation factor XII deficiency.

Age-Dependent Decrease in Serum Soluble Interferon-Gamma Receptor (sIFN-γR) in Healthy Japanese Individuals; Population Study of Serum sIFN-γR Level in Japanese

Abstract We planned to investigate the clinical significance of serum soluble interferon-gamma receptor (sIFN-γR) level in pediatric patients. The diagnostic application of the measurement of serum sIFN-γR level depends critically on the control value. However, there is no information of the control value of serum sIFN-γR for children. In the present study, we determined the serum sIFN-γR level of healthy Japanese children using an ELISA. The serum sIFN-γR level of children (0–14 years old) was significantly higher than that of adults (over 15 years old) (p < 0.01, n = 104). Thus, it is recommended that, when the serum sIFN-γR level of patients is evaluated, it should be compared against age-matched controls. We also preliminarily applied this assay as a diagnostic parameter for the patients with diarrhea positive (D +) hemolytic uremic syndrome (HUS).

Performance in adolescents of the two Japanese serum creatinine based estimated glomerular filtration rate equations, for adults and paediatric patients: A study of the Japan Renal Biopsy Registry and Japan Kidney Disease Registry from 2007 to 2013

There are two different Japanese serum creatinine‐based equations for estimated glomerular filtration rate (eGFR), for adults and paediatric patients, with both equations deemed applicable to 18‐year‐old subjects. This study assessed the relative accuracy of the two equations in assessing eGFR in patients aged 18 years with chronic kidney disease. A total of 3042 patients (1679 males and 1363 females), aged 2–20 years, who were registered in the Japan Renal Biopsy Registry or the Japan Kidney Disease Registry between 2007 and 2013 were evaluated. eGFR values derived from formulas for children (Uemuras formula) and adults (the 3‐variable Japanese formula) were calculated and compared, especially in patients aged 18 years. At all ages, but especially at younger ages, eGFR was significantly higher when calculated with the adult than the paediatric formula. This finding was also observed in 18‐year‐old adolescents with eGFR <90 mL/min per 1.73 m2 (P = 0.026). However, the mean difference between the two calculated eGFRs was only 2.79 mL/min per 1.73 m2. These findings indicate that both creatinine‐based equations used to calculate eGFR rate in Japanese children and adults with chronic kidney disease could be used to determine eGFR in 18‐year‐old subjects, with the difference between the two within permissible levels for clinical use.

Urinary Evaluation of the Balance Between the Soluble Interferon-Gamma Receptor (IFN-γR1) and the Interleukin-4 Receptor (IL-4Rα) in Children with Vesicoureteral Reflux

Abstract We planned to investigate the urinary soluble cytokine receptor profile in patients with vesico-ureteric reflux (VUR). The urine levels of soluble interferon-γ receptor R1 (sIFN-γR) and soluble interleukin-4 receptor α (sIL-4R) were measured using an ELISA technique. The urine levels of sIFN-γR in the patients with VUR were significantly higher than those in the healthy controls (p < 0.001). On the other hand, although the urine sIL-4R levels in the patients with VUR were also higher than those in the controls, there were no significant differences between them. The urinary soluble receptor levels did not correlate with the clinical severity of VUR. These results suggest that there may be an immunological basis to VUR complicatedly.

Age-dependent decreases in serum soluble interleukin-1 receptor type I (sIL-1RI) in healthy individuals: a population study of serum sIL-1RI levels in Japanese subjects.

The levels of several soluble cytokine receptors in body fluids of healthy individuals change with age. Clinical application of the measurement of the serum soluble interleukin‐1 receptor type I (sIL‐1RI) level depends critically on the samples used as the controls. At present, there is no information regarding the levels of serum sIL‐1RI in healthy subjects. The purpose of this study is to reveal the age‐related changes that occur in the serum sIL‐1RIlevels of healthy individuals. We determined the serum sIL‐1RI levels of healthy Japanese children using ELISA. The serum sIL‐1RI level of children (0–14 years) was significantly higher than that of adults (more than 15 years) (P=0.0138, n=90). Thus, it is recommended that when the serum sIL‐1RI level of patients is evaluated, it should be compared against age‐matched controls. J. Clin. Lab. Anal. 23:175–178, 2009.

Urinary evaluation of the balance between soluble interferon-gamma receptor (IFN-γR1) and interleukin-4 receptor (IL-4Rα)

To elucidate the usefulness of the simultaneous analysis of multiple kinds of soluble cytokine receptors in urine specimens, we determined the levels of both the soluble interferon-gamma receptor alpha chain (sIFN-gammaR1, Th1-type cytokine receptor) and the soluble interleukin 4-receptor alpha chain (sIL-4Ralpha, Th2-type cytokine receptor) in the urine of healthy subjects as reference values and preliminarily applied this method to evaluate patients with diarrhea positive (D+) hemolytic uremic syndrome (HUS) as the diagnostic parameters. The urinary sIFN-gammaR levels of children were significantly lower than those of adults (p < 0.01, n = 107). On the other hand, there was no significant difference between the urine sIL-4R levels of adults and children. Statistical correlation between sIFN-gammaR and sIL-4R values was not observed (p = 0.705). On the day of onset of HUS, the urine sIFN-gammaR levels of the patients (n = 6) with HUS were higher than those of the healthy control group (n = 67) (p < 0.01); however, there was no significant difference in the sIL-4R levels between both groups. The urine evaluation of the balance between the soluble cytokine receptors might be informative for the immune states of HUS patients.

Detection of interleukin-12 (IL-12)-secreting cells in normal mice with enzyme-linked immunospot (ELISPOT) assay.

Abstract To assess mice interleukin-12 (IL-12)-secreting cells at a single cell level, we have developed a murine IL-12 specific enzyme-linked immunospot (ELISPOT) assay. the application of the newly developed method clearly showed the frequency of IL-12-secreting cells in the resident peritoneal exuded cells was higher than other organs of normal DBA/1J mouse. Moreover, we determined the frequency of IL-12-secreting cells in the spleens of five strains of inbred mice, and found the incidence of IL-12 secretors in the strain C57BL/6 to be greatest, and significantly greater than four of the others. These results are compatible with the predicted evidence, supporting this ELISPOT assay for IL-12-secreting cells is accurate. the procedure provides a useful tool for investigating complicated immune responses at a single cell level.