Yumi Mizuno

Association of vascular endothelial growth factor (VEGF) and VEGF receptor gene polymorphisms with coronary artery lesions of Kawasaki disease.

We analyzed the genetic polymorphisms of vascular endothelial growth factor (VEGF) and its receptors [Fms-related tyrosine kinase-1, kinase insert domain receptor (KDR)] in Japanese patients with Kawasaki disease (KD) and normal control subjects to examine whether these genes would contribute to the KD occurrence and/or the development of coronary artery lesion (CAL) in KD. We found that the frequency of G allele of VEGF g.−634 G>C single-nucleotide polymorphism in the promoter region was significantly higher in KD patients with CAL than in those without CAL (p = 0.012) or control subjects (p = 0.021) because of a significantly higher frequency of the GG genotype in KD patients with CAL. In addition, the frequency of the A1 allele with 11 AC repeats of KDR g.+4422(AC)11–14 dinucleotide repeat polymorphism in intron 2 was significantly higher in KD patients with CAL than in those without CAL (p = 0.013) or control subjects (p = 0.040) as a result of a significantly higher frequency of the A1A1 genotype in KD with CAL patients. The multivariate analysis of clinical features and genotypes of the two polymorphisms showed that the A1A1 genotype of KDR g.+4422(AC)11–14 polymorphism was an independent risk factor for the development of CAL with the highest odds ratio among several clinical parameters (odds ratio 6.76; 95% confidence interval 1.05–43.48). Dual luciferase assay demonstrated that the A1 allele with KDR g.+4422(AC)11 repeats showed a weaker silencer function than the A2 allele with 12 AC repeats. These findings suggested that VEGF and its receptor, KDR, genes contributed to the development of CAL in KD patients.

Oversecretion of IL‐18 in haemophagocytic lymphohistiocytosis: a novel marker of disease activity

We investigated the significance of interleukin (IL)‐18 levels in the pathophysiology of haemophagocytic lymphohistiocytosis (HLH). IL‐18 levels were significantly elevated in all nine patients with active HLH compared with those of healthy controls. Serial determination of IL‐18 levels in three cases, showed a gradual decrease compared with those of IL‐12, interferon (IFN)‐γ or soluble Fas ligand (sFasL) in the course of clinical improvement, and seemed to be elevated until complete disappearance of disease activity. IL‐18 and IFN‐γ (CC 0.711, P = 0.018), and IFN‐γ and sFasL (CC 0.849, P = 0.0049) levels were significantly correlated. On the other hand, correlation between IL‐12 and IFN‐γ, IL‐18 and sFasL, or IL‐18 and IL‐12 was not observed. IL‐18, IFN‐γ and sFasL levels significantly correlated with disease activity such as fever and alanine transaminase (ALT) levels. IL‐18 mRNA expression was enhanced in spleen, but not in peripheral blood mononuclear cells (MNC), bone marrow MNC, liver from patients of active HLH, or the tumour from a patient with lymphoma‐associated haemophagocytic syndrome (LAHS). These results suggest that IL‐18 may play important roles in the pathogenesis of HLH, particularly through induction of Th1 cells. IL‐18 measurement may be useful for the diagnosis and for the detection of smouldering disease activity.

Th1 and Th1-inducing cytokines in Salmonella infection

Thl and Thl‐inducing cytokines and T cell responses were investigated in human salmonellosis. Serum IFN‐γ, IL‐12 and IL‐18 levels were increased significantly in patients with salmonellosis. The increase in serum IL‐15 and IL‐18 levels was more significant and prolonged in patients with the systemic form of salmonellosis than in those with the gastroenteric form. The serum IFN‐γ level was correlated significantly with IL‐12 and IL18 levels, and the IL‐15 level was correlated significantly with IL‐18. Upon stimulation with Salmonella in vitro, mononuclear cells from salmonellosis patients produced significantly higher amounts of IFN‐γ and IL‐12 compared with those from healthy controls. Anti‐IL‐12 moAb or anti‐IL18 MoAb significantly inhibited Salmonella‐induced IFN‐γ production in vitro. γδ T cells expressed significantly higher levels of IFN‐γ mRNA in salmonellosis patients than in healthy controls. The results suggest that Th1‐inducing cytokines appear to be involved in the in vivo response against Salmonella infection, promoting IFN‐γ production by αβ and γδ T cells which plays a protective role against Salmonella.

Unique activation status of peripheral blood mononuclear cells at acute phase of Kawasaki disease

Although Kawasaki disease (KD) is characterized by a marked activation of the immune system with elevations of serum proinflammatory cytokines and chemokines at acute phase, the major sources for these chemical mediators remain controversial. We analysed the activation status of peripheral blood mononuclear cells (PBMCs) by flow cytometry, DNA microarray and quantitative reverse transcription–polymerase chain reaction. The proportions of CD69+ cells in both natural killer cells and γδT cells at acute‐phase KD were significantly higher than those at convalescent‐phase KD. Microarray analysis revealed that five genes such as NAIP, IPAF, S100A9, FCGR1A and GCA up‐regulated in acute‐phase KD and the pathways involved in acute phase KD were related closely to the innate immune system. The relative expression levels of damage‐associated molecular pattern molecule (DAMP) (S100A9 and S100A12) genes in PBMCs at acute‐phase KD were significantly higher than those at convalescent‐phase KD, while those of TNFA, IL1B and IL6 genes were not significantly different between KD patients and healthy controls. Intracellular production of tumour necrosis factor‐α, interlaukin‐10 and interferon‐γ in PBMCs was not observed in KD patients. The present data have indicated that PBMCs showed a unique activation status with high expression of DAMP genes but low expression of proinflammatory cytokine genes, and that the innate immune system appears to play a role in the pathogenesis and pathophysiology of KD.

Association of IL12RB1 polymorphisms with susceptibility to and severity of tuberculosis in Japanese: a gene-based association analysis of 21 candidate genes

Tuberculosis (TB) is the second commonest cause of death from infectious disease after HIV/AIDS worldwide. Association studies have revealed that host genetic factors, such as human leukocyte antigen and solute carrier family 11 member A1 (NRAMP1), play roles in susceptibility to TB. To identify host genetic factors involved in the susceptibility to TB in Japanese, we performed a gene‐based association analysis of 21 candidate genes on 87 TB patients and 265 controls using marker single nucleotide polymorphisms (SNPs). For the genes with two or more marker SNPs exhibiting significant allele association, we subsequently analysed the association between adjacent coding SNPs (cSNPs) and TB. Among a total of 118 marker SNPs, 3 of IL1B and 2 of IL12RB1 showed association with TB. Non‐synomymous cSNPs were not identified in IL1B. Association studies on four non‐synomymous cSNPs of IL12RB1 (641A/G, 1094T/C, 1132C/G, 1573G/A) in linkage disequilibrium showed that three of them (641A/G, 1094T/C, 1132C/G) were significantly associated with the development of TB. Haplotype analysis on the four cSNPs demonstrated that frequency of ATGG haplotype was significantly lower in TB patients than in controls. When TB patients were divided into two subgroups according to the severity of lung disease, advanced subgroup showed a prominent association with 641A/G, 1094T/C and 1132C/G SNPs. These data suggested that genetic variants of IL12RB1, at least in part, confer genetic susceptibility to TB, and are associated with the progression of the disease, in Japanese.

Genetic Analysis of MMP Gene Polymorphisms in Patients With Kawasaki Disease

Kawasaki disease (KD) is an acute febrile disorder characterized by systemic vasculitis primarily occurring in coronary arteries. Matrix metalloproteinases (MMPs) have been considered to play pathophysiologic roles in the development of coronary artery lesions (CALs); therefore, an evaluation of the genetic contributions of the MMP genes to the development of CALs in KD patients would be beneficial for the prediction of CAL formation. We focused on the known functional single nucleotide polymorphisms (SNPs) in the MMP genes (MMP-2-735C>T, MMP-3-1612 5A/6A, MMP-9-1562C>T, MMP-12-82A>G, and MMP-13-77A>G) and performed the association study between these SNPs and CAL formation in KD. The study population consisted of 44 KD patients with CALs and 92 without CALs and 175 healthy controls. As a result, allele and genotype frequencies of MMP-13-77A>G showed significant differences between KD patients with CALs and without CALs (p = 0.00989 and p = 0.00551, respectively). The estimated frequencies of the G-C haplotype in the MMP-13 gene promoter were significantly lower in KD patients with CALs than in those without CALs. There was no association between other MMP genes and CAL formation. In conclusion, the genetic evaluation by association study demonstrated that the MMP-13 gene, at least in part, contributed to the development of CALs in KD.

Th1 and Th2 cytokine production is suppressed at the level of transcriptional regulation in Kawasaki disease

To clarify the functional state of T cells in Kawasaki disease, we analysed mRNA expression levels of Th1/Th2 cytokines (IFN‐γ and IL‐4) along with Th1/Th2‐inducing transcription factors, T‐bet and GATA‐3, which play pivotal roles in the development of Th1 and Th2 cells, respectively. By real‐time PCR, IFN‐γ mRNA levels in peripheral blood mononuclear cells (PBMNC) were significantly decreased in Kawasaki disease patients compared with those with measles, and tended to be lower than those in healthy controls. T‐bet mRNA levels were significantly decreased in patients with Kawasaki disease compared with healthy controls. In addition, IL‐4 and GATA‐3 mRNA levels were significantly decreased in Kawasaki disease compared with healthy controls. Regulatory cytokine mRNA levels (TGF‐β and IL‐10) were also decreased in Kawasaki disease. The mRNA levels of IFN‐γ showed a significant positive correlation with those of T‐bet in Kawasaki disease. These results suggest that the suppressed function of Th1 and Th2, associated with the suppression of both T‐bet and GATA‐3 gene expression, may be one of the immunological characteristics of Kawasaki disease.

Increased serum levels of interferon-γ-inducible protein 10 and monokine induced by gamma interferon in patients with haemophagocytic lymphohistiocytosis

We measured serum interferon‐gamma‐inducible protein 10 (IP‐10) and monokine induced by gamma interferon (MIG) levels to investigate the role of these molecules in the pathophysiology of haemophagocytic lymphohistiocytosis (HLH). Serum IP‐10 and MIG levels were significantly increased in patients with active HLH compared with those of healthy controls. Serum MIG levels decreased gradually during the course of disease in a patient who recovered without therapy. On the other hand, rapid reduction of MIG and IP‐10 levels was observed after chemotherapy in a patient with severe HLH. IP‐10 and MIG mRNA expression was enhanced in liver and spleen, and IP‐10 mRNA expression was enhanced in bone marrow in the patients, suggesting activated macrophages that infiltrated in these organs as one of the main producers of these cytokines. Serum IP‐10 and MIG levels showed a significant correlation with serum IFN‐γ levels. In addition, these chemokines had a significant correlation with fever and serum LDH levels, which are clinical indicators of disease activity of HLH. These results suggest that IP‐10 and MIG which are produced by activated macrophages by the stimulation of IFN‐γ, play an important role in the pathophysiology of HLH, by recruitment of activated Th1 cells into the tissues or organs.

Neurotrophin-3 Levels in Cerebrospinal Fluid From Children With Bacterial Meningitis, Viral Meningitis, or Encephalitis

Neurotrophin-3 levels were measured in the cerebrospinal fluid of 35 patients with bacterial meningitis, viral meningitis, or encephalitis by two-site enzyme immunoassay. Elevated cerebrospinal fluid levels of neurotrophin-3 were demonstrated in 8 of 18 patients with bacterial meningitis. Follow-up examination of the eight patients at the convalescent stage showed diminished cerebrospinal fluid levels of neurotrophin-3. In contrast, none of the 17 patients with viral meningitis or encephalitis showed an elevation of neurotrophin-3 levels in cerebrospinal fluid. No relationships were observed between neurotrophin-3 levels and cerebrospinal fluid cell numbers, cerebrospinal fluid protein levels, serum C-reactive protein concentrations, or outcome in bacterial meningitis. Since neurotrophin-3 is involved in the survival of neurons and the modulation of the immune system, neurotrophin-3 could play a neuroprotective or immunomodulatory role in bacterial meningitis. (J Child Neurol 2000;15:19-21).

Recurrent parotid swelling in children: clinical features useful for differential diagnosis of Sjögren's syndrome

Immunological evaluations were performed in 59 children with at least five episodes of parotid swelling. Autoantibody(ies) was transiently or persistently detected in 12 (20%) of 59 patients with recurrent parotitis. Three of the 12 children with autoantibodies were diagnosed as having Sjögrens syndrome. The mean age at onset of parotid swelling in Sjögrens syndrome was significantly higher than that of recurrent parotitis of unknown etiology. The present study and the review of the literature suggest that patients with the onset of parotid swelling at age five years or over deserve screening for underlying systemic immune disorders.