Autoimmune lymphoproliferative syndrome-like disease with somatic KRAS mutation.

Autoimmune lymphoproliferative syndrome (ALPS) is classically defined as a disease with defective FAS-mediated apoptosis (type I-III). Germline NRAS mutation was recently identified in type IV ALPS. We report 2 cases with ALPS-like disease with somatic KRAS mutation. Both cases were characterized by prominent autoimmune cytopenia and lymphoadenopathy/splenomegaly. These patients did not satisfy the diagnostic criteria for ALPS or juvenile myelomonocytic leukemia and are probably defined as a new disease entity of RAS-associated ALPS-like disease (RALD).

Human microRNA-1245 down-regulates the NKG2D receptor in natural killer cells and impairs NKG2D-mediated functions.

BACKGROUND: NKG2D is an activating receptor expressed by natural killer and T cells, which have crucial functions in tumor and microbial immunosurveillance. Several cytokines have been identified as modulators of NKG2D receptor expression. However, little is known about NKG2D gene regulation. In this study, we found that microRNA 1245 attenuated the expression of NKG2D in natural killer cells. DESIGN AND METHODS: We investigated the potential interactions between the 3'-untranslated region of the NKG2D gene and microRNA as well as their functional roles in the regulation of NKG2D expression and cytotoxicity in natural killer cells. RESULTS: Transforming growth factor-ß1, a major negative regulator of NKG2D expression, post-transcriptionally up-regulated mature microRNA-1245 expression, thus down-regulating NKG2D expression and impairing NKG2D-mediated immune responses in natural killer cells. Conversely, microRNA-1245 down-regulation significantly increased the expression of NKG2D expression in natural killer cells, resulting in more efficient NKG2D-mediated cytotoxicity. CONCLUSIONS These results reveal a novel NKG2D regulatory pathway mediated by microRNA-1245, which may represent one of the mechanisms used by transforming growth factor-ß1 to attenuate NKG2D expression in natural killer cells.

HLA-class II and class I genotypes among Japanese children with Type 1A diabetes and their families.

OBJECTIVE: To determine the HLA-DRB1, DQB1, DPB1, A, C, and B genotypes among Japanese children with autoimmune type 1 diabetes. METHODS: Four hundred and thirty patients who were GADAb and/or IA-2Ab-positive (Type 1A) were recruited from 37 medical centers as part of a nationwide multicenter collaborative study. DNA samples from 83 siblings of the children with Type 1A diabetes and 149 parent-child trios were also analyzed. A case-control study and a transmission disequilibrium test (TDT) were then performed. RESULTS: The susceptible and protective DRB1 and DQB1 alleles and haplotypes were confirmed. DPB1 alleles unique to the Japanese population and those common to multiple ethnic groups were also present. A linkage disequilibrium (LD) analysis showed both susceptible and protective haplotypes. The TDT did not reveal any alleles that were transmitted preferentially from the mother or father to children with Type 1A. Homozygosity for DRB1-09:01-DQB1-03:03 and heterozygosity for DRB1-04:05-DQB1-04:01 and DRB1-08:02-DQB1-03:02 were associated with an extremely high risk of Type 1A. A comparison of children with Type 1A and their parents and siblings suggested a dose effect of susceptible DRB1-DQB1 haplotypes and an effect of protective alleles on immunological pathogenesis. DRB1-09:01 appeared to be strongly associated with an early onset in preschool children with Type 1A diabetes. CONCLUSIONS: This study demonstrated the characteristic association of HLA-class II and class I genes with Type 1A diabetes among Japanese children. A TDT did not reveal the genomic imprinting of HLA-class II and class I genes in Type 1A diabetes.

[Monitoring of Epstein-Barr virus-infected cells by flow cytometer permits early diagnosis and evaluation of disease progression in EBV-associated hemophagocytic lymphohistiocytosis].

Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis (EBV-HLH) is characterized by clonal expansion of EBV-infected CD8(+)T-cells. We have recently demonstrated that detection of a clonally expanded population of EBV-infected CD8(+)T-cells with CD5 down-regulation was a useful tool to distinguish EBV-HLH from EBV-related disorders such as severe infectious mononucleosis. A 5-year-old girl who presented with fever, pancytopenia and liver dysfunction was diagnosed by this method in addition to conventional diagnostic tests. Further, EBV-infected cells were identified as CD5(-)HLA-DR(+) TCR V ß3(+) CD8(+)T cells, an increase or decrease of which over time reflected the disease severity in this patient. Treatment of patients with EBV-HLH varies from steroid alone to intensive chemotherapy or hematopoietic stem cell transplantation. Easy monitoring of EBV-infected cells by using flow cytometry over time may provide useful information to choose an appropriate treatment for each individual patient with EBV-HLH.

Effectiveness of four doses of pertussis vaccine during infancy diminished in elementary school age: A test-negative case-control study in Japan.

OBJECTIVE: The Japanese national immunization program recommends that children receive 4 doses of acellular pertussis vaccine between 3 months and 2 years of age. Nevertheless, the number of pertussis cases is increasing in elementary school children aged 6-12 years. Therefore, a test-negative case-control study was conducted to assess the effectiveness of the pertussis vaccine program. METHODS: Subjects included children aged ≥3 months who visited a collaborating hospital due to pertussis-specific cough between October 2017 and November 2019. All subjects underwent diagnostic tests for pertussis, and those diagnosed as positive were regarded as cases. Subjects diagnosed as pertussis-negative were classified as controls. Vaccination history was collected using a questionnaire administered to parents with reference to immunization records. Logistic regression models were employed to calculate the odds ratio (OR) and 95% confidence interval for laboratory-confirmed pertussis. RESULTS: Of 187 recruited subjects (120 cases and 67 controls), questionnaire responses were obtained for 145 subjects (95 cases and 50 controls). Compared with unvaccinated subjects, the vaccine effectiveness (VE) of 4 doses was 70% among all subjects and reached to 90% with marginal significance among subjects under 6 years of age. However, among school-aged subjects, the VE was not suggestive of protection against pertussis (VE: 8%). For vaccinees given 4 doses, the OR for developing pertussis increased significantly with longer duration since the fourth dose (compared with <4.5 years, OR of 6.0-8.2 years = 5.74; OR of ≥8.3 years = 3.88; P for trend by duration < 0.01). CONCLUSION: Effectiveness of administering 4 doses of pertussis vaccine during infancy decreases with time passed since the fourth dose. This regimen does not protect school-aged children against pertussis.

Distinct cytokine profiles of systemic-onset juvenile idiopathic arthritis-associated macrophage activation syndrome with particular emphasis on the role of interleukin-18 in its pathogenesis.

OBJECTIVES: To compare the pro-inflammatory cytokine profiles and the cytokine kinetics in patients with secondary macrophage activation syndrome (MAS) due to systemic-onset juvenile idiopathic arthritis (s-JIA) and in both active and inactive disease states of s-JIA (but no MAS), with those demonstrated in EBV-induced haemophagocytic lymphohistiocytosis (HLH) and Kawasaki disease (KD), and to investigate the significance of IL-18 in the pathogenesis of s-JIA. METHODS: Five patients with MAS complicating s-JIA (MAS/s-JIA), 10 with HLH due to EBV infection (EBV-HLH), 22 with KD and 28 healthy controls were analysed. Cytokine concentrations (IL-18, IL-6, neopterin and TNF-alpha receptor Types I and II) were quantified in serum by ELISA. Results were compared with clinical features of MAS/s-JIA, including ferritin concentrations. RESULTS: Serum IL-18 concentrations in MAS/s-JIA patients were significantly higher than those in EBV-HLH or KD patients (P < 0.05). Serum IL-6 concentrations in KD patients were significantly higher than those in EBV-HLH or MAS/s-JIA patients. Serum neopterin concentrations in EBV-HLH patients were significantly higher than those in MAS/s-JIA or KD patients. Serum IL-18 correlated positively with the following measurements of disease activity: CRP, ferritin, lactate dehydrogenase and other cytokines (P < 0.05). Serum concentrations of IL-18 in s-JIA patients remained elevated in the inactive phase of disease, whereas clinical parameters and other cytokines normalized. CONCLUSIONS: IL-18 may be an important mediator in s-JIA. Although serum Il-18 concentrations correlated with markers of the disease activity, IL-18 concentrations remained elevated even when other markers of disease activity normalized. Serum IL-18 concentration may be a promising indicator of the disease activity. The cytokine release pattern in MAS/HLH is different among patients with different aetiologies. Monitoring the cytokine profile, including IL-18, may be useful for differentiation of MAS/HLH and evaluation of disease activity in s-JIA.

Detection of T lymphocytes with a second-site mutation in skin lesions of atypical X-linked severe combined immunodeficiency mimicking Omenn syndrome.

X-linked severe combined immunodeficiency (XSCID) is caused by mutations of the common gamma chain (gammac) and usually characterized by the absence of T and natural killer (NK) cells. Here, we report an atypical case of XSCID presenting with autologous T and NK cells and Omenn syndrome-like manifestations. The patient carried a splice-site mutation (IVS1+5G>A) that caused most of the mRNA to be incorrectly spliced but produced normally spliced transcript in lesser amount, leading to residual gammac expression and development of T and NK cells. The skin biopsy specimen showed massive infiltration of revertant T cells. Those T cells were found to have a second-site mutation and result in complete restoration of correct splicing. These findings suggest that the clinical spectrum of XSCID is quite broad and includes atypical cases mimicking Omenn syndrome, and highlight the importance of revertant mosaicism as a possible cause for variable phenotypic expression.

[Role of T cells in the pathogenesis of type I diabetes mellitus: structure analysis of complementarity determining region 3 of circulating T cells].

Type 1 diabetes mellitus (type 1 DM) is the disease of insulin deficiency due to the destruction of islet cells of the pancreas, presumably through the pathogenic process mediated by autoreactive T cells. In many autoimmune diseases, oligoclonal expansion of autoreactive T cells have been reported recently. It is also suggested that proliferation of T cell clones which recognize pancreatic beta cell antigen are involved in the pathogenesis of type 1 DM. In this study, the diversity of T cell receptor (TCR) structures were evaluated in patients with type 1 DM by analyzing TCR Vbeta repertoire and complementarity determining region 3 (CDR3) size distributions of circulating T cells. Increase of specific TCR Vbeta repertoires was often observed in patients with positive anti-glutamic acid decarboxylase antibody, and this tendency was more evident among CD8+ T cells than in CD4+ T cells. Reductions of CDR3 sizes were frequently seen among CD8+ T cells from patients whose onset was within 10 years. These results suggested that selective expansion of CD8+ T cell clones play roles in the pathogenesis of type 1 DM.

Nucleotide substitutions in CD101, the human homolog of a diabetes susceptibility gene in non-obese diabetic mouse, in patients with type 1 diabetes.

AIMS/INTRODUCTION: Although genome-wide association studies have identified more than 50 susceptibility genes for type 1 diabetes, low-frequency risk variants could remain unrecognized. The present study aimed to identify novel type 1 diabetes susceptibility genes by newly established methods. MATERIALS AND METHODS: We carried out whole-exome sequencing and genome-wide copy-number analysis for a Japanese family consisting of two patients with type 1 diabetes and three unaffected relatives. Further mutation screening was carried out for 127 sporadic cases. The functional consequences of identified substitutions were evaluated by in silico analyses and fluorescence-activated cell sorting of blood samples. RESULTS: Whole-exome sequencing and genome-wide copy-number analysis of familial cases showed co-segregation of the p.V863L substitution in CD101, the human homolog of an autoimmune diabetes gene in the non-obese diabetic mouse, with type 1 diabetes. Mutation screening of CD101 in 127 sporadic cases detected the p.V678L and p.T944R substitutions in two patients. The p.V863L, p.V678L and p.T944R substitutions were absent or extremely rare in the general population, and were assessed as 'probably/possibly damaging' by in silico analyses. CD101 expression on monocytes, granulocytes and myeloid dendritic cells of mutation-positive patients was weaker than that of control individuals. CONCLUSIONS: These results raise the possibility that CD101 is a susceptibility gene for type 1 diabetes.

Rituximab therapy for Epstein-Barr virus-related chronic hepatitis following living donor kidney transplantation.

A patient who underwent living donor kidney transplantation was infected with Epstein-Barr virus (EBV) that resulted in persistent EBV infection and EBV-associated chronic hepatitis, determined by abnormally elevated anti-EBV antibody titers and high frequency of EBV-infected B lymphocytes. Despite decreases in immunosuppressant doses, persistent EBV infection and chronic hepatitis persisted for several years. Therapy using anti-CD20 monoclonal antibody (rituximab) virtually eliminated peripheral B lymphocytes and EBV-encoded small RNA 1 (EBER-1)-positive cells. Moreover, hepatic enzyme levels normalized and histological findings indicated marked improvement in hepatic inflammation. Although peripheral CD20(+) B lymphocyte and EBER-1-positive cell levels began to increase 4 months after the end of therapy, the number of EBER-1-positive cells remained very low, and liver function test results remained within normal ranges. The present case illustrates the significance of early diagnosis, monitoring of viral load, and vigorous management of EBV-related disorders associated with organ transplantation.

Early diagnosis of Danon disease: Flow cytometric detection of lysosome-associated membrane protein-2-negative leukocytes.

INTRODUCTION: Danon disease is an extremely rare X-linked dominant disorder characterized by progressive cardiomyopathy, muscle weakness, and mild mental retardation. Most cases harbor nonsense, frameshift, or splice-site mutations in LAMP2 that result in lysosome-associated membrane protein-2 (LAMP-2) deficiency and lysosomal defects. The identification of LAMP2 mutations makes it possible to detect female carriers with significant cardiomyopathy. Therefore, it is of paramount importance to develop useful carrier detection methods. METHODS: To screen for diminished LAMP-2 expression among female patients with progressive cardiomyopathy, we developed a flow cytometric method to detect LAMP-2-deficient leukocytes. RESULTS: In healthy controls, all circulating leukocyte populations, including granulocytes, monocytes, and lymphocytes, expressed significant levels of LAMP-2. In contrast, cells from a male patient with Danon disease lacked detectable LAMP-2. His younger twin sisters showed reduced levels of LAMP-2 expression with characteristic bimodal fluorescence intensity patterns. The percentage of LAMP-2-negative cells in the asymptomatic sibling was nearly the same as that in the symptomatic sibling. CONCLUSION: We developed a flow cytometric assay for LAMP-2 expression that can serve as a rapid primary screening method to detect carriers of LAMP-2 deficiencies. This assay will narrow the target population before subjecting patients to more laborious and expensive gene mutation analysis.

Epstein-Barr virus-associated T-/natural killer cell lymphoproliferative diseases.

Epstein-Barr virus (EBV) is a ubiquitous virus that infects the majority of the world population by adulthood. The major target for infection is the B lymphocyte, and acute infection causes vigorous EBV-specific killer T-cell responses exemplified clinically by acute infectious mononucleosis (IM). EBV infection usually persists latently life-long without eliciting any clinical symptoms. Rarely, active EBV infection is prolonged, with abnormal expansions of EBV-infected T or NK cells, conditions collectively defined here as EBV-associated T/NK lymphoproliferative diseases. Hemophagocytic lymphohistiocytosis (HLH), chronic active EBV infection (CAEBV), NK lymphoma/leukemia, and T-cell lymphoma are entities included in this category. Hypersensitivity to mosquito bite (HMB) represents a unique syndrome characterized by expansion of EBV-infected NK cells in the peripheral circulation and within the inflammatory skin lesions induced by mosquito bites. Target cell specificity, defects in host immune responses, and strain differences of EBV may account for ectopic EBV infections and for the unique clinical presentations characteristic of each illness.

Cell type specific infection of Epstein-Barr virus (EBV) in EBV-associated hemophagocytic lymphohistiocytosis and chronic active EBV infection.

While Epstein-Barr virus (EBV) tropism in B cells and nasopharygeal epithelial cells in the normal host has been demonstrated, recently the role of its infection into non-B cell populations has been suggested to play a pivotal role in the pathogenesis of several EBV-related hematological as well as non-hematological diseases. Ectopic EBV infection in T cells or natural killer (NK) cells has been reported in EBV-associated hematological diseases, such as acute fulminant EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH) and chronic active EBV infection (CAEBV). Recent advances in the analysis of EBV infection in lymphocyte subpopulations have clarified the differential virus-cell interaction within these EBV-related disorders. EBV infection was predominantly found in CD8(+) T-cells from EBV-HLH, and in CD4(+) T-cells or NK cells from CAEBV, while the majority of EBV infected cells were found in B cells from acute infectious mononucleosis (IM). Different virus-cell interactions between acute EBV-HLH and CAEBV have indicated different pathogenic mechanisms against EBV infection between the two EBV-associated diseases, accounting for the difference in clinical manifestations between the two diseases.

Heme oxygenase-1 production by peripheral blood monocytes during acute inflammatory illnesses of children.

Monocytes play key roles both in innate and adaptive antigen-specific immunity and they constitute critical components of the immune responses. Although most of the monocyte-derived cytokines exhibit proinflammatory functions in vivo, heme oxygenase-1 (HO-1), an inducible heme-degrading enzyme, exerts potent anti-inflammatory effect through production of carbon monoxide and bilirubin. We compared HO-1 production by monocytes in vivo in various acute inflammatory illnesses and in normal controls. Freshly isolated monocytes produced little HO-1 as detected by immunohistochemistry, but it was rapidly induced in vitro upon stimulation. HO-1 production by monocytes was selective because it was not induced in other leukocyte populations, including granulocytes and lymphocytes. Monocytes from acute inflammatory illnesses, such as Kawasaki disease and acute infectious diseases, viral or bacterial, produced significant levels of HO-1, as detected by flow cytometry, immunohistochemistry, and reverse transcription polymerase chain reaction. Quantitative analysis of HO-1 mRNA expression by real-time polymerase chain reaction revealed that monocytes from controls exhibited low, but significant levels of HO-1 mRNA, indicating that circulating monocytes produce HO-1 constantly, in response to basal level of oxidative stress encountered daily. Significantly elevated HO-1 mRNA levels seen in acute inflammatory illnesses suggest that monocyte HO-1 production serve as potent anti-inflammatory agent to control excessive cell or tissue injury in the presence of oxidative stress and cytokinemia.

Antimicrobial activities of cefditoren against respiratory pathogens isolated from children in Japan.

There is an increasing spread and incidence of penicillin-resistant bacteria that are becoming less susceptible to commonly prescribed oral antimicrobials, including extended-spectrum cephalosporins. Against this background, we undertook this study to determine the prevalence of penicillin resistance in Streptococcus pneumoniae and the in-vitro activity of oral antimitrobials. Between April 1996 and December 1997, in 245 children with respiratory tract infections (bronchitis in 61, pharyngitis in 115, and tonsillitis in 69), 119 strains of Haemophilus influenzae, 89 strains of Streptococcus pyogenes, 61 strains of Streptococcus pneumoniae, 36 strains of Staphylococcus aureus, and 34 strains of Moraxella catarrhalis were isolated from the pharynx. The antimicrobial susceptibility of these isolates was assessed by a broth microdilution method. The isolation incidence of penicillin-intermediately resistant S. pneumoniae (PISP) and penicillin-highly resistant S. pneumoniae (PRSP) was 59.0% and 13.1%, respectively. Most strains of PISP and PRSP were highly resistant to cefaclor, cefpodoxime, cefteram, cefdinir, clarithromycin, ampicillin, and minocycline, but susceptibile to ofloxacin and cefditoren (CDTR). The in-vitro activity of CDTR was superior to that of other cephalosporins, such as cefaclor, cefdinir, and cefpodoxime, when tested against both the beta-lactamase-positive and -negative H. influenzae isolated. CDTR was also active against all the other strains, including methicillin-sensitive S. aureus, S. pyogenes, and M. catarrhalis. This study suggested that CDTR was a useful oral antibiotic for pediatric respiratory tract infections.

Autoimmune lymphoproliferative syndrome mimicking chronic active Epstein-Barr virus infection.

Chronic active Epstein-Barr virus infection (CAEBV) is defined as a systemic EBV-associated lymphoproliferative disease characterized by fever, lymphadenopathy, and splenomegaly in apparently immunocompetent persons. Recent studies have revealed that EBV infects T or natural killer cells in most patients with CAEBV; the etiology of CAEBV, however, remains unknown. Autoimmune lymphoproliferative disorder (ALPS) is an inherited disorder associated with defects in apoptosis, and clinically characterized by lymphadenopathy, splenomegaly, hypergammaglobulinemia, and autoimmune disease. ALPS is most often associated with mutations in the FAS gene, which is an apoptosis-signaling receptor important for homeostasis of the immune system. Based on the clinical similarity between ALPS and CAEBV with respect to lymphoproliferation, we have examined the possibility of the co-occurrence of ALPS in patients with a diagnosis of CAEBV. In this study, we have identified FAS gene mutations in three Japanese patients with lymphadenopathy, hepatosplenomegaly, and unusual EBV infection, who were diagnosed with CAEBV. These observations, which indicate that the clinical development of ALPS may be associated with EBV infection, alert us to a potential diagnostic pitfall of CAEBV.