Heterozygous Mutations in OAS1 Cause Infantile-Onset Pulmonary Alveolar Proteinosis with Hypogammaglobulinemia.

Pulmonary alveolar proteinosis (PAP) is characterized by accumulation of a surfactant-like substance in alveolar spaces and hypoxemic respiratory failure. Genetic PAP (GPAP) is caused by mutations in genes encoding surfactant proteins or genes encoding a surfactant phospholipid transporter in alveolar type II epithelial cells. GPAP is also caused by mutations in genes whose products are implicated in surfactant catabolism in alveolar macrophages (AMs). We performed whole-exome sequence analysis in a family affected by infantile-onset PAP with hypogammaglobulinemia without causative mutations in genes associated with PAP: SFTPB, SFTPC, ABCA3, CSF2RA, CSF2RB, and GATA2. We identified a heterozygous missense variation in OAS1, encoding 2,'5'-oligoadenylate synthetase 1 (OAS1) in three affected siblings, but not in unaffected family members. Deep sequence analysis with next-generation sequencing indicated 3.81% mosaicism of this variant in DNA from their mother's peripheral blood leukocytes, suggesting that PAP observed in this family could be inherited as an autosomal-dominant trait from the mother. We identified two additional de novo heterozygous missense variations of OAS1 in two unrelated simplex individuals also manifesting infantile-onset PAP with hypogammaglobulinemia. PAP in the two simplex individuals resolved after hematopoietic stem cell transplantation, indicating that OAS1 dysfunction is associated with impaired surfactant catabolism due to the defects in AMs.

The possession of exon 2 or exon 3 variants in the MEFV gene promotes inflammasome activation in Japanese patients with familial Mediterranean fever with a heterozygous exon 10 mutation.

OBJECTIVES: The modification and pathogenesis of MEFV exon 2 or 3 variants in familial Mediterranean fever (FMF) remains unclear. We compared the clinical and laboratory characteristics between the coexistence and noncoexistence of MEFV exon 2 or 3 variants in patients with FMF that had a heterozygous MEFV exon 10 mutation. METHODS: We excluded patients with FMF that had two MEFV exon 10 mutations in one or more alleles and/or MEFV mutations in exons other than in exons 2, 3, or 10. Finally, we reviewed 131 Japanese patients with FMF that had a heterozygous MEFV exon 10 mutation, and they were divided into the groups with and without MEFV exon 2 or 3 variants of 97 and 34, respectively. RESULTS: All patients with MEFV exon 2 variants had either E148Q and/or L110P variants, none of patients had exon 3 variants. In the univariate analysis, the group with variants had significantly earlier onset, a higher percentage of thoracic pain with febrile attacks, a higher frequency of attack, and a higher IL-18 level at remission compared to the group without variants (all, p<0.05). Importantly, multivariate analyses showed that the coexistence of MEFV exon 2 variants was independently and significantly associated with earlier onset of FMF and thoracic pain (both, p<0.05). CONCLUSIONS: Our results suggested that coexistence of MEFV exon 2 variants have additional effects on manifestations of FMF with MEFV exon 10 mutations. Our findings highlighted the modifications and pathogenesis of such MEFV variants in FMF.

Next-generation sequencing of the whole MEFV gene in Japanese patients with familial Mediterranean fever: a case-control association study.

OBJECTIVES: We aimed to identify the whole nucleotide sequence of the Mediterranean fever (MEFV) gene in familial Mediterranean fever (FMF) and reveal novel single nucleotide variants (SNVs) associated with the susceptibility of FMF. METHODS: SeqCap capturing technique followed by Illumina next-generation sequencing have been used to assess two hundred SNVs in the whole region of MEFV in 266 Japanese patients with FMF and 288 ethnically matched controls. We performed an association analysis using these SNVs to identify genetic variants that predispose to FMF. RESULTS: We identified the two most significant SNVs [rs28940578; M694I in exon 10, odds ratio (OR) = 153, p=2.47×10-21 and rs3743930; E148Q in exon 2, OR = 1.65, p<0.0005]. Stratified analysis identified rs28940578 as a risk allele in typical FMF. Haplotype AG, defined by rs401298 and rs28940578, was the most significant and prevalent among patients with typical FMF compared with controls (22.4% vs. 0%, respectively; OR = 137, p=1.44×10-31). Haplotype GTC, defined by rs11466018, rs224231, and rs401877, was the most significant among patients with typical FMF without the rs28940578 mutation compared with controls (15.9% vs. 6%, respectively; OR = 12.4, p=0.004). CONCLUSIONS: rs28940578 is associated with the highest risk in typical FMF cases. This is consistent with results from previous studies in Japan. We found a novel MEFV gene haplotype that confers susceptibility of FMF among typical FMF without the rs28940578 mutation. There were no relevant SNVs identified in MEFV among the atypical FMF group.

[AN ADULT CASE OF PERIODIC FEVER, APHTHOUS STOMATITIS, PHARYNGITIS, AND ADENITIS (PFAPA) SYNDROME DEVELOPED IN CHILDHOOD AND DIAGNOSED AFTER PROGRESS MORE THAN 30 YEARS].

We report an adult case of periodic fever, aphthous stomatitis, pharyngitis, and adenitis (PFAPA) syndrome, who had a tonsillectomy at 10 years old and relapsed later. An early 40's-year-old man had been suffering from recurrent fever attack once in 1-2 months during childhood. He was accompanied by fever which was persist for several days, aphthous stomatitis, tongued tonsillitis with moss, pharyngitis, and submandibular lymphadenitis with tenderness. He was not doing well during fare-up. At the time of admission, CRP level was 12.5mg/dl and the remarkably increased expression of CD64 on neutrophils was found. Bacterial infections and collagen diseases were excluded by the several examinations. We suspected PFAPA syndrome, and treated with cimetidine, but cimetidine was not effective. At the time of flare up, administration of prednisolone was remarkably effective. We diagnosed PFAPA syndrome on the basis of clinical courses. Genetic analysis of responsible gene of familial Mediterranean fever, MEFV showed E148Q heterozygous mutation in exon 2.Since an adult case of PFAPA syndrome is likely to be made misunderstanding for infectious recurrent pharyngitis, it is important to note that we should consider PFAPA syndrome as a differential diagnosis when we meet with the adult patient of recurrent fever.

Mosaicism of an ELANE Mutation in an Asymptomatic Mother.

PURPOSE: We report normal neutrophil count in a mother, who carries the same ELANE mutation as her daughter with severe congenital neutropenia. We hypothesized that the mother possessed wild- and mutant-type clones and the wild-type clones could generate neutrophils, whereas the mutant clones could not. METHODS: We confirmed mutant variant ratio by sequence signals and measured the frequency of the mutant allele by subcloning in various cell types. We established the ELANE-mutated and non-mutated induced pluripotent stem cells (iPSCs) from the mother's T cells and compared granulopoiesis between these iPSCs. RESULTS: In the sequence analysis of isolated peripheral blood (PB), nail and hair, the mutant variant was detected in approximately 40-60% of lymphocytes, monocytes, hematopoietic progenitor cells, and hair as well as in a small percentage of nail, but in none of the neutrophils. In the subcloning analysis of extracted DNA from CD3+ and CD34+ cells, the mutant allele was identified in 37.5% and 38.1%, respectively. We reprogrammed the mother's PB cells and established the ELANE-mutated and non-mutated iPSCs. Granulopoiesis from mutated iPSCs revealed little sensitivity to granulocyte colony-stimulating factor in comparison with non-mutated iPSCs. CONCLUSIONS: These observations strongly suggest that mutant-carrying neutrophils did not appear in the mother's PB because mutated clones could not differentiate into neutrophils. The mother's normal hematological phenotype could be explained by the perseverance of normal, non-mutated granulopoiesis.

MicroRNA-204-3p inhibits lipopolysaccharide-induced cytokines in familial Mediterranean fever via the phosphoinositide 3-kinase γ pathway.

Objective: We sought to identify the microRNA (miRNA) profile and potential biomarkers in FMF and to clarify their gene targets to elucidate the pathogenesis of FMF. Methods: We performed an miRNA microarray using serum from FMF patients in attack and in remission. We then examined the expression of miRNAs in macrophages derived from THP-1 cells stimulated with toll-like receptor (TLR) ligands. Macrophages derived from THP-1 cells transfected with pre-miRNA were stimulated with lipopolysaccharides (LPSs) for the quantification of inflammatory cytokine production. To identify the target genes, we overexpressed their miRNA and performed a complementary DNA microarray. Transfection with reporter construct and the precursor miRNA was performed to confirm the suppression of target mRNA. Results: We found that miR-204-3p was greatly decreased in the serum from FMF patients in attack. The expression of miR-204-3p was suppressed by LPS stimulation in the macrophages derived from THP-1 cells and the inhibition of miR-204-3p significantly induced the production of TLR4-related cytokines. The bioinformatic analysis showed that miR-204-3p is predicted to target genes implicated in the TLR pathway through the regulation of PI3Kγ signalling. The reporter assay revealed that miR-204-3p directly suppressed the luciferase activity of 3'-UTR of PIK3CG reporter construct. The inhibition of PI3Kγ resulted in decreased amounts of IL-6 and IL-12p40 in monocytes from FMF patients. Conclusion: These data suggest that serum miR-204-3p has potential as a useful biomarker in FMF patients and that miR-204-3p serves as a suppressor of inflammatory cytokine production in FMF by targeting the PI3Kγ pathway.

A Novel In-Frame Deletion in the Leucine Zipper Domain of C/EBPε Leads to Neutrophil-Specific Granule Deficiency.

Neutrophil-specific granule deficiency (SGD) is a rare autosomal recessive primary immunodeficiency characterized by neutrophil dysfunction, bilobed neutrophil nuclei and lack of neutrophil-specific granules. Defects in a myeloid-specific transcription factor, CCAAT/enhancer binding protein-ε (C/EBPε), have been identified in two cases in which homozygous frameshift mutations led to loss of the leucine zipper domain. In this study, we report a 55-y-old woman affected with SGD caused by a novel homozygous 2-aa deletion (ΔRS) in the leucine zipper domain of the C/EBPε gene. The patient showed characteristic neutrophil abnormalities and recurrent skin infections; however, there was no history of deep organ infections. Biochemical analysis revealed that, in contrast to the two frameshift mutations, the ΔRS mutant maintained normal cellular localization, DNA-binding activity, and dimerization, and all three mutants exhibited marked reduction in transcriptional activity. The ΔRS mutant was defective in its association with Gata1 and PU.1, as well as aberrant cooperative transcriptional activation of eosinophil major basic protein. Thus, the ΔRS likely impairs protein-protein interaction with other transcription factors, resulting in a loss of transcriptional activation. These results further support the importance of the leucine zipper domain of C/EBPε for its essential function, and indicate that multiple molecular mechanisms lead to SGD.

Acute Myeloid Leukemia in a Patient With X-linked Severe Combined Immunodeficiency.

Severe combined immunodeficiency (SCID) is a defect in the differentiation and function of T cells. An increased malignancy risk, mainly lymphatic malignancy, has been described in patients with SCID. We report a patient with X-linked SCID who developed acute myeloid leukemia, derived from the recipient with somatic NRAS mutation 4 months after cord blood transplantation (CBT). Loss of heterozygosity phenomenon of the recipient at 6q14 locus was observed at 2 months post-CBT and progressed to 6q deletion (6q-) chromosome abnormality. Somatic NRAS mutation was detected at 3 months post-CBT. Thus, 6q- and NRAS mutation were strongly associated with the leukemic transformation in our patient.

Familial Mediterranean fever with P369S/R408Q exon3 variant in pyrin presenting as symptoms of PFAPA.

Familial Mediterranean fever (FMF) can be classified into typical and incomplete/atypical types. Periodic fever, aphthous stomatitis, pharyngitis, cervical adenitis (PFAPA) syndrome-like symptoms have been found in atypical type carrying P369S-R408Q mutations in the responsible gene MEFV. A 28-year-old female with recurrent fever and her young sisters and mother, all of whom had tonsillectomy for tonsillitis, carried heterozygous alterations involving E148Q/P369S/R408Q. A diagnosis of atypical FMF, MEFV exon3 variants with PFAPA syndrome-like symptoms, was made.

Markedly elevated CD64 expression on neutrophils and monocytes as a biomarker for diagnosis and therapy assessment in Kawasaki disease.

OBJECTIVE: Kawasaki disease (KD) is the most commonly encountered inflammatory disease in children. However, its pathogenesis and diagnostic biomarkers have not been fully investigated. We examined the activation of neutrophils and monocytes in KD. METHODS: We studied the expression of the Fcγ-receptors CD64 and CD16 on neutrophils and monocytes in KD before and after the treatment with intravenous infusion of high dose immunoglobulin (IVIG). Bacterial infections were addressed as well. RESULTS: CD64 expression on neutrophils and monocytes was dramatically increased at the onset of KD flare-ups, but later decreased just after IVIG. Similarly, CD16-positive monocytes were observed at the onset and were less apparent after therapy. The addition of immunoglobulin did not block the expression of CD64 or CD16 in vitro. Serum G-CSF in the majority of patients, and IFN-γ in some patients, were elevated during flares but decreased after treatment. CONCLUSION: Our findings demonstrate that remarkable CD64 expression during KD flare-ups may serve as a biomarker for diagnosis. Evaluation of CD64 is also potentially useful for the determination of treatment efficacy in KD.

Nod2-Nodosome in a Cell-Free System: Implications in Pathogenesis and Drug Discovery for Blau Syndrome and Early-Onset Sarcoidosis.

Nucleotide-binding oligomerization domain-containing protein (Nod) 2 is an intracellular pattern recognition receptor, which recognizes muramyl dipeptide (N-Acetylmuramyl-L-Alanyl-D-Isoglutamine: MDP), a bacterial peptidoglycan component, and makes a NF-κB-activating complex called nodosome with adaptor protein RICK (RIP2/RIPK2). Nod2 mutants are associated with the autoinflammatory diseases, Blau syndrome (BS)/early-onset sarcoidosis (EOS). For drug discovery of BS/EOS, we tried to develop Nod2-nodosome in a cell-free system. FLAG-tagged RICK, biotinylated-Nod2, and BS/EOS-associated Nod2 mutants were synthesized, and proximity signals between FLAG-tagged and biotinylated proteins were detected by amplified luminescent proximity homogeneous assay (ALPHA). Upon incubation with MDP, the ALPHA signal of interaction between Nod2-WT and RICK was increased in a dose-dependent manner. The ALPHA signal of interaction between RICK and the BS/EOS-associated Nod2 mutants was more significantly increased than Nod2-WT. Notably, the ALPHA signal between Nod2-WT and RICK was increased upon incubation with MDP, but not when incubated with the same concentrations, L-alanine, D-isoglutamic acid, or the MDP-D-isoform. Thus, we successfully developed Nod2-nodosome in a cell-free system reflecting its function in vivo, and it can be useful for screening Nod2-nodosome-targeted therapeutic molecules for BS/EOS and granulomatous inflammatory diseases.

Dramatic Improvement in the Multifocal Positron Emission Tomography Findings of a Young Adult with Chronic Granulomatous Disease Following Allogeneic Hematopoietic Stem Cell Transplantation.

Chronic granulomatous disease (CGD) is a primary immunodeficiency caused by defects of nicotinamide adenine dinucleotide phosphate oxidase. Catalase-positive bacteria and fungi are phagocytosed, but persist within phagocytes, resulting in granulomatous inflammation. Although allogeneic hematopoietic stem cell transplantation (HSCT) is a curative treatment for CGD, HSCT sometimes leads to fatal outcomes related to the exacerbation of persistent infectious or post-infectious inflammatory diseases, particularly in adolescent and young adult patients with a history of recurrent infections and/or multiple granulomas in organs. Here, we present the case of a young adult with X-linked CGD in whom multiple lesions were found in lungs and lymph nodes on both computed tomography and positron emission tomography (PET) scans before allogeneic HSCT, but all the lesions disappeared only on PET scan 5 months after HSCT. Monitoring the activity of multiple pre-existing lesions with PET scan may be beneficial to adolescent and young adult CGD-patients undergoing allogeneic HSCT.

Dysregulated mature IL-1ß production in familial Mediterranean fever.

OBJECTIVE: The aim of this study was to analyse the role of circulating cleaved IL-1ß in patients with FMF. METHODS: We enrolled 20 patients with FMF (5 males and 15 females), 22 patients with RA (4 males and 18 females) and 22 healthy controls (6 males and 16 females). Serum levels of serum amyloid A (SAA) were measured by ELISA. We also determined whether IL-1ß was present as the cleaved form (p17) in the sera of FMF patients by immunoblotting using anti-cleaved IL-1ß antibody. RESULTS: Although SAA concentrations were elevated in the sera, there was no significant difference in these concentrations between FMF patients and RA patients. Immunoblot analysis demonstrated that the cleaved form of IL-1ß (p17) was present in sera from FMF patients during febrile attack periods, but not in healthy controls. Bands representing the cleaved form of IL-1ß were not detected in serum from FMF patients at non-febrile attack periods or remission periods under colchicine treatment. The amounts of cleaved IL-1ß (p17) were significantly higher in patients with FMF compared with those in patients with RA in the inflammatory phase. CONCLUSION: The cleaved form of IL-1ß is a valuable biomarker for monitoring disease activity and response to colchicine treatment in patients with FMF. It might be useful to discriminate FMF from other non-IL-1ß-mediated inflammatory disorders.

Clinical and laboratory features of fatal rapidly progressive interstitial lung disease associated with juvenile dermatomyositis.

OBJECTIVE: Rapidly progressive interstitial lung disease (RP-ILD) is a rare but potentially fatal complication of JDM. The aim of this study was to establish markers for the prediction and early diagnosis of RP-ILD associated with JDM. METHODS: The clinical records of 54 patients with JDM were retrospectively reviewed: 10 had RP-ILD (7 died, 3 survived), 19 had chronic ILD and 24 were without ILD. Routine tests included a high-resolution CT (HRCT) scan of the chest and measurement of serum levels of creatine phosphokinase, ferritin and Krebs von den Lungen-6 (KL-6). Anti-melanoma differentiation-associated gene 5 (MDA5) antibodies and IL-18 levels were measured by ELISA. RESULTS: No differences were found in the ratio of juvenile clinically amyopathic DM between the three groups. Initial chest HRCT scan findings were variable and could not distinguish between RP-ILD and chronic ILD. Anti-MDA5 antibodies were positive in all 8 patients with RP-ILD and 10 of 14 with chronic ILD, but none of the patients without ILD. Serum levels of anti-MDA5 antibody, ferritin, KL-6 and IL-18 were significantly higher in the RP-ILD group than in the chronic ILD and non-ILD groups. Serum levels of IL-18 positively correlated with serum KL-6 (R = 0.66, P < 0.001). CONCLUSION: High serum levels of IL-18, KL-6, ferritin and anti-MDA5 antibodies (e.g. >200 units by ELISA) are associated with RP-ILD. These can be used as an indication for early intensive treatment. Both alveolar macrophages and autoimmunity to MDA5 are possibly involved in the development of RP-ILD associated with JDM.

Clinical course in a patient with neutrophil-specific granule deficiency and rapid detection of neutrophil granules as a screening test.

PURPOSE: Neutrophil-specific granule deficiency (SGD) is a rare, congenital disorder characterized by atypical neutrophil structure and function that results in frequent and severe bacterial infections. However, the clinical course of patients with SGD have not been described in detail because of the scarcity of the disease. We present the clinical course of an adult patient with SGD and propose a method for making an early diagnosis of SGD. PATIENT AND METHODS: A32-year-old Japanese woman with SGD had a small impetigo lesion on her face and experienced the rapid spread of a facial abscess to a pulmonary abscess via the blood stream. We also analyzed the expression of neutrophil granule proteins in our patient compared with a healthy control by flow cytometry. RESULTS: We confirmed defects of several neutrophil granule proteins in our patient by flow cytometry. CONCLUSION: Severe bacterial infections sometimes occur and spread rapidly in SGD. Detection of neutrophil granules by flow cytometry is useful for a rapid diagnosis and a screening of SGD.

Successful cord blood transplantation after repeated transfusions of unmobilized neutrophils in addition to antifungal treatment in an infant with chronic granulomatous disease complicated by invasive pulmonary aspergillosis.

BACKGROUND: Chronic granulomatous disease (CGD) is a rare inherited primary immunodeficiency that affects phagocytic cells. CGD patients are susceptible to fungal infections, especially Aspergillus infections. The management of life-threatening Aspergillus infections in CGD is particularly difficult because some infections cannot be eradicated with standard antifungal treatments and, hence, result in death. CASE REPORT: A 2-week-old girl developed invasive pulmonary aspergillosis, which rapidly progressed to respiratory failure. Liposomal amphotericin B, micafungin, and voriconazole were not effective. At the age of 2 months, she was diagnosed with p67phox-deficient CGD. In addition to antifungal treatment, the patient received 21 granulocyte transfusions (GTX), which were obtained from 300- or 400-mL whole blood samples from healthy random donors who were not treated with granulocyte-colony-stimulating factor or dexamethasone. The median neutrophil count of the GTX was 1.88 × 10(8) /kg body weight. Rituximab was administered to reduce alloimmunization to human leukocyte antigens (HLA) after the eighth GTX, resulting in their absence of anti-HLA before and after cord blood transplantation (CBT). A marked improvement in her invasive pulmonary aspergillosis was achieved, although the first CBT was rejected. Complete hematopoietic recovery was obtained after the second CBT. CONCLUSION: Repeated GTX containing relatively low doses of neutrophils might be able to control severe Aspergillus infections in infants with CGD.

Defect of suppression of inflammasome-independent interleukin-8 secretion from SW982 synovial sarcoma cells by familial Mediterranean fever-derived pyrin mutations.

Familial Mediterranean fever (FMF) is a recessive inherited autoinflammatory syndrome. Patients with FMF have symptoms such as recurrent fever and abdominal pain, sometimes accompanied by arthralgia. Biopsy specimens have revealed substantial neutrophil infiltration into synovia. FMF patients have a mutation in the Mediterranean fever gene, encoding pyrin, which is known to regulate the inflammasome, a platform for processing interleukin (IL)-1ß. FMF patients heterozygous for E148Q mutation, heterozygous for M694I mutation, or combined heterozygous for E148Q and M694I mutations, which were found to be major mutations in an FMF study group in Japan, suffer from arthritis, the severity of which is likely to be lower than in FMF patients with M694V mutations. Expression plasmids of wild-type (WT) pyrin and mutated pyrin, such as E148Q, M694I, M694V, and E148Q+M694I, were constructed, and SW982 synovial sarcoma cells were transfected with these expression plasmids. IL-8 and IL-6 were spontaneously secreted from the culture supernatant of SW982 cells without any stimulation, whereas IL-1ß and TNF-α could not be detected even when stimulated with lipopolysaccharide. Notably, two inflammasome components, ASC and caspase-1, could not be detected in SW982 cells by Western blotting. IL-8 but not IL-6 secretion from SW982 cells was largely suppressed by WT pyrin, but less suppressed by mutated pyrin, which appeared to become weaker in the order of E148Q, M694I, E148Q+M694I, and M694V mutations. As for IL-8 and IL-6, similar results were obtained using stable THP-1 cells expressing the WT pyrin or mutated pyrins, such as M694V or E148Q, when stimulated by LPS. In addition, IL-8 secretion from mononuclear cells of FMF patients was significantly higher than that of healthy volunteers when incubated on a culture plate. Thus, our results suggest that IL-8 secretion from SW982 synovial sarcoma cells suppressed by pyrin independently of inflammasome is affected by pyrin mutations, which may reflect the activity in FMF arthritis.

Rapid detection of intracellular p47phox and p67phox by flow cytometry; useful screening tests for chronic granulomatous disease.

Chronic granulomatous disease (CGD) is caused by defects of NADPH oxidase. The diagnosis of CGD can be made by analysis of NADPH oxidase activity, however, identification of the CGD subgroups is required before performing mutation analysis. The membrane-bound subunits, gp91phox and p22phox, can be quickly analyzed by flow cytometry, unlike the cytosolic components, p47phox and p67phox. We evaluated the feasibility of flow cytometric detection of p47phox and p67phox with specific monoclonal antibodies in two patients with p47phox deficiency and 7 patients with p67phox deficiency. Consistent with previous observations, p47phox and p67phox were expressed in phagocytes and B cells, but not in T or natural killer cells, from normal controls. In contrast, patients with p47phox and p67phox deficiency showed markedly reduced levels of p47phox and p67phox, respectively. These techniques will be useful to rapidly assess the expression of the cytosolic components, p47phox and p67phox, and represents important secondary screening tests for CGD.

Long-term epidemiologic longitudinal study on the effect of vaccines on public inoculation.

Public vaccination policies in Japan for several viruses have achieved favorable results. To accurately evaluate their overall effectiveness, we conducted a 45- year epidemiological survey of measles, varicella and mumps cases at our clinic. The number of patients with measles was found to be significantly decreased with the single-dose vaccination provided at public expense. However, we also witnessed an increasing trend of infection at a later age. The vaccination rates for varicella and mumps were relatively low because of their optional availability in Japan, and thus they cannot be considered to confer public protection. Although localized to a particular region, our results show that it is important to increase the immunization rate of vaccines for large-scale protection against viral infections through public programs.