Development of immune and microbial environments is independently regulated in the mammary gland.

Breastfeeding is important for mammals, providing immunological and microbiological advantages to neonates, together with the nutritional supply from the mother. However, the mechanisms of this functional diversity in the mammary gland remain poorly characterized. Here, we show that, similar to the gastrointestinal tract, the mammary gland develops immune and microbial environments consisting of immunoglobulin A (IgA) and the microflora, respectively, both of which are important for protecting neonates and the mother from infectious diseases. The IgA production and microflora development are coordinated in the gastrointestinal tract but seem to be independently regulated in the mammary gland. In particular, the chemokine (C-C motif) ligand 28 and poly-Ig receptor, crucial molecules for the IgA production in milk, were expressed normally in germ-free lactating mice but were almost undetectable in postweaning mothers, regardless of the microflora presence. Our findings offer insights into potentially improving the quality of breastfeeding, using both immunological and microbiological approaches.

Detection of Merkel cell polyomavirus with a tumour-specific signature in non-small cell lung cancer.

BACKGROUND: We searched for a viral aetiology for non-small cell lung cancer (NSCLC), focusing on Merkel cell polyomavirus (MCPyV). METHODS: We analysed 112 Japanese cases of NSCLC for the presence of the MCPyV genome and the expressions of RNA transcripts and MCPyV-encoded antigen. We also conducted the first analysis of the molecular features of MCPyV in lung cancers. RESULTS: PCR revealed that 9 out of 32 squamous cell carcinomas (SCCs), 9 out of 45 adenocarcinomas (ACs), 1 out of 32 large-cell carcinomas, and 1 out of 3 pleomorphic carcinomas were positive for MCPyV DNA. Some MCPyV DNA-positive cancers expressed large T antigen (LT) RNA transcripts. Immunohistochemistry showed that MCPyV LT antigen was expressed in the tumour cells. The viral integration sites were identified in one SCC and one AC. One had both episomal and integrated/truncated forms. The other carried an integrated MCPyV genome with frameshift mutations in the LT gene. CONCLUSION: We have demonstrated the expression of a viral oncoprotein, the presence of integrated MCPyV, and a truncated LT gene with a preserved retinoblastoma tumour-suppressor protein-binding domain in NSCLCs. Although the viral prevalence was low, the tumour-specific molecular signatures support the possibility that MCPyV is partly associated with the pathogenesis of NSCLC in a subset of patients.

Effects of leg resistance training on arterial function in older men.

BACKGROUND: Little information is available on the effect of strength training on vascular function, particularly in older people. OBJECTIVE: To determine the effect of resistance training on arterial stiffness and endothelial function in older adults. METHOD: Eleven healthy men (mean (SEM) age 64 (1) years) performed 12 weeks of resistance training involving knee flexion and extension (three sets a day, two days a week). RESULTS: Resistance training increased maximal muscle power by 16% (p<0.0001). Arterial stiffness as assessed by aortic pulse wave velocity did not change with resistance training. Plasma concentration of nitric oxide (NO), measured as its stable end product (nitrite/nitrate), had increased (p<0.05) after resistance training (61.2 (10.4) v 39.6 (3.2) micromol/l). There was no change in plasma concentration of endothelin-1. CONCLUSION: The results suggest that short term resistance training may increase NO production without stiffening central arteries in healthy older men.

Association between polymorphisms in the SPINK5 gene and atopic dermatitis in the Japanese.

Atopy, which is characterized by increased levels of immunoglobulin E (IgE) against common environmental allergens, is considered the strongest predisposing factor for asthma and atopic dermatitis (AD). Mutations in the gene encoding serine protease inhibitor Kazal-type 5 (SPINK5) are responsible for Netherton syndrome, a rare skin disorder characterized by greatly elevated IgE levels with atopic manifestations. A recent study of Caucasian AD families showed that maternally derived alleles of the SPINK5 gene are associated with development of AD and asthma, suggesting the parent-of-origin effect for the development of atopic diseases in the SPINK5 gene. We studied the possible association of the SPINK5 gene for the development of atopic diseases by determining the genotypes of five polymorphisms in a Japanese population. Ttransmission disequilibrium tests revealed an association of SPINK5 polymorphisms with AD but not with asthma. Our data indicate that the SPINK5 gene is associated with AD across ethnicities.

Insertion/deletion coding polymorphisms in hHAVcr-1 are not associated with atopic asthma in the Japanese population.

Hepatitis A virus receptor (HAVcr-1) and T-cell immunoglobulin- and mucin-domain-containing molecule (TIM)-3 were recently implicated as asthma susceptibility genes in the study of congenic mice. In a genome-wide screen, we found strong evidence for linkage of atopic asthma with marker D5S820, located approximately 0.5 Mb from hHAVcr-1 and human TIM3. We screened for mutations in human HAVcr-1 (hHAVcr-1) and in TIM3 and found seven, including two insertion/deletion polymorphisms, in hHAVcr-1 and two in TIM3. We conducted transmission disequilibrium tests (TDTs) in families identified through children with atopic asthma. None of the hHAVcr-1 allele were transmitted preferentially to asthma-affected children (P>0.1). In quantitative TDT analysis, no association was observed between the log[total IgE] and either allele of the hHAVcr-1 polymorphism (P>0.1). The two TIM3 mutations were rare in the Japanese population, occurring in only one of 48 unrelated asthmatic subjects. Our results indicate that hHAVcr-1 polymorphisms are not likely to be associated with the development of atopy-related phenotypes in the Japanese population.

New polymorphisms of haematopoietic prostaglandin D synthase and human prostanoid DP receptor genes.

BACKGROUND: Prostaglandin D2 (PGD2), a major cyclo-oxygenase metabolite of arachidonic acid in mast cells, induces bronchoconstriction in the human lung. It has been reported that mice lacking PGD receptor fail to develop the bronchial hyper-responsiveness upon ovalbumin challenge, suggesting that PGD2 functions as a mediator of allergic asthma. OBJECTIVE: To determine if there are any mutations associated with the development of asthma in the haematopoietic prostaglandin D synthase (H-PGDS) gene and the human prostanoid DP receptor (PTGDR) gene. METHODS AND RESULTS: We screened the 5'flanking and coding regions of the H-PGDS gene and the PTGDR gene by direct sequence. We identified one variant in intron 2 (IVS2 + 11 A > C) and one variant in intron 3 (IVS3 + 13T > C) of the H-PGDS gene, and two variants in the 5'flanking region of the PTGDR gene (-197T > C and -2C > T). The IVS3 + 13T > C and -197T > C variants were rare, appearing only once in 48 subjects. transmission disequilibrium test (TDT) analysis of 144 asthmatic families revealed that the IVS2 + 11 A allele of the H-PGDS gene was significantly transmitted preferentially to asthma-affected children (P = 0.0056), but no association was observed between -2C/T polymorphism of the PTGDR gene and asthma (P > 0.05). CONCLUSION: Our results suggest that the IVS2 + 11A/C allele may be involved in the development of asthma in the Japanese population.

A genome-wide linkage analysis of orchard grass-sensitive childhood seasonal allergic rhinitis in Japanese families.

Seasonal allergic rhinitis (SAR) is an inflammatory disease of the nose and eyes that follows sensitization to air-born pollens. We conducted a genome-wide linkage screening of 48 Japanese families (188 members) with orchard grass (OG)-sensitive SAR children (67 affected sib-pairs) in a farming community in central Japan where OG was planted for apple farming and OG pollen is a major cause of SAR. We used the GENEHUNTER program to performed nonparametric multipoint linkage analysis for OG-sensitive SAR as a qualitative trait and for log total serum IgE levels and OG-RAST IgE levels as quantitative traits. Genotyping data of 400 microsatellite markers suggested linkage of SAR to chromosomes 1p36.2, 4q13.3, and 9q34.3 (P < 0.001), linkage of serum total IgE levels to 3p24.1, 5q33.1, 12p13.1, and 12q24.2 (P < 0.001), and linkage of OG-RAST IgE levels to 4p16.1, 11q14.3, and 16p12.3 (P < 0.001). Weak evidence for linkage of SAR to 5q33.1 was also observed (P = 0.01). All these regions, with the exception of 9q34.3, have been previously reported to be linked to asthma and/or atopy. These data suggest that, although loci linked to SAR are likely to be common to asthma, a strong contribution by specific gene(s) to OG-sensitive SAR is unlikely.

Identification of missense mutation in the IL12B gene: lack of association between IL12B polymorphisms and asthma and allergic rhinitis in the Japanese population.

Interleukin-12 (IL-12) is a macrophage-derived cytokine that modulates T lymphocyte responses and can suppress allergic inflammation. In a genome-wide screen, we found strong evidence for linkage of atopic asthma with marker D5S1352, located close to IL12B, with a maximum lod score of 4.34. We screened for mutation in IL12B and found three novel (-4475-4insG, Glu186Asp and Ser226Asn) variants and one previously reported (1188A>C) variant in IL12B, and conducted transmission disequilibrium tests in families identified through children with atopic asthma or allergic rhinitis. Frequencies of the Asn226 and 1188C alleles in the parents were 0.04 and 0.5, respectively. Preferential transmission of either the Ser226/Asn226 or 1188A/C allele to asthma-affected or rhinitis-affected children was not observed (P > 0.1) and was not associated significantly with total serum IgE level (P > 0.1). Our results indicate that polymorphisms in IL12B are not likely to be associated with the development of atopy-related phenotypes.

Haplotypes of the 5' region of the IL-4 gene and SNPs in the intergene sequence between the IL-4 and IL-13 genes are associated with atopic asthma.

IL-4 and IL-13 are important in IgE synthesis and allergic inflammation. Therefore, genes encoding IL-4 and IL-13 are candidates for predisposition to asthma and atopy. A recent study in the YAC transgenic mouse has revealed that one of the conserved noncoding sequences (CNS-1) between IL-4 and IL-13 influences the expression of IL-4, IL-5, and IL-13, suggesting that CNS-1 acts as a coordinate regulator of these genes. This investigation screened for mutations in the 13-kb region between IL-4 and IL-13, which includes the human equivalent of the murine CNS-1. Four single nucleotide polymorphisms (SNPs) were found in the region between IL-4 and IL-13 (IL-4-IL-13SNP1, IL-4-IL-13SNP2, IL-4-IL-13SNP3, and IL-4-IL-13SNP4). There was no mutation in the human CNS-1. We genotyped these and other previously reported polymorphisms in IL-4 and IL-13 using asthmatic families, and examined association by transmission disequilibrium test. Two-locus haplotype analysis revealed that haplotypes composed of the IL-4 RP2del, IL-4 +33T, or IL-4 -589T alleles and either IL-4-IL-13SNP3G or IL-4-IL-13SNP4C are transmitted significantly to asthma-affected children (p = 0.002). This data suggests that haplotypes composed of the 5' region polymorphisms in the IL-4 gene and SNPs in the intergene sequence between IL-4 and IL-13 influence the development of asthma.

Association between a new polymorphism in the activation-induced cytidine deaminase gene and atopic asthma and the regulation of total serum IgE levels.

BACKGROUND: Activation-induced cytidine deaminase (AICDA) is a recently identified RNA-editing deaminase that plays an important role in class-switching. Defects in AICDA result in a hyper-IgM phenotype and lack of IgG, IgA, and IgE in both human beings and mice. OBJECTIVE: The aim of this study was to determine whether the AICDA gene is related to regulation of total serum IgE and development of atopic asthma. METHODS: We screened for polymorphisms in the 5;-flanking and coding regions of the AICDA gene in subjects with atopic asthma and analyzed the effect of these polymorphisms on the development of atopic asthma and on total serum IgE levels in Japanese asthmatic families. RESULTS: We identified 3 novel polymorphisms (5923A/G, 7888C/T, and 8578A/C) and 1 rare variant (Arg25Cys) in the AICDA gene. Transmission disequilibrium testing showed that the 7888C allele was transmitted preferentially to asthma-affected children (P =.007). Mean log [total serum IgE] levels of parents with the 7888C/7888C, 7888C/7888T, and 7888T/7888T genotypes were 2.12, 1.99, and 1.77, respectively, and a significant association was observed between the genotypes (P =.02). In RT-PCR experiments, we found 2 novel splice variants of AICDA, one lacking all of exon 4 (variant 1; 367 base pairs) and the other lacking the first 30 base pairs of exon 4 (variant 2; 453 base pairs). These variants were not associated with the 7888C/T polymorphism. CONCLUSION: The 7888C/T polymorphism might be associated with the pathogenesis of atopic asthma and the regulation of total serum IgE levels.

Detection of human herpesvirus 6 and JC virus in progressive multifocal leukoencephalopathy complicating follicular lymphoma.

Progressive multifocal leukoencephalopathy (PML), a demyelinating infectious disease caused by JC virus (JCV), occurs almost exclusively in immunocompromised patients usually with malignant diseases. We report here a Japanese female with follicular lymphoma who subsequently developed PML. In addition to JCV, human herpesvirus 6 (HHV-6) was detected in the affected brain lesions of the patient by polymerase chain reaction and by in situ hybridization. HHV-6, recognized as a neurotropic virus, is known to be reactivated during immunosuppression and can cause fatal complications such as encephalitis/encephalopathy. It is likely that impaired immunity associated with lymphoma and the additional immunosuppression following cytopenia-inducing chemotherapies predisposed the patient to reactivated HHV-6 infection. Although it remains to be clarified whether HHV-6 plays an important role as a co-agent with JCV in causing demyelination of the brain, our observation alerts physicians to the possible association of HHV-6 with the pathogenesis of PML.

Relationship between Respiratory Movements and Energy Efficiency in the Post-Exercise Recovery Phase.

The breathing movement is highly automated but it can be voluntarily controlled in some situations. Compared with period of exercise and post-exercise, subjects could control breathing more voluntarily in the post-exercise recovery phase. So, we analyzed relationship between the strategy for controlling the breathing movement pattern and energy efficiency in this phase. Fifteen healthy men (mean age, 22.1 years) were subjected to exercise with a bicycle ergometer at 100 W for 5 minutes, then respiratory pattern was measured four times for 5 minutes after exercise. The measurement items were 1) chest expansion, 2) abdomen expansion, 3) tidal volume, 4) respiration rate, 5) minute ventilation, 6) oxygen intake, and 7) heart rate. The items 1) and 2) were measured by a three-dimensional motion analysis system, and the items 3) through 7) were measured by a respiratory gas analyzer. As for the breathing movement pattern, there were differences among the subjects in terms of the rates of increase in chest expansion and in respiration rate after exercise. The subjects were classified into 4 groups according to these differences. The group, in which respiration rate was increased, showed the marked increase in minute ventilation and oxygen intake, and the rapid recovery of heart rate. The group, in which chest expansion rate was increased, on the other hand, showed small increase in minute ventilation and oxygen intake, and the slow recovery of heart rate. The breathing strategies which are beneficial to energy efficiency exist, however, healthy men do not always select the beneficial strategies.

Translocation (10;11)(p13;q13) and MLL gene rearrangement in a case of AML (M5a) with aggressive leukemia cutis.

A male patient with a secondary acute monocytic leukemia whose leukemia cells had a t(10;11)(p13;q13) chromosomal abnormality is described. Gene analysis disclosed that the patient's leukemia cells had MLL gene rearrangement. His leukemia cells responded poorly to chemotherapy, and the patient developed an unusual aggressive leukemia cutis. A t(10;11)(p13;q13) chromosomal abnormality that expresses MLL gene rearrangement has not been reported previously in secondary leukemia.

Analysis of the Smad2 gene in hematological malignancies.

A total of 34 leukemia and lymphoma samples (17 clinical samples and 17 cell lines) were analyzed for mutations of the Smad2 gene by reverse transcriptase-polymerase chain reaction single strand conformation polymorphism (RT-PCR-SSCP) analysis. Nine of the 34 samples had 18q chromosomal abnormalities. No shifted bands were detected in any of the hematological malignancies. Our results suggest that resistance to cell growth inhibitory effects of TGF-beta in hematological malignancies is not due to alterations of the Smad2 gene.

Human herpesvirus 6 (HHV-6)-positive Burkitt's lymphoma: establishment of a novel cell line infected with HHV-6.

Human herpesvirus 6 (HHV-6) DNA has been detected in several human lymphoproliferative disorders. We report a case of HHV-6-infected Burkitt's lymphoma, from which a cell line, designated Katata, has been established. Katata cells had an immature B-cell phenotype with an L3 morphology and carried a t(8;14)(q24;q32) chromosomal abnormality. The HHV-6 DNA sequences were detected in both the patient's tumor cells and Katata cell line by polymerase chain reaction using three sets of primers that target different regions of HHV-6 DNA. The presence of HHV-6 DNA in Katata cells was also shown by Southern blot hybridization with the BamHI fragment of HHV-6. It is likely that the virus is in a latent state, since (1) virion-associated protein was not expressed in Katata cells, (2) transcriptional level of the immediate-early gene was very low, and (3) no viral particles were observed by electron microscopy. Katata cells were highly tumorigenic in nude mice and the tumor cells also contained HHV-6 DNA. We have successfully obtained several clonal lines by allowing the cells to form colonies in soft agarose and by the limiting dilution method. HHV-6 DNA was detectable in all 13 clones analyzed, suggesting that virtually all Katata cells are infected with HHV-6. This is the first report of a case of HHV-6+ Burkitt's lymphoma in the absence of Epstein-Barr virus. Furthermore, there has been no report of lymphoma cell lines that are persistently and nonproductively infected with HHV-6. The Katata Burkitt's lymphoma cell line, therefore, would provide a useful tool for studies of the mechanisms of HHV-6 latency and reactivation.

Establishment of a clonal cell line producing granulocyte colony-stimulating factor and parathyroid hormone-related protein from a lung cancer patient with leukocytosis and hypercalcemia.

Squamous cell lung carcinoma cells obtained from a patient who presented with leukocytosis and hypercalcemia were transplanted into nude mice and a serially transplantable cell line, OKa-N-1, was established. The nude mice transplanted with OKa-N-1 cells displayed leukocytosis and hypercalcemia. Serum levels of granulocyte colony-stimulating factor (G-CSF) and parathyroid hormone-related protein (PTHrP) were both elevated in these mice. In vitro cultivation of this tumor cell line gave rise to a clonal cell line, OKa-C-1. Nude mice transplanted with the OKa-C-1 cell line also showed leukocytosis and hypercalcemia with high serum G-CSF and PTHrP levels. The culture supernatant of OKa-C-1 contained high levels of G-CSF and PTHrP. Immunohistochemical studies showed the expression of PTHrP in OKa-C-1 cells. Reverse transcription polymerase chain reaction revealed the presence of G-CSF and PTHrP mRNA in this cell line. Dexamethasone treatment inhibited the transcription of G-CSF and PTHrP genes. This new human squamous carcinoma cell line, OKa-C-1, would be useful for studying the mechanism of simultaneous production of G-CSF and PTHrP and their control in cancer patients with leukocytosis and hypercalcemia.

Direct transplantation of chronic myelogenous leukemia cells into nude mice and establishment of a leukemic stem cell (Ph1+, CD34+) line dependent on mouse bone marrow stromal cells in vitro.

Peripheral blood cells from a female patient with Ph1-positive chronic myelogenous leukemia (CML) in blast crisis were serially transplanted in BALB/c nude mice for 16 passages. This in vivo cell line, designated CML-N-1, had Ph1 chromosome abnormality and BCR gene rearrangement. The cells expressed CD11b, CD13, CD33, CD34, CD38, and HLA-DR antigens until the 11th passage and subcutaneous tumors produced by these passages were composed of admixtures of immature and maturing cells that differentiated to basophils when cultured in vitro. From the 12th passage on, the tumors became composed mainly of immature cells expressing CD13, CD34, and HLA-DR, and no longer differentiated to basophils even upon in vitro culture. In contrast to the vigorous proliferation in vivo, CML-N-1 cells from any passage failed to proliferate in vitro under standard liquid culture conditions with or without growth factors, such as granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor, monocyte colony-stimulating factor, interleukin 3, interleukin 6 and stem cell factor. However, a continuously growing cell line, designated CML-C-1, was established by culturing CML-N-1 cells on feeder layers of mouse bone marrow stromal cells. This mouse bone marrow stromal cell-dependent cell line showed immature cell morphology and expressed early myeloid phenotype positive for CD13, CD34, and HLA-DR. These results indicate that mouse bone marrow stromal cells provide a certain growth factor(s) active on human leukemia cells.