Effects of dietary uridine 5'-monophosphate on immune responses in newborn calves.

When compared with normal milk, bovine colostrum contains a large amount of uridine 5'-monophosphate (UMP) and its derivatives. In the present study, we carried out 2 experiments to determine the effects of dietary UMP (2 g/d) on the immune status of newborn calves. In Exp. 1, newborn Holstein bull calves were fed milk replacer alone (control group) or milk replacer supplemented with UMP (UMP group) from d 4 to 10 after birth. The increase in interferon-gamma concentration by peripheral blood mononuclear cells (PBMC) on d 24 tended to be greater in the UMP group than in the control group (P = 0.06). The IgA concentration of the ileal mucosa was greater in the UMP group than in the control group (P < 0.05), although there was no difference between groups in the jejunal mucosa. In Exp. 2, newborn Holstein bull calves were fed milk replacer alone (control group) or milk replacer supplemented with UMP (UMP group) from d 4 to 56 after birth. The proliferation of PBMC was greater in the UMP group than in the control group on d 14, 28, and 42 (P < 0.01). The increase in interferon-gamma concentration by PBMC was greater in the UMP group than in the control group on d 28 and 42 (P < 0.05). From these results, we concluded that dietary UMP affected the immune responses of newborn calves.

Recurrent herpes simplex virus infection in a patient with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy associated with L29P and IVS9-1G>C compound heterozygous autoimmune regulator gene mutations.

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) has characteristic clinical features with organ-specific autoimmune polyendocrine diseases and candidiasis, caused by the mutations of autoimmune regulator (AIRE) gene. Although almost all patients are complicated with mucocutaneous candidiasis, no apparent susceptibility to other infections has yet been reported. We herein report that a patient with APECED suffered from recurrent herpes simplex virus type 1 (HSV-1) infection after severe primary herpetic stomatitis, associated with sequential HSV-1 isolates of the same genomic profile, consistent with endogeneous recurrence. Thus, not only candidiasis but also HSV infection should receive more attention in patients with APECED, with treatment being administered accordingly.

Dietary fructooligosaccharides up-regulate immunoglobulin A response and polymeric immunoglobulin receptor expression in intestines of infant mice.

We examined whether or not dietary fructooligosaccharides (FOS) in infancy can have a beneficial effect on the mucosal immune system. Newborn BALB/c mice, accompanied by their dams until 21 days of age, were fed either a control diet based on casein [FOS- diet group] or a FOS- diet supplemented with 5% (w/w) FOS [FOS+ diet group]. Total IgA levels in tissue extracts from the intestines of mice in the FOS+ diet group at 38 days of age were about twofold higher (P < 0.05) than those in the FOS- diet group in the jejunum, ileum and colon. Ileal and colonic polymeric immunoglobulin receptor (pIgR) expression in the FOS+ diet group at 36 days of age was 1.5-fold higher than in the FOS- diet group (P < 0.05). Consistent with these results, the ileal IgA secretion rate of the FOS+ diet group at 37 days of age was twofold higher than that of the FOS- diet group (P < 0.05). Moreover, the percentage of B220(+)IgA+ cells in Peyer's patches (PP) was significantly higher in the FOS+ diet group than in the FOS- diet group (6.2%versus 4.3%, P < 0.05), suggesting that isotype switching from IgM to IgA in PP B cells might be enhanced in vivo. Taken together, our findings suggest that dietary FOS increases the intestinal IgA response and pIgR expression in the small intestine as well as the colon in infant mice.

Association study between CD30 and CD30 ligand genes and type 1 diabetes in the Japanese population.

CD30-CD30 ligand (CD30L) signal transduction appears to protect against autoimmune diabetes by preventing expansion of autoreactive T cells and suppressing Th1-cytokine response. The purpose of this study was to determine whether CD30 or CD30L genes serve as a novel susceptibility gene for type 1 diabetes in humans. We screened CD30 and CD30L genes for polymorphisms in Japanese patients with type 1 diabetes and control subjects. Then, association studies were performed between each of the identified polymorphisms and type 1 diabetes. Direct-sequencing analysis of the CD30 and CD30L genes revealed four polymorphisms: one in the CD30 gene (-201G/A from the transcription start site), and three in the CD30L gene [CA repeat in the promoter, 276G/A in the exon 3, -73T/C in the intron 3 (IVS3 -73T/C)]. Association studies revealed no association between the CD30 and CD30L genes and type 1 diabetes in the whole population. In the female and male subpopulations, however, the frequency of (CA)(9) allele of the CD30L gene promoter or T allele of IVS3 -73T/C polymorphism in the CD30L gene was slightly higher in female patients with type 1 diabetes than that in control females. In conclusion, we could not find significant association between CD30 or CD30L genes and type 1 diabetes, but (CA)(9) allele in the promotor or T allele of -73T/C in intron 3 in CD30L gene might play a minor role in the pathogenesis of type 1 diabetes, only in the Japanese female population.

Association studies of CTLA-4, CD28, and ICOS gene polymorphisms with type 1 diabetes in the Japanese population.

Co-stimulatory molecules of CD28, cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), and the newly identified inducible co-stimulator (ICOS) are expressed on cell surfaces and provide regulatory signals for T-cell activation. Their genes are candidate susceptibility genes for type 1 diabetes because they co-localize to Chromosome 2q33 with the IDDM12 locus. After determining the genomic structure and screening for polymorphisms of the ICOS gene, we performed association studies between newly identified polymorphisms of the ICOS gene, together with known polymorphisms of CD28 and CTLA-4 genes, and type 1 diabetes. The 49A/G dimorphism in exon 1 and the (AT)n in the 3' untranslated region of the CTLA-4 gene were significantly associated with type 1 diabetes. Evaluation of the CTLA-4 49A-3'(AT)n 86-bp haplotype frequency in patients and controls confirmed the results from the analysis of each polymorphic site. Dimorphism in intron 3 of the CD28 gene was associated with type 1 diabetes only in the early-onset group. In contrast, there was no association with the microsatellite polymorphisms in the ICOS gene or dimorphisms in the promotor region of CTLA-4. Of the three genes encoding co-stimulatory molecules, the CTLA-4 gene appears to confer risks for the development of type 1 diabetes.

Dietary nucleotides increase the proportion of a TCR gammadelta+ subset of intraepithelial lymphocytes (IEL) and IL-7 production by intestinal epithelial cells (IEC); implications for modification of cellular and molecular cross-talk between IEL and IEC by dietary nucleotides.

We have investigated the effects of dietary nucleotides on intraepithelial lymphocytes (IEL) and intestinal epithelial cells (IEC) in weanling mice. The proportion of T-cell receptor (TCR) gammadelta+ IEL in BALB/c mice fed a diet supplemented with nucleotides (NT(+) diet) was significantly higher than that in mice fed the nucleotide-free diet, while the proportion of TCR alphabeta+ IEL in NT(+) diet-fed mice was significantly decreased. The change of the TCR alphabeta+/TCR gammadelta+ ratio was mainly observed in a CD8 alphaalpha+ subset of IEL. IEC from NT(+) diet-fed mice produced a higher level of IL-7, which is important in the development of TCR gammadelta+ IEL, than those from control diet-fed mice. The expression levels of IL-7 and IL-2 receptors on IEL were not different between the two dietary groups. Our findings suggest that the increased population of a TCR gammadelta+ IEL subset by feeding nucleotides may be caused by the increased production of IL-7 by IEC.

Suppression of insulitis and diabetes in B cell-deficient mice treated with streptozocin: B cells are essential for the TCR clonotype spreading of islet-infiltrating T cells.

In order to clarify the role of B cells in the development of insulitis and diabetes, B cell-deficient (B(-)) mice treated with streptozocin (STZ) were studied. The extent of insulitis and the cumulative incidence of diabetes were significantly suppressed in B(-) mice (P < 0.0001), indicating that B cells are crucial for the progression of insulitis and diabetes. Accumulation of both CD4(+) T cells and B cells was observed in islets of B(+) mice, while CD4(+) T cells but not B cells were found in B(-) mice. A few CD8(+) T cells and macrophages were detectable in both types of mice. The immunohistochemical study did not reveal any change in the subpopulations of infiltrating lymphocytes except for the absence of B cells in the B(-) mice. TCR V(beta) gene repertoire usage of islet-infiltrating T cells was restricted to some extent in the B(+) or B(-) mice, but there was no significant difference between the B(+) and B(-) mice, suggesting that the initial islet-reactive T cell response can occur in the absence of B cells. In contrast, TCR clonotype spreading of islet-infiltrating T cells was significantly suppressed in B(-) mice compared with B(+) mice (P < 0.0001). These data suggest that initial priming of T cells is not impaired and TCR V(beta) repertoire usage is not limited by the lack of B cells, while B cells are important essentially for the spreading of islet-infiltrating clonal T cells in autoimmune diabetic mice induced with STZ.

Dietary nucleotides can up-regulate antigen-specific Th1 immune responses and suppress antigen-specific IgE responses in mice.

BACKGROUND: It has been reported that dietary nucleotides enhance T helper cell activities. In this study, we have determined the effects of dietary nucleotides on antigen-specific Th1 and Th2 responses and IgE responses. METHODS: Ovalbumin (OVA)-specific T cell receptor (TCR) transgenic (OVA-TCR Tg) mice, 3 weeks old, were fed a nucleotide-free diet (NT(-) diet) or the NT(-) diet supplemented with dietary nucleotides (NT(+) diet) for 4 weeks. Cytokine production by spleen cells and macrophages obtained from these mice was measured in vitro. BALB/c mice, 3 weeks old, immunized intraperitoneally with OVA adsorbed onto alum, were fed the NT(-) diet or the NT(+) diet for 4 weeks. Serum levels of antigen-specific antibodies in the BALB/c mice were determined by ELISA. RESULTS: The level of production of antigen-specific interferon-gamma by spleen cells was significantly higher in the OVA-TCR Tg mice fed the NT(+) diet than in the control mice. The levels of secretion of bioactive IL-12 by spleen cells and peritoneal macrophages were also significantly increased in the NT(+) diet group. The serum OVA-specific IgE level was significantly decreased in BALB/c mice fed the NT(+) diet compared with those fed the NT(-) diet. CONCLUSION: These results show that dietary nucleotides up-regulate the antigen-specific Th1 immune response through the enhancement of IL-12 production and suppress the antigen-specific IgE response.

Accelerated secretion of mutant beta-lactoglobulin in Saccharomyces cerevisiae resulting from a single amino acid substitution.

Transformed yeasts producing a mutant form of bovine beta-lactoglobulin (beta-LG), W19Y, in which Trp(19) was replaced with Tyr, were shown to secrete 6 times more than those producing wild type beta-LG. Northern blot analysis suggested that the enhanced level of secretion was not the result of upregulated transcription of W19Y. The ratio of the amount of W19Y secreted into the supernatant to the amount of W19Y remaining inside the cells was much larger than that in the case of wild type beta-LG as shown by immunoblot analysis. A pulse/chase experiment revealed that the speed of secretion of W19Y was significantly accelerated, compared to wild type beta-LG. These results indicated that W19Y was more efficiently and rapidly transported in the course of secretion than wild type beta-LG. Our previous study showed that the DeltaG of unfolding of W19Y in water is 6.9 kcal/mol smaller than that of wild type beta-LG. Furthermore, immunoblot analysis of intracellular beta-LG under non-reducing conditions indicated that W19Y as well as wild type beta-LG maintained a specific folded structure inside the yeast cells, whereas other non-secretable mutant beta-LGs with Phe or Ala at position 19 (W19F and W19A, respectively) did not. These data suggest that low molecular stability and the maintenance of a specific folded structure inside the yeast cells are prerequisites for efficient and rapid secretion. W19Y was more efficiently secreted than wild type beta-LG also in transformed ern1 mutant yeast cells expressing only a basal level of BiP which is considered to function in quality control in the endoplasmic reticulum (ER) by playing an important role in determining the secretion efficiency of secretory proteins. Thus, the reason for the enhanced secretion of W19Y is considered to be that the improved folding ability of W19Y can allow the half-life of the W19Y-BiP complex to become shorter than that of the wild type beta-LG-BiP complex, leading to faster translocation of W19Y into transport vesicles, or that W19Y can fold in a BiP-independent manner in the ER of the yeast cells. Our findings demonstrate that the amount of protein secreted can be improved by alteration of a single amino acid residue crucial for its structure.

Establishment of an embryonic stem (ES) cell line derived from a non-obese diabetic (NOD) mouse: in vivo differentiation into lymphocytes and potential for germ line transmission.

A non-obese diabetic (NOD) mouse-derived embryonic stem (ES) cell line has been stably maintained in an undifferentiated state with a characteristic ES cell-like morphology, expressing the stem cell marker alkaline phosphatase, and displaying a normal diploid karyotype. After injecting the NOD-ES cells into blastocysts, chimeric mice were obtained. Small but significant numbers of lymphocytes expressed the NOD-derived MHC allele. When a chimeric mouse was mated with C57BL/6 mice, an agouti mouse was obtained, having the NOD-derived H-2 I-A(beta)g7 haplotype. Thus, an NOD-ES cell line which can differentiate into lymphocytes with potential for germ line transmission was successfully established.

Strain dependency of the immunopotentiating activity of Lactobacillus delbrueckii subsp. bulgaricus.

To obtain strains of Lactobacillus delbrueckii subsp. bulgaricus with high immunopotentiating activity, we screened 90 strains of this bacterial species for the proliferative response of murine spleen and beta-lactoglobulin-primed lymph node cells. In this screening, certain strains showed strong immunopotentiating activity. Among them, strain 1023 had the strongest mitogenic activity for murine Peyer's patch (PP) cells. Furthermore, strain 1023 induced IgA antibody production by PP cells as strongly as Bifidobacterium longum 6001, which had adjuvant activity when orally administered. Also in the assays using immune cells from human-flora-associated mice a few strains including 1023 showed strong immunopotentiating activity comparable to B. longum 6001. These results suggest that L. delbrueckii subsp. bulgaricus strains such as 1023 may be useful for the production of fermented milk with a more beneficial effect on the systemic and mucosal immune system.

Association of polymorphism in the NeuroD/BETA2 gene with type 1 diabetes in the Japanese.

NeuroD/BETA2, a transcription factor of the insulin gene, also plays an important role in the development of pancreatic beta-cells. Recently, the NeuroD/BETA2 gene has been mapped to the long arm of human chromosome 2 (2q32) where the IDDM7 gene has previously been mapped, implying its involvement in diabetes. To identify mutations in the NeuroD/BETA2 gene that may predispose patients to develop diabetes, we studied the gene in 50 Japanese subjects with diabetes (4 with type 1 and 46 with type 2) by the polymerase chain reaction (PCR) followed by single-strand conformation polymorphism and sequencing analyses. Further analysis was performed in 392 Japanese subjects (60 with type 1 and 158 with type 2 diabetes and 174 healthy control subjects) by mismatch PCR restriction fragment length polymorphism. We found a DNA polymorphism of the NeuroD/BETA2 gene. A nucleotide G-to-A transition results in the substitution of alanine to threonine at codon 45 (Ala45Thr). The frequencies of heterozygotes for the Ala45Thr variant were 9.8% in the control subjects, 9.5% in the patients with type 2 diabetes, and 25.0% in the patients with type 1 diabetes, a significant difference (P = 0.006). Because the variant of the NeuroD/BETA2 gene (Ala45Thr) is associated with type 1 but not type 2 diabetes, it may be implicated in the loss of pancreatic beta-cells in type 1 diabetes.

CD95 (Fas) ligand expression of Epstein-Barr virus (EBV)-infected lymphocytes: a possible mechanism of immune evasion in chronic active EBV infection.

The Epstein-Barr virus (EBV) induces infectious mononucleosis (IM) and can be associated with chronic active EBV infection (CAEBV). Cytotoxic T lymphocytes (CTL) play an important role in excluding EBV-infected cells. Two cytotoxic mechanisms of CTL have been demonstrated: one perforin/granzyme-based and the other Fas (CD95)/Fas ligand (FasL)-based. To clarify these two pathways in CAEBV, we analyzed six patients with CAEBV and four patients with IM using immunohistochemical staining of the lymph nodes. In both CAEBV and IM, CD8+ T-cells increased in number, but CD56+ natural killer cells were rare. In four of six cases with CAEBV, approximately half the lymphocytes were positive for T cell-restricted intracellular antigens (TIA-1), which were recognized by the cytolytic granules of CTL. In IM, the number of TIA-1 positive cells was smaller than that in CAEBV. Fas-positive lymphocytes were frequently encountered in both CAEBV and IM. However, FasL-positive lymphocytes increased in three of six patients with CAEBV, but not in patients with IM. Except for one case with CAEBV, the number of perforin- and/or granzyme-positive cells was small in number in both CAEBV and IM cases. In double-staining FasL and EBV in situ hybridization, FasL-positive EBV-infected lymphocytes were detected in CAEBV but not in IM. In CAEBV, the Fas/FasL pathway and not perforin pathways appears to play an important role in the pathogenesis. The data suggest that EBV-infected lymphocytes may evade immune attack through the expression of FasL.

Proliferation of CD3+ B220- single-positive normal T cells was suppressed in B-cell-deficient lpr mice.

It is known that lpr mice develop systemic lymphadenopathy and lupus erythematosus-like autoimmune disease that are associated with the accumulation of CD4- CD8- (double-negative; DN) CD3+ B220+ abnormal T cells as well as normal mature CD4+ or CD8+ single-positive (SP) CD3+ T cells. In order to clarify the role of B cells in the lymphoproliferation and autoimmunity of lpr mice, we created B-cell-deficient C57BL/6 (B6) lpr mice (B6lpr/lpr microMT/microMT) by crossing B6lpr/lpr mice with B6 microMT/microMT mice in which the B-cell development was arrested at pre-B stage owing to a targeted disruption of the immunoglobulin mu heavy-chain gene locus. In the B-cell-deficient B6-lpr mice, both lymphadenopathy and splenomegaly were markedly suppressed. Although the accumulation of both CD3+ B220- SP normal T cells and CD3+ B220+ DN abnormal T cells was inhibited in the B-cell-deficient lpr mice, the decrease in numbers of CD3+ B220- SP normal T cells occurred more strikingly than that of the CD3+ B220+ DN abnormal T cells. Glomerulonephritis did not develop in the B-cell-deficient lpr mice over 40 weeks. The present results indicate that the B cells thus play a crucial role in the extensive proliferation of normal CD3+ B220- mature SP T cells rather than the accumulation of abnormal DN T cells.

Beta2-glycoprotein I-dependent and -independent anticardiolipin antibody in non-obese diabetic (NOD) mice.

In the present study we investigate whether or not anticardiolipin antibody (aCL) is produced in NOD mice, which is a representative animal model of insulin-dependent diabetes mellitus (IDDM). We found that IgG class aCL appeared in 6.9% of non-diabetic NOD mice at weeks 5-15. The rates increased with age to 31.6% at weeks 16-25 and 71.9% at weeks 26-35. In addition, the titre and incidence of aCL were higher in diabetic mice than in non-diabetic mice. It was also found that aCL in NOD mice involved beta2-glycoprotein I (beta2-GPI)-dependent and -independent aCL, when beta2-GPI was added to the aCL assay system. The IgG subclass of both beta2-GPI-dependent and -independent aCL belonged exclusively to IgG2a. In addition, immunohistochemical studies revealed the predominant accumulation of IgG2a- or IgG3-positive B lymphocytes within insulitis. These results suggest that the autoimmunity in NOD mice may thus be associated with Th1 predominant immunological response. In conclusion, aCL with multiple antigenic specificity were produced in NOD mice along with the development of insulitis and diabetes. NOD mice should thus be added to the list of animal models possessing antiphospholipid antibody.

Intradermal administration of viral vaccines.

Intradermal administration maybe useful in lowering the cost of vaccination against hepatitis B significantly, and may also be helpful for the rapid induction of antibodies, reversing non-responsiveness, improving postexposure prophylaxis and immunising immunocompromised people. In addition, delayed type hypersensitivity skin reaction to the vaccine could serve as a useful marker for the acquisition of T helper type 1 immunoreactivity in vivo. However, there are some disadvantages when using intradermal vaccinations, including the requirement for skilful administration, the absence of approval from licensing authorities, the development of local skin reactions and a lower antibody response when 1/10 of the standard vaccine dose is used. This requires that appropriate vaccination regimens, including the correct vaccine dosage, and vaccination schedule are followed. In the future, a similar vaccination strategy might also be applied for the prevention and control of other infectious diseases. Copyright 1998 John Wiley & Sons, Ltd.

Clinicopathological study of severe chronic active Epstein-Barr virus infection that developed in association with lymphoproliferative disorder and/or hemophagocytic syndrome.

Chronic active Epstein-Barr virus (CAEBV) infection has been previously reported to be sometimes associated with an aggressive clinical course. However, the role of EBV in the CAEBV is not well clarified. A retrospective study was performed on nine adult and five child patients (eight males and six females). Histologically, at first admission, the presence of neoplastic lesions could not be confirmed. The lymph nodes in half of all cases revealed paracortical hyperplasia with transformed lymphocytes (hyperplastic type). Half of the cases showed non-suppurative necrosis and an increased number of histiocytes with phagocytosis (histiocytic type). Activated histiocytes with lymphokine positivity were frequently detected in the histiocytic type. In the phenotypical study, 10 of the examined 11 cases showed increased numbers of natural killer (NK) cells and/or CD8-positive T lymphocytes. In situ hybridization (ISH) showed EBV-infected lymphoid cells, but the number of EBV-infected cells varied. Double-labeling immunochemistry/ISH demonstrated EBV-infected T cells, including NK cells, but not B cells. In addition, three cases showed a monoclonal dissemination of EBV terminal repetitive sequence (TR), and two cases showed oligoclonal dissemination. From those findings, monoclonal, oligoclonal and polyclonal populations of EBV-infected T or NK cells were considered to be present in CAEBV states. During the clinical course, 12 of the 14 cases died within 5 years. Six cases died from EBV-associated hematopoietic tumors (histiocytic tumor, T cell lymphoma, B cell lymphoma, plasmacytoma, and NK cell leukemia); one from non-EBV-associated acute myelogenous leukemia, and five due to hemophagocytic syndrome. The examined EBV-associated hematopoietic tumors showed monoclonal EBV terminal repetitive sequences. There is a possibility that the monoclonal dissemination of EBV-infected cells develops from oligoclonal or polyclonal EBV-infected cells. And active histiocytes with lymphokine positivity were frequently detected in the cases with histologically histiocytic type. These findings seem to be related with the causes of death due to hemophagocytic syndrome.

Dentatorubral pallidoluysian atrophy (DRPLA) protein is cleaved by caspase-3 during apoptosis.

Dentatorubral pallidoluysian atrophy (DRPLA) is an autosomal dominant neurodegenerative disorder. It is associated with an abnormal CAG repeat expansion resulting in formation of a protein with an elongated polyglutamine stretch. However, neither the physiological roles of the DRPLA gene product nor molecular mechanisms of its pathogenesis have yet been elucidated. Here we report that the DRPLA protein is cleaved at a site near the N terminus during apoptosis induced by VP-16, staurosporine, or glucocorticoid. Moreover, the in vitro translated DRPLA protein is cleaved by recombinant caspase-3, a member of the cysteine protease family, which is thought to be a main executioner of apoptosis. Using mutant DRPLA proteins, the cleavage site was identified as 106DSLDG110. The cleavage, however, was not modulated by the length of the polyglutamine stretch. These findings suggest that the DRPLA protein is one of the physiological substrates of caspase-3, and its cleavage may result in structural and biochemical alterations associated with apoptosis.