Motion-Induced Transition of Positronium through a Static Periodic Magnetic Field in the Sub-THz Region.

Atoms moving in a static periodic field experience a time-dependent oscillating field in their own rest frame. By tuning the frequency, an atomic transition can be induced. So far, this type of transition has been demonstrated in the EUV region or at higher frequencies by crystalline fields and in the microwave region by artificial fields. Here, we present the observation of the transition of positronium (Ps) in the sub-THz region by using an energy-tunable Ps beam with a multilayered magnetic grating. This grating produces a microsized periodic field, whose amplitude corresponds to a huge energy flux of ∼100 MW cm^{-2}, resulting in the efficient magnetic dipole transition.

T cell receptor V gene usage of islet beta cell-reactive T cells is not restricted in non-obese diabetic mice.

Five islet-reactive T cell clones were established from islet-infiltrating T cells of non-obese diabetic (NOD) mice. All clones expressed CD4, but not CD8, and responded to islet cells from various strains of mice in the context of I-ANOD. They could induce insulitis when transferred into disease-resistant I-E+ transgenic NOD mice. The T cell receptor (TCR) sequences utilized by the clones were determined. Their usage of TCR V and J segments was not restricted but was rather diverse. One of the clones utilized V beta 16. The expression of V beta 16 was significantly reduced in I-E+ transgenic NOD, suggesting the possibility that the islet-reactive T cell clone expressing V beta 16 may be deleted or inactivated by I-E molecules. This clone might be one of the candidates that triggers insulitis.

Induction and properties of cytoplasmic factor(s) which enhance nuclear nonhistone protein phosphorylation in lymphocytes stimulated by anti-Ig.

An increased in vitro phosphorylation of nonhistone nuclear proteins (NHP) was observed in the nuclei isolated from rabbit lymphocytes which had been stimulated with anti-Ig for 4 h. No concomitant increase of phosphorylation in histones or 0.14 M NaCl-soluble proteins was observed. The increase of in vitro phosphorylation of NHP was also observed in the nuclei isolated from nonstimulated cells when these nuclei were preincubated for 2 h with cell-free extracts from anti-Ig-stimulated cells. The active substance in cell-free extracts was maximally induced when lymphocytes were stimulated with anti-Ig for 2 h. The induction of an increased phosphorylation of NHP in nonstimulated nuclei with the cell-free extracts was not due to decrease of the adenosine triphosphate pool in the extracts from anti-Ig-stimulated cells. The active substance in cell-free extracts was not NHP-protein kinase itself, but it probably activated NHP-protein kinase in quiescent nuclei. The active substance was nondialyzable and probably protein. It was resistant against heating at 56 degrees C for 30 min, but the activity was completely destroyed by heating at 90 degrees C for 30 min. The active substance may be responsible for the transduction of the membrane-mediated signals given through Ig receptors to nuclei.

Impairment of T cell development in deltaEF1 mutant mice.

Using the method of gene targeting in mouse embryonic stem cells, regulatory function of deltaEF1, a zinc finger and homeodomain-containing transcription factor, was investigated in vivo by generating the deltaEF1 mutant mice. The mutated allele of deltaEF1 produced a truncated form of the deltaEF1 protein lacking a zinc finger cluster proximal to COOH terminus. The homozygous deltaEF1 mutant mice had poorly developed thymi with no distinction of cortex and medulla. Analysis of the mutant thymocyte showed reduction of the total cell number by two orders of magnitude accompanying the impaired thymocyte development. The early stage intrathymic c-kit+ T precursor cells were largely depleted. The following thymocyte development also seemed to be affected as assessed by the distorted composition of CD4- or CD8-expressing cells. The mutant thymocyte showed elevated alpha4 integrin expression, which might be related to the T cell defect in the mutant mice. In the peripheral lymph node tissue of the mutant mice, the CD4-CD8+ single positive cells were significantly reduced relative to CD4+CD8-single positive cells. In contrast to T cells, other hematopoietic lineages appeared to be normal. The data indicated that deltaEF1 is involved in regulation of T cell development at multiple stages.

Fc epsilon receptor, a specific differentiation marker transiently expressed on mature B cells before isotype switching.

The expression of Fc epsilon R on human lymphocytes was studied with the anti-Fc epsilon R mAbs. Fc epsilon R was expressed on most mu+,delta+ circulating B cells, whereas T cells did not express Fc epsilon R even in patients with hyper-IgE syndrome. B cells with gamma, alpha, or epsilon phenotype did not express Fc epsilon R, moreover its expression could not be induced, suggesting that the Fc epsilon R expression was correlated with isotype switching. mu+delta+ B cells in bone marrow did not express Fc epsilon R, but PHA-sup (supernatant from PHA-stimulated cell cultures) could induce its expression, and the addition of IgE augmented this induction. Recombinant IL-2, IL-1, IFN-gamma or -beta, or purified B cell differentiation factor (BSF-2 B cell-stimulatory factor 2) could not induce Fc epsilon R expression in bone marrow B cells. IFN-gamma inhibited the Fc epsilon R expression induced by PHA-sup, suggesting that the human counterpart of BSF-1 may be responsible for Fc epsilon R expression in bone marrow B cells. B cells from patients with common variable immunodeficiency and ataxia telangiectasia did not express Fc epsilon R, but PHA-sup could induce its expression, indicating that circulating B cells of these patients are at a differentiation stage similar to B cells in bone marrow. The study showed that Fc epsilon R is a B cell-specific differentiation marker, the expression of which is restricted to a defined stage of B cell differentiation.

Strain dependency of B and T lymphoma development in immunoglobulin heavy chain enhancer (E mu)-myc transgenic mice.

The transgenic mice were produced by injecting eggs of B6 and C3H/HeJ mice with the human E mu-myc gene. Preferential development of B lymphomas was observed in the B6 transgenic mice, whereas the C3H/HeJ transgenic mice developed mostly T lymphomas. The phenotypic activation of B lineage cells but not of T lineage cells was detected in the prelymphomatous transgenic mice of both strains. The transgene was similarly expressed in B and T cells of the transgenic mice of both strains. These results suggest that a high incidence of T lymphomas in the C3H/HeJ transgenic mice may not be due to the preferential activation of or the preferential E mu-myc expression in T lymphocytes. When the bone marrow or fetal liver cells from the prelymphomatous transgenic mice of both strains were transferred into irradiated normal C3H/HeJ mice, most of the recipients developed T lymphomas. Moreover, even when irradiated B6 mice received the hematopoietic stem cells from the prelymphomatous B6 transgenic mice, the incidence of T lymphoma increased up to 50%. These findings suggest that B6 and C3H/HeJ mice might provide the environment that supports the development or growth of B and T lymphomas, respectively, and that such an environment could be modified by irradiation of the mice.

CD40-CD40 ligand interactions are critical in T-B cooperation but not for other anti-viral CD4+ T cell functions.

CD40-CD40 ligand (CD40L) interaction is required for the generation of antibody responses to T-dependent antigens as well as for the development of germinal centers and memory B cells. The role of the CD40-CD40L interaction in the induction of antigen-specific. Th cells and in mediating Th cell effector functions other than cognate help for B cells is less well understood. Using CD40- and CD40L-deficient mice together with lymphocytic choriomeningitis virus and vesicular stomatitis virus as viral model antigens, this study corroborates earlier findings that no lg isotype switching of virus-specific antibodies was measurable upon infection of CD40- or CD40L-deficient mice. In contrast, in vivo induction of virus-specific CD4+ T cells measured by proliferation and cytokine secretion of primed virus-specific Th cells in vitro was not crucially dependent on the CD40-CD40L interaction. In addition, virus-specific Th cells primed in a CD40-deficient environment, adoptively transferred into CD40-competent recipients, were able to mediate lg isotype switch. Th-mediated effector functions distinct from and in addition to T-B collaboration were analyzed in CD40- and CD40L-deficient and normal mice: (a) local inflammatory reactions upon LCMV infection mediated by LCMV-specific Th cells were not dependent on a functional CD40-CD40L interaction, (b) cytokine-mediated protection by CD4+ T cells primed by vesicular stomatitis virus against a challenge infection with recombinant vaccinia virus expressing the glycoprotein of vesicular stomatitis virus was found to be equivalent in CD40L-deficient and normal mice. Thus, CD40-CD40L interaction plays a crucial role in T-B interactions for Th-dependent activation of B cells but not, or to a much lesser extent, in T cell activation, antigen-specific Th cell responses in vitro, and for interleukin-mediated Th cell effector functions in vivo.

Mimicry of CD40 signals by Epstein-Barr virus LMP1 in B lymphocyte responses.

The effect of the Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) on the activation and differentiation of normal B cells was investigated. B cells of transgenic mice expressing LMP1 under the control of immunoglobulin promoter/enhancer displayed enhanced expression of activation antigens and spontaneously proliferated and produced antibody. Humoral immune responses of LMP1 transgenic mice in CD40-deficient or normal backgrounds revealed that LMP1 mimics CD40 signals to induce extrafollicular B cell differentiation but, unlike CD40, blocks germinal center formation. Thus, these specific properties of LMP1 may determine the site of primary B cell infection and the state of infection in the natural course of EBV infection, whereas subsequent loss of LMP1 expression may affect the site of persistent latent infection.

Aeroallergen-induced eosinophilic inflammation, lung damage, and airways hyperreactivity in mice can occur independently of IL-4 and allergen-specific immunoglobulins.

In this investigation we have used a mouse model containing certain phenotypic characteristics consistent with asthma and IL-4- and CD40-deficient mice to establish the role of this cytokine and allergen-specific immunoglobulins in the initiation of airways hyperreactivity and morphological changes to the airways in responses to aeroallergen challenge. Sensitization and aerosol challenge of mice with ovalbumin resulted in a severe airways inflammatory response which directly correlated with the induction of extensive airways damage and airways hyperreactivity to beta-methacholine. Inflammatory infiltrates were primarily characterized by the presence of CD4+ T cells and eosinophils. In IL-4-deficient mice, the recruitment of airways eosinophils was impaired, but not abolished in response to aeroallergen. Moreover, the characteristic airways damage and hyperreactivity normally resulting from allergen inhalation were not attenuated. Induction of these structural and functional changes to the airways occurred in the absence of ovalbumin-specific IgE and IgG1, but IgG2a and IgG3 were detected in the sera of IL-4-deficient mice. CD4+ T cells isolated from both wild-type and IL-4-deficient mice given ovalbumin produced significant levels of IL-5 after in vitro stimulation. Treatment of IL-4-deficient mice with anti-IL-5 mAb before aeroallergen challenge abolished blood and airways eosinophilia, lung damage, and airways hyperreactivity. These results indicate that IL-4 is not essential for the development of IL-5-producing CD4+ T cells or for the induction of eosinophilic inflammation and airways damage and hyperreactivity. In response to sensitization and aerosol challenge, CD40-deficient mice did not produce ovalbumin-specific IgE, IgG isotypes, or IgA, and airways inflammation and hyperreactivity were not attenuated. Our results suggest that allergic airways disease can occur via pathways which operate independently of IL-4 and allergen-specific immunoglobulins. Activation of these pathways is intimately associated with IL-5 and eosinophilic inflammation. Such pathways may play a substantive role in the etiology of asthma.

A case of human B cell leukemia that implicates an autocrine mechanism in the abnormal growth of Leu 1 B cells.

Production of B cell growth factor (BCGF) from B-chronic lymphocytic leukemia (B-CLL) cells was demonstrated. Freshly isolated monoclonal B-CLL cells expressed surface mu, delta, B1, and Leu 1, but not Ba (an antigen expressed only on activated B cells). Upon stimulation with anti-IgM, they secreted BCGF, which could act on anti-IgM-stimulated autologous leukemic cells as well as anti-IgM-stimulated normal B cells. Cell lines established from these leukemic cells also constitutively secreted BCGF. The BCGF from B-CLL cells or established cell lines induced neither proliferation nor enhanced HLA-DR expression in resting B cells. These results show the presence of B cell-derived BCGF, which is distinct from BSF-1 and effective only on activated B cells. They also suggest that an autocrine mechanism may operate in the growth of B-CLL cells.

Prevention of adoptively transferred diabetes in nonobese diabetic mice with IL-10-transduced islet-specific Th1 lymphocytes. A gene therapy model for autoimmune diabetes.

Four pancreatic islet-specific CD4+ helper T (Th) 1 (Th1) clones and two Th1 clones transduced with an SRalpha promoter-linked murine IL-10 (mIL-10) cDNA of 2.0-6.0 x 10(6) cells were adoptively transferred to nonobese diabetic (NOD) mice at age 8 d. Cyclophosphamide (CY) was administered at age 37 d (plus CY), and the incidence of diabetes and the histological grade of insulitis were examined at age 47 d. After the adoptive transfer of IL-10-transduced Th1 cells, polymerase chain reaction (PCR) and reverse-transcription (RT)-PCR detected the neo gene and the retrovirus vector-mediated IL-10 mRNA in situ in recipient islets, respectively. RT-PCR detected the decrease of IFN-gamma mRNA relative to IL-10 mRNA in IL-10-transduced Th1 clones in vitro and also in recipient islets. All four wild type Th1 clones plus CY induced the insulitis grade of 2.75 and diabetes in 66% of recipient NOD mice. IL-10-transduced two Th1 clones plus CY induced periinsulitis with the grade of 1.43 and diabetes in 8.0%. The 1:1 mixture of wild type Th1 cells and IL-10-transduced Th1 cells plus CY induced periinsulitis with the grade of 1.85 and diabetes in 20%. The suppression of diabetes through decreasing IFN-gamma mRNA by the tissue-specific delivery of IL-10 to pancreatic islets with IL-10-transduced Th1 cells affords us the starting basis to develop the gene therapy for autoimmune diabetes.

Abrogation of autoimmune diabetes in nonobese diabetic mice and protection against effector lymphocytes by transgenic paracrine TGF-beta1.

Paracrine effect of transforming growth factor-beta1 (TGF-beta1) on autoimmune insulitis and diabetes was studied by transgenic production of the active form of porcine TGF-beta1 (pTGF-beta1) in pancreatic islet (islet) alpha cells in nonobese diabetic (NOD) mice under the control of rat glucagon promoter (RGP) (NOD-RGP-TGF-beta1). None of 27 NOD-RGP-TGF- beta1 mice developed diabetes by 45 wk of age, in contrast to 40 and 71% in male and female nontransgenic mice, respectively. None of the NOD-RGP-TGF-beta1 mice developed diabetes after cyclophosphamide (CY) administration. Adoptive transfer of splenocytes of NOD-RGP-TGF-beta1 mice to neonatal NOD mice did not transfer diabetes after CY administration. Adoptive transfer of three types of diabetogenic lymphocytes to NOD-RGP-TGF-beta1 and nontransgenic mice after CY administration led to the lower incidence of diabetes in NOD-RGP-TGF-beta1 mice versus that in nontransgenic mice: 29 vs. 77% for diabetogenic splenocytes, 25 vs. 75% for islet beta cell-specific Th1 clone cells, and 0 vs. 50% for islet beta cell-specific CD8(+) clone cells, respectively. Based on these, it is concluded that autoimmune diabetes in NOD mice is not a systemic disease and it can be completely prevented by the paracrine TGF-beta1 in the islet compartment through protection against CD4(+) and CD8(+) effector lymphocytes.

Protective immunity induced by tumor vaccines requires interaction between CD40 and its ligand, CD154.

Interactions between CD40 and its ligand, CD154 (CD40L, gp39), have been shown to play a central role in the regulation of humoral immunity. Recent evidence suggests that this ligand-receptor pair also plays an important role in the induction of cell-mediated immune responses, including those directed against viral pathogens, intracellular parasites, and alloantigens. The contribution of this ligand-receptor pair to the development of protective immunity against syngeneic tumors was evaluated by blocking the in vivo function of CD154 or by studying tumor resistance in mice genetically deficient in CD40 expression (CD40-/-). In the former case, anti-CD154 monoclonal antibody treatment inhibited the generation of protective immune responses after the administration of three potent tumor vaccines: irradiated MCA 105, MCA 105 admixed with Corynebacterium parvum adjuvant, and irradiated B16 melanoma cells transduced with the gene for granulocyte macrophage colony-stimulating factor. Confirmation of the role of CD40/CD154 interactions in tumor immunity was provided by the overt tumor susceptibility in CD40-deficient mice as compared to that in CD40+/+ mice. In this case, wild-type but not CD40-deficient mice could be readily protected against live TS/A tumor challenge by preimmunization with TS/A admixed with C. parvum. These findings suggest a critical role for CD40/CD154 interactions in the induction of cellular immunity by tumor vaccines and may have important implications for future approaches to cell-based cancer therapies.

Induction of 19S IgM secretion in a murine pre-B cell line, 70Z/3, by cell hybridization with non-secreting myeloma cells.

Somatic cell hybrids were prepared between the TK-deficient variant of murine pre-B cell line, 70Z/3, cells and the HGPRT-deficient variant of non-secreting myeloma cells. Several hybrid clones which secreted IgM but did not express surface IgM were isolated. LPS stimulation did not induce the expression of surface IgM. SDS-PAGE analysis indicated that the IgM secreted by one of the hybrid clones was a 19S pentamer and that the size of its mu-chains was the same as that of mu-chains from MOPC 104E myeloma IgM. Two-dimensional gel electrophoresis of biosynthetically labeled immunoglobulin showed that the same pattern was obtained with kappa-chains from two hybrid clones and from the LPS-induced 70Z/3 cells. The result showed that cell hybridization could induce L-chain synthesis in the pre-B cell line. Anti-idiotypic antibody against the secreted IgM was prepared and it was shown that the surface IgM expressed on all LPS-stimulated 70Z/3 cells bore the same idiotype. Those results indicated that the specificity commitment has already occurred in 70Z/3 cells.

Functional interaction between beta 2-adrenoceptor agonists and interleukin-4 in the regulation of CD23 expression and release and IgE production in human.

Normal human peripheral blood mononuclear cells (PBMC) produced IgE when stimulated with IL-4. In the present report it was shown that beta 2-adrenoceptor agonists, salbutamol and fenoterol, potentiated the IL-4-induced IgE production without significantly affecting the expression of the low affinity receptor for IgE at the cell surface of monocytes and B lymphocytes. However, beta 2-adrenoceptor agonists were shown to enhance at day 7 the IL-4-induced release of the soluble form of CD23 (sCD23) by PBMC. This effect was specific since a beta-adrenoceptor antagonist, D,L-propranolol, inhibited the IL-4-induced IgE production by these cells. Alternatively, the beta 2-adrenoceptor agonists inhibited the production by these cells of interferon-gamma (IFN-gamma) but did not affect the production of IL-4 when stimulated with phytohemagglutinin A + a phorbol ester. These data suggest that beta 2-adrenoceptor agonists influence the IL-4-induced IgE production in humans by enhancing the release of sCD23 and inhibiting the production of endogenous IFN-gamma. In addition to the effect on the IL-4-induced IgE production it was shown that beta 2-adrenoceptor agonists potentiated the effect of IL-4 on a human promonocytic cell line, U 937, by enhancing CD23 expression and release and by inducing the differentiation of these cells into monocyte-like cells. Taken together, these data indicate that beta 2-adrenoceptor agonists potentiated the effect of IL-4 and that this functional interaction is different considering the cell-lineage and the stage of differentiation of these cells.

Epoc-1: a POU-domain gene expressed in murine epidermal basal cells and thymic stromal cells.

POU-domain transcription factors are known as developmental regulators which control organ development and cell phenotypes. In order to clarify the roles of POU-domain transcription factors in cell differentiation, we cloned a novel POU family gene, Epoc-1, from a murine thymus cDNA library. The amino acid (aa) sequence of the POU-specific domain of Epoc-1 is almost identical to those of Oct-1 and Oct-2. However, within the POU-homeodomain, 13 out of 60 aa differ between Epoc-1 and Oct-2. Recombinant Epoc-1 products were found to bind specifically to the octamer sequence. Epoc-1 was found to be expressed in skin, thymus, stomach and testis. In situ hybridization experiments and RNase protection assays indicated that Epoc-1 is expressed in the epidermal basal cells of the skin, which contain stem cells unipotent for keratinocyte differentiation and in thymic stromal elements. These results suggest that Epoc-1 might be one of the developmental regulators which controls epidermal development and thymic organogenesis.

Introduction of human gamma 1 immunoglobulin genes into fertilized mouse eggs.

A rearranged human gamma 1 immunoglobulin gene was introduced into fertilized mouse eggs. The phage Ch4A-VCE-gamma 1 was constructed by ligating an EcoRI and BglII fragment of pBR322-CESSV(CE-1) containing the VDJ region with an EcoRI and BamHI fragment of Ch4A-HIg gamma 1-10 containing the gamma 1 constant region. About 200 copies of Ch4A-VCE-gamma 1 genes were introduced into fertilized mouse eggs. Of 489 eggs injected with these genes, 319 survived and were transferred to oviducts of foster mothers. Thirtyeight mice were born and were screened for the presence of human gamma 1 immunoglobulin genes by Southern blot hybridization. Five of these 38 mice had integrated human gamma 1 immunoglobulin genes. None of the human gamma 1 copies in each mouse had undergone deletions or rearrangements as judged by the Southern blotting patterns for several restriction enzymes. Human gamma 1 gene was present in several different tissues. All the mice tested so far transmit the human gamma 1 gene to a fraction of their offspring in an autosomal dominant manner. Spleen cells from transgenic mice were analyzed for immunoglobulin production by reverse plaque assay or immunofluorescence staining of cytoplasmic immunoglobulin, but synthesis and secretion of human gamma 1 chains could not be detected. No human gamma 1 immunoglobulin mRNA was detected in the liver and spleen of a transgenic mouse. The presence of the human gamma 1 immunoglobulin gene appeared to have no effect on the expression of endogenous mouse immunoglobulin genes.

Protection against colitis by CD100-dependent modulation of intraepithelial γδ T lymphocyte function.

Intraepithelial γδ T lymphocytes (γδ IEL) have important roles in repair of tissue damage at epithelial sites, such as skin and intestine. Molecules that orchestrate these γδ T-cell functions are not well defined. Recently, interaction of the semaphorin CD100 on skin γδ T cells with plexin B2 on keratinocytes was shown to be important for effective γδ T-cell function in the epidermis, which raised the possibility that CD100 may exert similar functions in the intestinal tract. In this study, we find that CD100 is expressed on all IEL, and plexin B2 is present on all epithelial cells of the mouse colon. Using the dextran sulfate sodium (DSS) mouse model of colitis, disease severity is significantly exacerbated in CD100-deficient (CD100(-/-)) mice, with increased colon ulceration and mucosal infiltration with inflammatory cells. The severe colitis in CD100(-/-) mice is attributable to the failure of the colon epithelium to mount a proliferative response to damage. Unlike wild-type γδ IEL, γδ IEL from CD100(-/-) mice fail to produce keratinocyte growth factor-1 (KGF-1) in response to DSS treatment. Administration of recombinant KGF-1 to CD100(-/-) animals ameliorates disease and reverses colitis susceptibility. These results demonstrate that CD100-mediated signals are critical for effective activation of γδ IEL to produce growth factors, including KGF-1, that are required for healing of the colon epithelium during colitis.

Neuroimmune semaphorin 4A downregulates the severity of allergic response.

To define the role of semaphorin 4A (Sema4A) in allergic response, we employed Sema4A⁻/⁻ and wild-type (WT) mice in the experimental model of ovalbumin (OVA)-induced allergic airway inflammation. We observed a selective increase in eosinophilic airway infiltration accompanied by bronchial epithelial cell hyperplasia in allergen-treated Sema4A⁻/⁻ mice relative to WT mice. This enhanced inflammatory response was associated with a selective increase in bronchoalveolar lavage (BAL) interleukin 13 (IL-13) content, augmented airway hyperreactivity, and lower regulatory T cell (Treg) numbers. In vivo allergen-primed Sema4A⁻/⁻ CD4+ T cells were more effective in transferring T helper type 2 (Th2) response to naive mice as compared with WT CD4+ T cells. T-cell proliferation and IL-13 productions in OVA323₋339-restimulated Sema4A⁻/⁻ cell cultures were upregulated. Generated bone marrow chimeras showed an equal importance of both lung-resident cell and inflammatory cell Sema4A expression in optimal disease regulation. These data provide a new insight into Sema4A biology and define Sema4A as an important regulator of Th2-driven lung pathophysiology.