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To assess the frequency of radiographically evident drug-induced sarcoidosis-like reaction (DISR) in patients treated with anti-cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) therapy, anti-programmed cell death protein 1 (PD-1) therapy, or a combination of both in a single centre.The images and medical records of 457 patients with metastatic melanoma or head and neck cancer treated with either anti-CTLA-4 therapy, anti-PD-1 therapy, or a combination of both at University of California medical centre were reviewed retrospectively and the incidence of radiological manifestations of DISR was assessed among these treatment groups.Radiological manifestations of DISR were found in 19/457 patients (4.1%). The mean interval from the initiation of immunotherapy to development of DISR was 5.5 months (range 2.3-13.5 months). Mean interval from radiological detection of DISR to imaging evidence of resolution was 5.8 months (range 1.6-18.3 months). Three patients out of 81 (3.7%), 11/297 (3.7%), and 5/79 (6.3%) developed sarcoidosis-like reaction after treatment with anti-CTLA-4 antibody, anti-PD-1 antibody, and a combination of both, respectively. Most patients with DISR were asymptomatic and did not require systemic therapy. Most patients did not demonstrate concomitant increased maximum standardised uptake value (SUVmax) in other organs on their integrated 2-[In the present retrospective study of patients treated with immune checkpoint inhibitors (ICIs), DISR occurred in approximately 3.7% of patients treated with either anti-CTLA-4 or anti-PD-1 antibody and 6.3% of patients treated with a combination of both.

Interleukin 10 (IL-10) is an antiinflammatory cytokine, but also promotes B cell responses and plays a pathogenic role in systemic lupus erythematosus (SLE). CD4(+)CCR6(+)IL-7R(+)T cells from human tonsils produced IL-10 following stimulation by naïve B cells, which promoted B cell immunoglobulin G (IgG) production. These tonsillar CCR6(+)B helper T cells were phenotypically distinct from follicular helper T (T(FH)) cells and lacked BCL6 expression. In peripheral blood, a CCR6(+)T cell population with similar characteristics was identified, which lacked Th17- and T(FH)-associated gene signatures and differentiation-associated surface markers. CD4(+)CCR6(+)T cells expressing IL-10, but not IL-17, were also detectable in the spleens of cytokine reporter mice. They provided help for IgG production in vivo, and expanded systemically in pristane-induced lupus-like disease. In SLE patients, CD4(+)CCR6(+)IL-7R(+)T cells were associated with the presence of pathogenic anti-dsDNA (double-stranded DNA) antibodies, and provided spontaneous help for autoantibody production ex vivo. Strikingly, IL-10–producing CCR6(+)T cells were highly abundant in lymph nodes of SLE patients, and colocalized with B cells at the margins of follicles. In conclusion, we identified a previously uncharacterized population of extrafollicular B helper T cells, which produced IL-10 and could play a prominent pathogenic role in SLE.

Isolates of the take-all fungus, Gaeumannomyces graminis var. avenae, which affects oats, wheat and other grasses, and of G.g. var. tritici, which preferentially affects wheat, rye and barley, contain a high proportion of repeated sequences. Total DNA from 57 fungal isolates collected from many locations and different cereal hosts, and scored for virulence on wheat, rye and oats, revealed many restriction fragment length polymorphisms. These RFLPs were observed either by staining the DNA directly, by hybridization to radioactively labelled total fungal DNA, or by hybridization with labelled wheat ribosomal DNA. With only a few exceptions, the isolates with the same preferred cereal hosts showed more similar patterns of restriction fragments than isolates that had different pathogenicity properties on cereal hosts, irrespective of the geographical origins of the isolates. This was even the case for R isolates of G.g. var. tritici that were virulent on wheat and rye compared with N isolates that were virulent only on wheat. Isolates were identified by hybridizing DNA from infected root samples with 32P-labelled total fungal DNA. The restriction fragment polymorphisms involving families of repeated sequence can therefore be used as a predictive assay for host preference of an isolate, and have probably arisen by host selection of fungal lineages. The variation between isolates in different pathogenicity groups suggests that there is little gene flow between isolates that can infect different hosts, even though they can coexist in the same field.

Glutamic acid decarboxylase (GAD65) has been implicated as a targeted self antigen in the immune destruction of pancreatic beta cells. T cell responses to GAD65 peptides have been detected in both patients with type I diabetes and in the non-obese diabetic (NOD) mouse. To establish which GAD65 epitopes are important in the immunopathogenesis of disease we initially compared T cell responses to GAD65 epitopes in conditions of disease susceptibility and protection. T cell responses to GAD65 peptides were measured in monozygotic twin pairs selected on the basis of disease discordance and T cell recognition of immunogenic regions of GAD65. Peptides of interest were then used to immunize susceptible NOD mice and H2-E transgenic NOD mice which are protected from diabetes. A differential response to the epitope GAD65 521-535 discriminated diabetic from non-diabetic human twins as well as susceptible from protected mice. This epitope as well as GAD 505-519 induces T cell responses despite binding the type I diabetes associated HLA-DQA1*0301/DQB1*0302 product with low affinity. Since DQ-restricted T cell responses are difficult to study in humans, HLA-DQ8 transgenic mice were then used: GAD epitopes 521-535 and 505-519 induced responses in DQ8 transgenic mice and T cell lines were established. Long-term T cell lines against GAD 505-519 were HLA-DQ restricted, and responded to peptide with a strong IFN-gamma and IL-10 response. The findings implicate GAD 521-535 as a possible target peptide in pathogenesis and are compatible with a model whereby self-reactive T cells specific for low-affinity peptide-MHC complexes may escape thymic negative selection.

Murine Lyme borreliosis is characterized by arthritis and carditis that are most severe at 2 to 3 wk, then regress during the course of persistent infection. Borrelia burgdorferi-specific Abs and CD4+ T cells have been implicated in the resolution phase of arthritis. Therefore, MHC class II transactivator (CIITA)-deficient mice that do not express conventional class II molecules and lack the normal CD4 repertoire were used to investigate the role of MHC class II-mediated responses in Lyme disease. The development of arthritis and carditis, and the resolution of arthritis, were similar in CIITA-deficient and control C57/BL6 mice. In contrast, the resolution of carditis was delayed in CIITA-deficient animals compared with controls. Moreover, CIITA-deficient mice developed B. burgdorferi-specific IgG2b Abs, and sera from these animals passively protected naive C3H/HeN mice from challenge inoculation and cleared B. burgdorferi from 2 day-infected C.B.17 SCID mice. These data suggest that CD4+ T cells and MHC class II-mediated responses are not required for the generation of protective Abs or the regression of arthritis, but may be important in the resolution of Lyme carditis in mice.

In wheat-rye hybrids the nucleolus organizer regions (NORs), the sites of ribosomal RNA genes, from rye are suppressed. Wheat and wheat-rye hybrid genetic stocks containing different numbers of wheat and rye nucleolus organizers, as well as addition lines and rye-barley hybrids, were used in Southern hybridization experiments to determine the cause of nucleolar dominance and suppression in cereal hybrids. Based on the use of restriction endonucleases that cleave near the ends of the spacer unit and an additional, methylation-sensitive enzyme, HpaII, which does not recognize the CCGG restriction site if the internal C is methylated, an indirect method of assaying NOR expression was established. The results indicated that cleavage by the HpaII enzyme of the rye NOR sequences, is reduced when major NORs from other cereals were present. The reduction in the number of rye rRNA genes containing an unmethylated CCGG site in the promoter was associated with the suppression of the rye nucleolus. These results are consistent with a model in which promoter and upstream regulatory repeats of ribosomal RNA genes compete for limited concentrations of regulatory proteins, and genes that are methylated at key binding sites fail to engage these regulatory proteins and thus remain inactive.

Acquiring the ability to selectively produce IFN-gamma or IL-4 is a fundamental property of the immune system and enables T cell subsets (Th1, Th2) to deliver their effector functions. To create an experimental system to examine regulation of critical promoter elements within the IL-4 gene, we have prepared and analyzed transgenic mice expressing the luciferase gene under the control of the distal NF-AT binding site from the IL-4 promoter. This site is immediately adjacent to an AP-1 site, and this NF-AT/AP-1 composite site is termed either P1 or PubB. The distal NF-AT site we have used to prepare these reporter transgenic mice lacks the AP-1 binding site but contains the NF-AT binding site (P1(NF-AT)) These transgenic mice were also intercrossed with transgenic mice expressing a single MHC class II-restricted TCR. Expression of transcriptional activity under the control of P1(NF-AT) was observed only in effector T cells, and not naive T cells, after stimulation with Ag or polyclonal stimuli. By contrast, gel mobility shift assays showed that nuclear extracts from both naive and effector T cells contained NF-AT, which could effectively bind to the P1(NF-AT) element. IL-4-stimulated Th2 differentiation did not increase the TCR responsiveness of the P1(NF-AT) element by more than 2-fold but increased production of IL-4 protein by more than 10-fold. These data suggest that factors interacting with the P1(NF-AT) element regulate transcriptional activity in a naive/effector T cell-specific manner but not in a Th1/Th2-specific manner. Th1/Th2-specific regulation of the composite P1 element may result from regulation of transacting factors that bind to the AP-1 portion of this element.

Most inbred strains of mice are susceptible to Leishmania amazonensis infection. We have examined the mechanism(s) underlying this generalized susceptibility using mice deficient in T cell development or in the expression of either MHC class I or class II. In contrast to wild-type C57BL/6 (B6) mice that uniformly developed large ulcerating lesions, mice lacking functional CD4+ T cells (due to targeted disruption of genes for either MHC class II trans-activator or I-A beta) showed no signs of lesion development for up to 12 to 14 wk postinfection and contained significantly lower numbers of parasites in lesions. Similarly, both B6 nude and RAG2 -/- mice failed to develop lesions. However, RAG2 -/- mice reconstituted with naive wild-type CD4+ T cells and beta2m -/- mice did develop lesions. Lesions of MHC class II -/- mice contained minimal numbers of CD8+ T cells, a marked reduction of monocytes/macrophages, and evident extracellular parasites. The inability to mount an inflammatory response in MHC class II -/- mice correlated with the failure to produce lymphokines that lead to the recruitment of monocytes/granulocytes. These results demonstrate that CD4+ T cells are the primary lymphocyte subset that mediates cellular infiltration, lesion pathology, and therefore, susceptibility to L. amazonensis infection. The disease-promoting CD4+ T cells in L. amazonensis-infected mice have the characteristics of Th1 cells. The striking differences in the course of infection between MHC class II -/- mice infected with L. amazonensis and Leishmania major suggest that these parasites may have adapted different strategies regarding the CD4-dependent immune response.

It has previously been reported that Th1 CD4 T cell populations can be converted to IL-4 producers, whereas Th2 populations are refractory to IL-12-mediated IFN-gamma production. We have recently shown that CD30 is a marker for the IL-4 response and have therefore used CD30 here to study Th1 and Th2 commitment. We show that Th2 cell populations normally show a stable phenotype and fail to respond to IL-12 because of endogenous IL-4 production. IFN-gamma abrogates this antagonistic effect of IL-4 and permits the conversion of Th2 populations to IFN-gamma producers by IL-12. In the complete absence of IL-4, however, IFN-gamma is not required for this transformation, and Th1 cells generated by IL-12 become committed to the Th1 pathway and lose the ability to respond to IL-4. Thus, the balance between local IL-4 and IFN-gamma in an immune response is a key factor in determining the outcome of the CD4 effector T cell response.

Prior studies have implicated CD30 as a marker for Th2 cells, but the mechanism that underlies this correlation was unknown. We show here that CD30 was expressed on activated CD4+ T cells in the presence of IL-4. In the absence of endogenously produced IL-4, however, even Th2 lineage cells lost CD30 expression. Thus, CD30 is not an intrinsic marker of Th2 cells, but is inducible by IL-4. CD30 was also found to be down-regulated by IFN-gamma. Committed Th1 effector cells do not express CD30, although differentiating Th1 lineage cells temporarily express CD30. The transient expression of CD30 on differentiating Th1 lineage cells was mainly the result of endogenously produced IL-4 induced by IL-12. Culture of IL-12-primed cells under conditions that reverse the phenotype (Ag plus IL-4) resulted in two cell populations based upon their ability to express CD30. One population responded to IL-4 upon restimulation and became a CD30-positive, Th0-like cell population, while the other remained CD30 negative and synthesized only IFN-gamma. Thus, CD30 expressed on CD4+ T cells reflected the ability of CD4+ T cells to respond to IL-4.

To explore the mechanisms by which alpha beta T cells and gamma delta T cells regulate systemic autoimmunity, lupus-prone mice were rendered deficient in CD40 ligand and/or alpha beta T cells by intercrossing CD40L -/- and TCR-alpha -/- knockouts, generating CD40L-intact or -deficient (CD40L+ or CD40L-), alpha beta T cell-intact or -deficient (alpha beta+ or alpha beta-) MRL-lpr/lpr animals. As expected, CD40L+ alpha beta+ mice developed high titer autoantibodies along with severe renal and cutaneous disease. CD40L+ alpha beta- animals developed lower levels of autoantibodies, accompanied by less severe or delayed renal and cutaneous disease. CD40L- alpha beta+ mice developed even lower titers of autoantibodies and less severe renal disease yet developed cutaneous lesions indistinguishable from those of CD40L+ alpha beta+ disease. Most surprisingly, CD40L- alpha beta- animals developed higher levels of some autoantibodies than did CD40L- alpha beta+ mice and developed renal disease similar in severity to CD40L+ alpha beta- counterparts; however, they failed to develop skin disease. Thus, disruption of CD40L and alpha beta T cells provides a novel dissection of the physiology and pathology of murine lupus; while these data confirm previous findings demonstrating a role for CD40L-dependent, alpha beta T cell-dependent mechanisms in autoantibody production and renal disease in murine lupus, they also: 1) establish that alpha beta T cells may drive autoimmune skin disease by a CD40L-independent mechanism; 2) identify a role for CD40L in non-alpha beta T cell-dependent autoantibody production and autoimmune skin disease; and 3) suggest a role for alpha beta T cells in the down-regulation of autoimmunity driven by other T cells. Thus, both alpha beta and non-alpha beta T cells, such as gamma delta T cells, regulate systemic autoimmunity by CD40L-dependent and -independent mechanisms.

IL-1 beta-converting enzyme (ICE) cleaves pro-IL-1 beta to the mature, released form. Although other proteases can process pro-IL-1 beta, ICE-deficient (ICE -/-) mice do not release mature IL-1 beta in response to endotoxin. The purpose of our study was to investigate the response of ICE -/- mice in two models of local inflammation, turpentine-induced tissue damage and zymosan-induced peritonitis. No differences were observed in the development of the systemic acute phase response after turpentine administration between wild-type and ICE -/- mice, but this response was completely impaired in IL-1 beta -/- mice. Accordingly, the levels of mature IL-1 beta produced in response to turpentine did not differ between wild-type and ICE -/- mice. In contrast, following zymosan-induced peritonitis, the levels of mature IL-1 beta were significantly lower in ICE -/- mice. This was associated with a 50% decrease in cellular infiltrate in ICE -/- mice compared with that in wild-type controls. The reduced production of zymosan-induced mature IL-1 beta in ICE -/- mice was also observed from cultured peritoneal or spleen cells. Our results demonstrate that in turpentine-induced tissue necrosis, precursor IL-1 beta is processed by non-ICE proteases, but in complement-mediated inflammation, ICE participates in the processing of the IL-1 beta precursor.

To investigate whether the immunosuppressive neuropeptide calcitonin gene-related peptide (CGRP) was a potential candidate for tissue-specific gene therapy, we engineered nonobese diabetic (NOD) mice to produce CGRP in beta cells by placing the modified calcitonin gene under the control of the rat insulin promoter. CGRP inhibits CD4 T cell production of the cytokines that have been implicated in the pathogeny of type I diabetes. Three transgene-positive mouse lines were obtained, two of which expressed immunoreactive CGRP in beta cells (NOD-CGRP mice). Isolated islets from one of these two transgene-positive founders produced active CGRP, whereas islets from transgene-negative littermates did not. The production of CGRP by beta cells prevented insulin-dependent diabetes mellitus in male and reduced its incidence by 63% in female mice. This prevention was due to a local immunosuppressive effect of CGRP as no difference was detected between NOD-CGRP and NOD littermate lymph node, spleen, and thymus cells by either FACS analysis or proliferative response to stimulation by Ag, alloantigen or anti-CD3. These data suggest that CGRP is a potential therapeutic molecule to prevent or treat diabetes and possibly other diseases and conditions in which immune cells are involved. These data also suggest that endogenous CGRP concentrated in sensory nerve endings may regulate locally the immune response, further strengthening the importance of the functional neuroimmune link.

Naive CD4+ T cells initially transcribe both IL-4 and IFN-gamma when stimulated with either the mitogen Con A or Ag in the presence of IL-4 or IL-12 and, therefore, appear uncommitted as to the pathway of differentiation they will follow. However, when stimulated either with Con A or with Ag in the presence of IL-4, CD4+ T cells become primed to follow the Th2 differentiation pathway, and we show now that by 48 h of culture in this environment these cells extinguish IFN-gamma gene transcription. Likewise, priming in the presence of IL-12 leads to the development of Th1 cells, which switch off the expression of the IL-4 gene. To clarify the Th1 differentiation pathway, we performed ablation studies using IL-4 thymidine kinase transgenic mice. When the antiviral drug ganciclovir was added 1 day after primary stimulation in the presence of IL-12, IFN-gamma- and IL-4-producing cells were ablated. In contrast, when ganciclovir was added 2 days after primary stimulation, IL-4-producing cells, but not IFN-gamma-producing cells, were ablated. Thus, our studies show that by 48 h after activation, Th1 or Th2 cells have already become polarized to the differentiation pathway that they will follow. As the differentiation toward Th1 and Th2 effector cells proceeds, substantial amounts of IFN-gamma and IL-4 mRNA accumulate, while the mRNAs of the corresponding lineage (i.e., IFN-gamma in the case of Th2 cells, and IL-4 in the case of Th1 cells) diminish to undetectable levels. IL-4R is up-regulated during T cell differentiation by a mechanism mediated mainly by IL-4. The fact that IL-12 priming does not suppress IL-4-dependent IL-4R up-regulation shows that both IL-4 mRNA and cytokine are produced by IL-12-primed naive CD4+ T cells during differentiation into Th1 cells. Naive CD4+ T cells, therefore, begin as uncommitted cells which express both Th1 and Th2 cytokines that rapidly extinguish the expression of the inappropriate cytokine as the commitment toward the effector lineages is made.

Alloreactive T cells require costimulatory signals via CD40 ligand (CD40L). The tissue-destructive properties of allogeneic CD4+ but not CD8+ T cells were inhibited by anti-CD40L mAb. Fewer CD4+ thoracic duct lymphocytes (TDL) were obtained in mAb-treated recipients. Kinetic studies revealed that CD4+ T cell expansion was reduced or delayed which may account, in part, for the partial graft-vs-host disease protective effect of anti-CD40L mAb. TDL were found to have diminished anti-host-specific proliferative responses. The frequency of donor TDL and splenocytes that expressed the Th1 cytokines IL-2, IL-12 p40, and IFN-gamma mRNA was markedly diminished in mAb-treated recipients, demonstrating that Th1-driven alloresponses were susceptible to CD40L targeting. Perforin mRNA-expressing T cells were undetectable in mAb-treated recipients, consistent with reduced in vivo lethality after the adoptive transfer of allogeneic CD4+ T cells. Similar findings were observed in both B cell-replete or -deficient recipients, indicating that allogeneic T cell expansion and priming can be sustained by a non-B cell, CD40+ host cell population. Mice receiving CD40L-deficient allogeneic CD4+ T cells had survival rates comparable to the rates of those given anti-CD40L mAb treatment. Because anti-CD40L mAb also was found to prevent host anti-donor-mediated marrow allograft rejection, in vivo blockade of CD40L-CD40 interactions may provide a highly beneficial approach to improving the outcome of allogeneic bone marrow transplantation.

Lyme disease, due to infection with the tick-borne spirochete Borrelia burgdorferi, is a multisystem disorder that can lead to chronic disabling symptoms. Abs to the outer surface protein A (OspA) of B. burgdorferi provide protection against infection, and OspA is now the basis of a first generation recombinant vaccine undergoing phase III efficacy studies. Recent studies have suggested that T cells reactive with N-terminal epitopes in OspA could contribute to the development of treatment-resistant Lyme arthritis. In the present studies, we use the murine model of Lyme borreliosis to define an OspA T cell epitope located in the carboxyl terminus that accelerates anti-OspA IgG production after infection. In addition, we show that this T cell epitope is elicited by immunization with rOspA or with a truncated form of OspA that contains the B cell epitope targeted by protective OspA mAb. Polyclonal antisera to the truncated OspA fragment can protect mice from challenge infection. These results are the first demonstration of a B. burgdorferi-specific peptide that elicits a biologically important T cell response in vivo and have implications for the design of a second generation OspA-based subunit vaccine.

Pancreatic islet transplantation represents a potential treatment for insulin-dependent diabetes mellitus. One approach to circumvent the requirement for recipient immune suppression is to reduce or eliminate the immunogenicity of the donor graft prior to transplantation. In this study, we have examined the relative contributions of graft MHC Ag expression and donor-derived costimulatory (CoS) activity to the rejection of islet allografts. Depletion of donor hemopoietic APCs from islet tissue facilitated long-term allograft survival even when donor class I MHC Ag expression was greatly increased by IFN-gamma treatment prior to grafting. Conversely, islet allografts from transgenic mice expressing the CoS molecule B7-1 (CD80) on islet beta cells were acutely rejected even when hemopoietic APCs were eliminated. Thus, B7-1 is sufficient to confer the capacity of islet parenchymal cells to stimulate allorejection. Taken together, these results point towards donor-derived CoS activity as a primary target of intervention therapy to modulate tissue immunogenicity.

An allelic rDNA variant from the Nor-B2 locus of 'Bezostaya' wheat that forms an especially active nucleolus was cloned and characterized. It carries an unusually large intergenic spacer compared with rDNA units in most other wheat genotypes. The additional intergenic length is in the array of 135-bp A repeats and not in other internal repeats. These A repeats have sequences nearly identical to other A repeats described for other alleles. It is suggested therefore that the more active Nor-B2 locus of 'Bezostaya' may be due to the constituent rDNA units possessing a larger array of A repeats. Key words : ribosomal DNA, nucleolar organizer region, A and B repeats, allelic, spacer length variants.

Passive immunization with murine or human Abs to outer surface protein A (OspA) can protect mice against Borrelia burgdorferi, but OspA Abs elicited during natural infection in mice or humans are unable to clear the spirochete from the infected host. To examine Ab binding by OspA during the course of human infection, we amplified the operon encoding full-length ospA and ospB from synovial fluids of a patient with chronic Lyme arthritis, the first such recoveries from human material, at four separate time points over 4.5 mo, and expressed OspA in Escherichia coli. OspA mAbs that passively protected mice from infection did not bind one of the expressed OspAs, because of a deletion in ospA that resulted in a frame shift and premature stop codon near the carboxyl terminus. However, expressed OspA from a later synovial fluid sample did not contain this deletion. Thus, although altered forms of OspA, which potentially can influence host immune effectiveness, do occur in the human host, they cannot be the only factors responsible for microbial persistence.

WIS 2-1A, the first retrotransposon found in wheat, has been recently studied and characterized. Southern hybridization experiments utilizing several species from the Triticeae revealed the presence of homologous sequences in all the taxa tested, showing high levels of interspecific variability and almost no intraspecific differentiation. Further experiments using in situ hybridization in several species showed that the retroposon was almost completely dispersed throughout the genomes tested. These results suggested that WIS 2-1A is an ancient element that probably was present in the unknown common ancestor of the Triticeae and that only under rare circumstances does it become active. DNA fragments homologous to the WIS 2-1A reverse transcriptase gene were isolated from most of the Triticeae species using PCR. The fragments obtained were sequenced and analyzed. Even though the sequence alignment was consistent with the phylogenetic studies made in the past, the genus Thinopyrum showed new evidence for a possible horizontal propagation of the retroelement.Mention of a trade name or proprietary product does not constitute a guarantee, warranty or recommendation of the product by the U.S. Department of Agriculture or the University of Missouri and does not imply its approval to the exclusion of other products that may be suitableAll programs and services of the U.S. Department of Agriculture are offered on a non-discriminatory basis without regard to race, colour, national origin, religion, sex, age, marital status, or handicap.

Sequences homologous to the retro-element BIS-1 and the stem-loop repeat Hi-10 are present in the genomes of a number of cereal species. A detailed characterization of these elements indicated that they are non-randomly organized in the genomes of at least two of these species, namely barley and rye. In contrast to the BIS-1 retro-elements, the stem-loop repeats are also non-randomly organized into discrete domains in interphase nuclei from barley and rye. Features of the organization of these repeats along chromosomes and within interphase nuclei of rye, barley and rice are discussed.

The 84-, 91-, and 113-kDa proteins of the ISGF-3 alpha complex are phosphorylated on tyrosine residues upon alpha interferon (IFN-alpha) treatment and subsequently translocate to the nucleus together with a 48-kDa subunit. In this study, we investigated the presence and the functional status of ISGF-3 alpha subunits and Tyk-2 and JAK1 tyrosine kinases in mutant HeLa cells defective in the IFN-alpha/beta and -gamma response. Stable cell fusion analysis revealed a single complementation group among one class (class B) of mutants. The class B mutants contain detectable level of mRNA and proteins of the 84-, 91-, and 113-kDa proteins, but neither the protein nor mRNA is inducible by IFN-alpha or -gamma. The 91-kDa protein IFN-gamma-activated factor fails to be activated into a DNA-binding state after IFN-alpha or -gamma treatment. In addition, the 91-kDa protein is unable to localize in the nucleus after IFN-alpha and -gamma treatment, and the 113-kDa protein fails to translocate after IFN-alpha treatment. Immunoprecipitation studies document a failure of phosphorylation of the 84- or 91-kDa proteins after IFN-alpha or -gamma treatment. Similarly, no tyrosine-phosphorylated 113-kDa protein was detected after IFN-alpha treatment. The inability of class B mutants to phosphorylate the 84-, 91-, or 113-kDa protein on tyrosine residues correlated with the loss of biological response to IFN-alpha and -gamma. The genetic defect appears to be the absence of the tyrosine kinase JAK1. Our data therefore confirm a recent report that JAK1 plays a critical early signaling role for both IFN-alpha/beta and IFN-gamma systems.

A variety of cell types expressing MHC class II molecules is known to function as APC in vitro. We employed the Ig kappa gene enhancer and promoter to target the class II E alpha gene, and thereby I-E, exclusively to B cells to address their APC function in vivo. Although transgenic I-E was expressed on B lymphocytes, we unexpectedly obtained I-E on thymic medullary epithelium but not macrophages and at low frequency on dendritic cells. Using these transgenic mice, we constructed bone marrow irradiation chimeras with I-E expressed only on medullary epithelium, in order to determine the role of this cell type in tolerance by clonal deletion in the thymus. Although it is accepted that bm-derived cells play a primary role in deletion, and thymic epithelium can delete clones to a lesser degree, the role of cortical vs medullary thymic epithelium has not been directly dissected. We demonstrate that medullary epithelium alone can tolerize by partial deletion of I-E-reactive V beta 5+ T cells. These results indicate a role for medullary epithelium in deletion during the later stages of thymic development, and support the notion that positive and negative selection of developing T cells can occur in distinct temporal and anatomic compartments.

During early stages in their biogenesis, murine class I histocompatibility molecules interact transiently with a molecular chaperone of the endoplasmic reticulum designated p88. Using a series of mutant class I heavy chains we mapped the region of the heavy chain that interacts with p88. Domain deletion mutants of the H-2Db and H-2Kb molecules revealed that most of the extracellular portion of the heavy chain and the bulk of the cytoplasmic domain were not required for the association. However, replacement of the transmembrane segment and cytoplasmic domain with a glycosyl phosphatidylinositol anchor from Q7b resulted in a heavy chain that was incapable of interaction with p88. These results suggested that the primary site of interaction with p88 is within a region containing the transmembrane segment and several flanking amino acids of the class I heavy chain. This finding was supported by replacing the glycosyl phosphatidylinositol anchor of the noninteracting Q7b protein with segments of the Db heavy chain containing the putative interaction site and showing that the hybrids were capable of associating with p88. The apparent lack of interaction between segments of p88 and the class I heavy chain that are present within the lumen of the endoplasmic reticulum was also observed when the association between p88 and the alpha chain of the T cell receptor was examined. The full-length transmembrane alpha chain formed a complex with p88, whereas a soluble variant consisting of most of the luminal portion of the alpha chain exhibited only minimal interaction. Thus, p88 is capable of associating with nascent integral membrane proteins through transmembrane interactions that are unavailable to the major soluble chaperone of the endoplasmic reticulum, BiP (GRP78).

The actin gene family of Nicotiana tabacum has been partially characterised by Southern hybridisation and by isolating lambda EMBL4 recombinants from a genomic library having homology to the soybean actin gene, Sac3. The number of actin genes with homology to Sac3 is estimated at between 20 to 30, based on Southern hybridisation and library screening, though the total gene family may be larger. Twenty-four recombinant lambda clones were isolated, 18 had unique restriction profiles and from these, 2 clones, Tac9 and Tac25, were selected for further study. The region of Tac25 hybridizing to Sac3 was sequenced and shown to contain an open reading frame (ORF) with homology to actin. Partial sequencing of Tac9 revealed a sequence with homology to the third exon of Tac25 and Sac3. The two tobacco actin sequences were compared to other reported actin gene sequences; Tac25 was closely related to the allelic potato actins, Pac58 and Pac85, while Tac9 was more related to Pac79 than to other plant actins. Northern hybridisation analysis showed that while Tac9 detected actin transcripts in RNA from root, leaf, stigma and pollen, Tac25 transcripts were only detected in pollen RNA.

The murine immune response to Borrelia burgdorferi (Bb), the etiologic agent of Lyme disease, is characterized by the development of antibodies reactive with the outer surface protein (Osp) A. It has been demonstrated that passive immunization of mice with at least some Osp A antibodies, including an Osp A mAb (CIII.78) that binds to a conformational epitope in the carboxyl-terminus of Osp A, provides protection against Bb challenge. Active immunization of mice with Osp A also confers protection, making Osp A a candidate for a vaccine Ag. To determine the regions of the Osp A protein that can elicit protective immunity, we immunized boosted mice with overlapping recombinant truncated fragments of Osp A, then challenged them with Bb. All groups of mice developed IgG Osp A antibodies detectable by immunoblotting with sera diluted at least 5000-fold. As expected, vaccination with full-length recombinant Osp A protected mice from challenge infection. In contrast, none of the mice vaccinated with the truncated Osp A proteins demonstrated immunity, even those immunized with an Osp A fragment binding the neutralizing mAb CIII.78. Osp A antibodies contained in the truncated Osp A antisera also failed to immunoprecipitate in vitro translated full-length Osp A and did not bind, as demonstrated by indirect immunofluorescence, to live or acetone-fixed Bb. Taken together, these results suggest that neutralizing Osp A antibodies are induced by vaccination with the full-length recombinant Osp A protein but not by vaccination with recombinant fragments.

To understand the role of TNF in the regulation of inflammation and the development of autoimmune diseases such as insulin-dependent diabetes mellitus, we produced transgenic mice in which the synthesis of murine TNF-alpha was directed by the rat insulin II promoter. The expression of the TNF-alpha transgene was restricted to the pancreas, in contrast to TNF-beta expression from the same promoter, in which the transgene was expressed in the pancreas, kidney, and skin. The expression of TNF-alpha in the pancreas of transgenic mice resulted in an overwhelming insulitis, composed of CD4+ and CD8+ T cells and B220+ B cells, considerably greater than that of TNF-beta transgenics. Moreover, in contrast to the predominant peri-insulitis observed in TNF-beta transgenic mice, the majority of the infiltrate in the TNF-alpha transgenic mice was within the islet itself. These unique patterns of infiltration were observed in the F1 progeny of crosses with C57BL/6 as well as NOD. Both TNF-alpha and TNF-beta transgenic mice show elevated expression of leukocyte adhesion molecules VCAM-1 and ICAM-1 in islet endothelia and increased expression of MHC class I on islet cells. This inflammation did not result in reduced insulin content of the islets, nor did it lead to diabetes. These data suggest that additional stimuli are necessary to initiate the process of islet destruction.

Previously, it has been shown that polyclonal antibodies to Borrelia burgdorferi and some monoclonal antibodies (MAbs) to borrelia major surface proteins caused inhibition of adherence of the bacteria to cultured human umbilical vein endothelial (HUVE) cells. In this study, fragment antigen binding (Fab) molecules generated from the immunoglobulin G fraction of rabbit anti-recombinant OspA serum were found to inhibit the adherence of B. burgdorferi to HUVE cells by 73%. Subsequently, MAbs were generated for use in determining whether or how B. burgdorferi outer surface proteins (Osps) A and/or B are involved in mediating attachment to, and/or invasion of, HUVE cells by B. burgdorferi. Twenty-two MAbs were generated to borrelial proteins with apparent molecular masses (determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) of 19, 31 (OspA), 34 (OspB), and 35 kDa. Fab molecules from one anti-OspA MAb, 9B3D, demonstrated an inhibitory effect on bacterial association with HUVE cells. None of the other MAbs, including the other anti-OspA MAbs, showed an inhibitory effect on cell association of greater than 5%. This effect of Fab 9B3D was concentration dependent and plateaued at approximately 6 micrograms of Fab per ml (nearly 80% inhibition of the bacterial association with the monolayer). Penetration assays and cell association experiments performed by using immunofluorescence also suggested that the inhibitory action of 9B3D occurs at the level of adherence. MAb 9B3D recognized the OspA of every North American strain tested (n = 19) but only 3 [corrected] of 20 strains from western Europe, Russia, and Japan, suggesting that the North American strains and strains from other parts of the world may use different molecules and/or different OspA epitopes to interact with endothelial cells. Immunoblots of Escherichia coli expressing different OspA fusion peptides suggested that the 9B3D epitope resides in the carboxy-terminal half of OspA. MAb 9B3D promises to be a valuable tool for elucidating the domain or domains of OspA involved in the endothelial cell cytadherence of North American strains of B. burgdorferi.

WIS-2-1A, a 8624 bp insertion in the Glu-1A-2 locus of chromosome 1A of wheat, consists of two 1755 bp long terminal repeats enclosing a 5114 bp internal region. No long open reading frames could be found, but inspection of the predicted amino acid sequence showed regions with homology to retrotransposon structures, including a methionine tRNA initiator binding site, a nucleotide binding domain, a protease, an integrase and a polymerase. DNA replication errors have resulted in frame-shifts in the protein coding region, suggesting that retrotransposition of WIS-2-1A, if it occurs, must be mediated by trans-acting factors.

Mice vaccinated with outer surface protein A (OspA) from Borrelia burgdorferi strain N40 are protected from challenge with an intradermal syringe inoculum of B. burgdorferi strains N40, B31, and CD16. Vaccination experiments were done to determine if protection extended to strains 297 and 25015. We now show that OspA-N40 immunized mice are protected against challenge with strain 297, isolated from the cerebrospinal fluid of a patient with neuroborreliosis, but not against challenge with strain 25015, isolated from a tick in Millbrook, NY. The OspA gene from strain 25015 was therefore cloned and sequenced. The deduced OspA-25015 protein sequence differs from OspA-N40 at 40 of 273 amino acids. Furthermore, mice vaccinated with rOspA-25015 are protected from challenge with strain 25015 but not against strain N40. The results extend the usefulness of OspA as a vaccine candidate, but indicate that OspA can vary among strains of B. burgdorferi and that vaccination of mice with OspA-N40 does not protect against intradermal challenge with an inoculum of 10(4) strain 25015 spirochetes.

Genes encoding high molecular weight (HMW) glutenin, a wheat seed storage protein, are expressed only in the developing endosperm. It was previously demonstrated that sequences essential for endosperm-specific transcription reside within 436 base pairs upstream of the initiation codon for HMW glutenin translation. We have further analyzed this region by testing the ability of a series of truncated HMW glutenin promoter fragments to enhance transcription from an adjacent heterologous promoter. The activity of these hybrid promoters was determined by measuring the expression of a linked beta-glucuronidase (GUS) reporter gene in transgenic tobacco plants. An HMW glutenin promoter fragment spanning nucleotides -375 to -45 relative to the transcription start site was found to stimulate GUS expression in tobacco seeds when inserted in either orientation upstream of the heterologous promoter. Furthermore, this fragment could also potentiate transcription when located 3' to the GUS reporter gene. Stimulation of GUS gene expression in transgenic tobacco seeds did not occur until 9 days to 12 days after anthesis, coincident with the onset of storage protein synthesis in the developing tobacco and wheat seed, and was confined to the endosperm tissue. By testing progressively shorter promoter fragments, the enhancer element responsible for this pattern of expression was localized to a 40-base pair region some 170 base pairs upstream of the start site for HMW glutenin transcription.

Chromosome marking by cytosine methylation has been examined in two gene systems in wheat - at the loci encoding high molecular weight (BMW) glutenin sub-units (seed proteins) and ribosomal RNA. Variation in cytosine methylation occurs between progeny in highly inbred lines around the BMW glutenin locus. The variation is inherited through meiosis to F1 and F2 generations but occasionally a new variant arises. Specific cytosine residues lose their methyl group in the seed, the organ where the genes are expressed. Within the multigene family of ribosomal RNA genes, several subsets of genes can be defined based upon the cytosine methylation patterns. High activity of a ribosomal RNA gene locus is correlated with loss of methylation at specific cytosine residues, especially in the promoter and upstream regulatory regions. A model is described in which the subset of genes selected to be used are those to which specific regulatory proteins and transcription complexes bind most favourably. Binding of such proteins inhibits cytosine methylation and so marks the subset of genes for expression in subsequent cell generations. Examples are described where new types of RNA genes are introduced via sexual crosses that result in changes to the methylation patterns of the ribosomal RNA genes. The processes determining the changes begin, it is believed, in the fertilised egg.

T cell homeostasis is required for normal immune responses and prevention of pathological responses. Transforming growth factor beta (TGFbeta) plays an essential role in that regulation. Owing to its broad expression and inhibitory effects on multiple immune cell types, TGFbeta regulation is complex. Through recent advances in cell-specific targeting of TGFbeta signaling in vivo, the role of TGFbeta in T cell regulation is emerging. We demonstrated here a critical role for TGFbeta in regulating effector vs regulatory T cell homeostasis.

c-Jun N-terminal kinase (JNK) is one of the several main MAP kinase groups identified in mammals. Original studies by use of Jurkat T cells implicated JNK in T cell activation and interleukin (IL-2) expression. Recent advances using mouse genetic approaches have revealed novel functions of this pathway in primary mouse T cells. JNK is not essential for T-cell activation; instead, it is required for helper T differentiation into effector cells and their cytokine production. In this review, we summarize these advances in understanding the expression, function, and regulation of the JNK pathway in T-lymphocyte activation and differentiation.

Haemophilus influenzae type b capsular polysaccharide (PRP) conjugate vaccines, which are thought to induce T cell-dependent antibody production, induce protective responses after a single dose in individuals under 15 months of age. However, multiple doses of these vaccines are required to induce protective antibody responses in infants, with the exception of PRP conjugated to meningococcal outer membrane proteins (OMPC), which does so after a single dose. The basis for this difference is not fully understood, although others have proposed that OMPC and porins, the major protein component of OMPC, act as adjuvants or mitogens. In this report OMPC is shown to enhance CD40 ligand-mediated, T cell-dependent antibody production in mice. This paralleled the induction by OMPC of CD86, CD80 and CD40 costimulatory molecules on human neonatal and murine B cells and of Th1 cytokines. Neither porins nor lipopolysaccharide fully reproduced the effects of OMPC. These studies indicate that OMPC acts both as carrier and adjuvant, and thereby enhances T cell-dependent antibody responses in human infants.

The stress activated protein kinase pathway culminates in c-Jun phosphorylation mediated by the Jun Kinases (JNKs). The role of the JNK pathway in sympathetic neuronal death is unclear in that apoptosis is not inhibited by a dominant negative protein of one JNK kinase, SEK1, but is inhibited by CEP-1347, a compound known to inhibit this overall pathway but not JNKs per se. To evaluate directly the apoptotic role of the JNK isoform that is selectively expressed in neurons, JNK3, we isolated sympathetic neurons from JNK3-deficient mice and quantified nerve growth factor (NGF) deprivation-induced neuronal death, oxidative stress, c-Jun phosphorylation, and c-jun induction. Here, we report that oxidative stress in neurons from JNK3-deficient mice is normal after NGF deprivation. In contrast, NGF-deprivation-induced increases in the levels of phosphorylated c-Jun, c-jun, and apoptosis are each inhibited in JNK3-deficient mice. Overall, these results indicate that JNK3 plays a critical role in activation of c-Jun and apoptosis in a classic model of cell-autonomous programmed neuron death.

Previous studies have shown that c-Jun NH(2)-terminal kinase (JNK) belongs to the mitogen-activated protein kinase (MAPK) family of signal transduction components that are rapidly initiated and activated by many extracellular stimuli. However, the potential role of JNK in mediating tumor promotion and carcinogenesis is unclear. We show here that in JNK2-deficient (Jnk2(-/-)) mice, the multiplicity of papillomas induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) was lower than that in wild-type mice. Papillomas on wild-type mice grew rapidly and were well vascularized compared with Jnk2(-/-) mice. After the 12th week of TPA treatment, the mean number of tumors per mouse was 4.13-4.86 in wild-type mice but only 1.13-2.5 in Jnk2(-/-) mice. TPA induced phosphorylation of extracellular signal-regulated kinases and activator protein-1 DNA binding activity in wild-type mice, but the phosphorylation of extracellular signal-regulated kinases and activator protein-1 DNA binding were inhibited in Jnk2(-/-) mice. These data suggest that JNK2 is critical in the tumor promotion process.

Systemic hyporesponsiveness occurs following oral administration of antigen (oral tolerance) and involves the uptake and processing of antigen by the gut-associated lymphoid tissue (GALT), which includes Peyer's patches (PP) lamina propria lymphocytes and mesenteric lymph nodes (MLN). Animals with targeted mutations of genes in the tumor necrosis factor (TNF) family have differential defects in the development of peripheral lymphoid organs including PP and MLN, and provide a unique opportunity to investigate the role of GALT structures in the induction of oral tolerance. Oral tolerance could not be induced in TNF/lymphotoxin (LT) alpha-/- mice, which are devoid of both PP and MLN, although these animals could be tolerized by intraperitoneal administration of antigen, demonstrating the requirement for GALT for oral tolerance induction. LTbeta-/- mice and LTalpha/LTbeta+/- animals do not have PP but could be orally tolerized, as measured by IFN-gamma production and delayed-type hypersensitivity responses by administration of both low or high doses of ovalbumin. To further investigate the requirement for PP, we tested the progeny of LTbeta-receptor-IgG-fusion-protein (LTbetaRigG)-treated mice, which do not form PP but have an otherwise intact immune system. Although these animals had decreased fecal IgA production, they could be orally tolerized. Our results demonstrate that PP are not an absolute requirement for the induction of either high- or low-dose oral tolerance, although oral tolerance could not be induced in animals devoid of both PP and MLN.

We generated vascular cell adhesion molecule (VCAM)-1 “knock-in” mice and Cre recombinase transgenic mice to delete the VCAM-1 gene (vcam-1) in whole mice, thereby overcoming the embryonic lethality seen with conventional vcam-1–deficient mice. vcam-1 knock-in mice expressed normal levels of VCAM-1 but showed loss of VCAM-1 on endothelial and hematopoietic cells when interbred with a “TIE2Cre” transgene. Analysis of peripheral blood from conditional vcam-1–deficient mice revealed mild leukocytosis, including elevated immature B cell numbers. Conversely, the bone marrow (BM) had reduced immature B cell numbers, but normal numbers of pro-B cells. vcam-1–deficient mice also had reduced mature IgD+ B and T cells in BM and a greatly reduced capacity to support short-term migration of transferred B cells, CD4+ T cells, CD8+ T cells, and preactivated CD4+ T cells to the BM. Thus, we report an until now unappreciated dominant role for VCAM-1 in lymphocyte homing to BM.

Cell death is an important mechanism during the mammalian brain development, with documented roles in both morphogenetic and histiogenetic degeneration. The mammalian caspase family of cysteine-containing, aspartatespecific proteases was suggested to play a pivotal role in execution of developmental apoptosis due to its homology to the cell death gene ced-3 of the nematode C. elegans. We have used the gene targeting strategy to test the biological functions of several members of the caspase family. Our results indicate that caspase-9 and caspase-3 are essential for programmed cell death and normal mouse brain development. Moreover, caspase-9 and caspase-3 form a sequential cell death cascade, as the absence of caspase-9 abolishes the cytochrome c-mediated caspase-3 activation both in vivo and in vitro and exhibits similar phenotypes to those of caspase-3 deficiency. To test whether the activation of caspase-3 in the mammalian brain is also regulated by Bax and BclxL, homologues of the pro-apoptotic gene eg1-1 and antiapoptotic gene ced-9 in C. elegans, respectively, we conducted epistatic genetic analysis in caspase-3/bc1-x double mutants. The absence of caspase-3 rescued the ectopic cell death of post-mitotic neurons caused by the BclxL deficiency, indicating an evolutionary conserved cell death pathway. However, Bax and BclxL are expressed only in post-mitotic neurons, in contrast to caspase-3, which is activated in the proliferative population and specific brain regions associated with morphogenesis. Moreover, unlike caspase-3 or -9 deficiency, Bax-deficient mutants exhibited a normal amount of cell death in the early developing brain. These results indicate additional signaling pathways in early brain development, preventing a random, haphazard activation of caspases. The Jun N-terminal kinase (JNK) signaling pathway appears to be an important mechanism that regulates the brain region-specific activation of caspases. In the absence of Jnkl and Jnk2 genes, both encoding a somatic form of JNK, there is reduced apoptosis in the hindbrain, leading to neural tube defect and widespread cell death coupled with ectopic caspase-3 activation in the forebrain. Taken together, these results suggest complex regulation mechanisms and distinct functions of caspase activation in mammalian brain development.

After activation CD4(+) helper T cells differentiate into T-helper (Th) 1 or Th2 effector cells. These two subsets are characterized by their distinct cytokine expression pattern and the immune function they mediate. Over the past years, a number of factors have been identified to affect helper T cell lineage determination, including antigen receptor, coreceptors and, most importantly, cytokine environment. In this review, we also summarize recent advancement in understanding of transcriptional and signaling regulation of the differentiation process. This knowledge will become important in the future to develop means in treating immune disorders.

The mitogen-activated protein (MAP) kinase family members, which include the extracellular response kinases (ERK), p38, and c-Jun amino terminal kinases (JNK), play a role in mediating signals triggered by cytokines, growth factors, and environmental stress. JNK and p38 MAP kinases have been involved in inflammatory processes induced by a variety of stimuli, such as oxidative stress. Here, we describe the role of the JNK and p38 MAP kinase signaling pathways in the development of T cells in the thymus, and activation and differentiation of T cells in the peripheral immune system.