The nonobese diabetic (NOD) mouse develops spontaneous T-cell-dependent autoimmune diabetes. We tested here whether vaccination of NOD mice with a plasmid DNA encoding glutamic acid decarboxylase (GAD), an initial target islet antigen of autoimmune T cell repertoire, would modulate their diabetes. Our results showed that vaccination of young or old female NOD mice with the GAD-plasmid DNA, but not control-plasmid DNA, effectively prevented their diabetes, demonstrating that GAD-plasmid DNA vaccination is quite effective in abrogating diabetes even after the development of insulitis. The prevention of diabetes did not follow the induction of immunoregulatory Th2 cells but was dependent upon CD28/B7 costimulation. Our results suggest a potential for treating spontaneous autoimmune diabetes via DNA vaccination with plasmids encoding self-Ag.
Thirty-two children with Henoch–Schönlein purpura with or without renal symptoms were studied to characterize the IgA hyperglobulinemia observed in the serum of most patients. It was shown that only the IgA1 subclass concentration was increased. Secretory IgA and IgA to gliadin levels were frequently increased in serum, with a good correlation between them. Circulating IgA immune complexes were detected often and contained high activity to gliadin. In contrast, IgA activity to tetanus toxoid did not change. We failed to show any differences in renal involvement. These data suggest that elevation of serum IgA in Henoch–Schönlein purpura is due in part to a disturbance of the gut mucosal immune system, and the presence of circulating IgA immune complexes with dietary antigens can be postulated but cannot explain the occurrence of urinary symptoms.
Monosomic deletions of chromosome 22q11.2 are the leading cause of DiGeorge syndrome, velocardiofacial syndrome, and conotruncal anomaly face syndrome. DiGeorge syndrome was originally described as an immunodeficiency disorder secondary to impaired T cell production due to thymic aplasia or hypoplasia; however, the frequency of immunodeficiency in the other clinical syndromes associated with the chromosome 22q11.2 microdeletion has not been previously investigated. This study examines the frequency and severity of impaired T cell production and immunodeficiency in chromosome 22q11.2 deletion syndromes and the relationship of the immunodeficiency to specific phenotypic features. Sixty patients over 6 months of age with the characteristic chromosome 22q11.2 deletion underwent immunologic evaluations. Seventy-seven percent of patients with chromosome 22q11.2 deletions were found to have evidence of immunocompromise. The severity of the immunodeficiency did not correlate with any particular phenotypic feature, nor was it restricted to patients who were categorized as having DiGeorge syndrome. Therefore, impaired T cell production and impaired immunologic function are common in patients with deletions of chromosome 22q11.2. The presence or severity of the immunocompromise cannot be predicted based on other phenotypic features and each child should be individually assessed for immune function.
The effects of cocaine exposure upon the host's immune response is equivocal since a variety of studies have generated conflicting conclusions, often as the result of differences betweenin vitroand/or animal models and the actual conditions experienced in humans who are acutely abusing this drug. To further address this issue, we have studied a group of patients who were positive for cocaine or cocaine metabolites and we evaluated a variety of functional parameters of T-lymphocytes and other peripheral lymphoid cell populations, as well as immunophenotypic characteristics of these cells. When compared to normal controls and patients who were negative for cocaine, we found that the cocaine-positive patients had T-cell functional assays which were essentially normal, with the exception of a slight depression in PHA stimulation. Likewise, the immunophenotype of the peripheral blood lymphocytic populations showed normal percentages and numbers of their T cell subsets (CD4, CD8), NK cells, and B cells. Multicolor flow cytometry analysis revealed no difference in T cell subpopulations positive for the "memory" marker, CD62L. No correlation could be established between levels of cocaine or cocaine metabolites and any phenotypic, demographic, or functional parameter. In summary, these results demonstrate that individuals acutely exposed to cocaine do not show markedly altered T cell function or fluctuations in phenotypically identified cell populations. These studies imply that acute cocaine exposure does not predispose individuals to grossly apparent immunosuppression. However, the possibility that subtle, transient, or more specific changes in the immune system may be incurred by use of cocaine, particularly with chronic exposure, remains to be determined.
Despite identification of the CD1 family of molecules in the late 1970s, the function of CD1 was undetermined for more than a decade. Recent evidence has established that CD1 molecules comprise a novel lineage of antigen-presenting molecules, distinct from major histocompatibility complex (MHC) class I and class II molecules. Unlike the MHC molecules, which bind short peptides in their antigen-binding groove for presentation to either CD4+or CD8+T cells bearing αβ T cell receptors, the CD1 molecules appear to accommodate lipid and glycolipid antigens in their hydrophobic cavity for presentation to a wide variety of T cells, including double-negative αβ and γδ T cells and CD8+αβ T cells. By using a unique cytoplasmic signal, some CD1 molecules traffic to endosomal compartments for sampling mycobacteria-derived lipid antigens, and subsequently lipid antigen-loaded CD1 molecules are expressed on the cell surface to activate specific T cells. These CD1-restricted T cells kill mycobacteria-infected cells and secrete interferon-γ, indicating a potential role of CD1-mediated T cell responses in clearing mycobacterial infection. The identification of an MHC-independent antigen presentation pathway for nonpeptide antigens provides new insights into immunoregulation and host defense.
The nonintegrin receptor CD26, also known as dipeptidyl peptidase IV (DPP IV) is a transmembrane 110- to 120-kDa serine aminopeptidase glycoprotein with multiple functions, including cellular trafficking through extracellular matrix, and costimulatory potential during T cell activation, and is an influence upon T cell differentiation during their maturation in the thymus. In order to further define the expression and functional activity of this membrane exopeptidase in human thymus, we utilized a nondisruptive, cytofluorogenic assay which allowed simultaneous measurement of intracellular DPP IV activity using a fluorochrome-conjugated peptide substrate with surface staining of plasma membrane-associated T lymphocyte lineage antigens CD4 and CD8, as well as CD26. Human thymi were examined using the three-color assay, and significant differences in time-dependent DPP IV activity were found among the thymocyte subsets defined by their CD4/CD8 phenotype. In this regard, CD4(-)/CD8(-) thymocytes displayed the lowest DPP IV activity and had higher activity than the smaller-sized CD26(+) cells. Thymocytes containing greater percentages of apoptotic cells expressed lower DPP IV activity than viable cells. Thus, DPP IV appears to be upregulated as thymocytes mature and is reduced among thymocyte populations enriched for cells undergoing programmed cell death, suggesting that CD26-associated enzymatic activity is ontogenically controlled during T cell maturation and may be involved in thymic deletion of emerging clones.
Tolerance to experimental autoimmune myasthenia gravis by nasal administration of microgram amounts of acetylcholine receptor (AChR) has been reported. To elucidate the mechanisms behind tolerance induction via the respiratory tract and the involvement of CD4+T cells, we established AChR-specific CD4+CD8−T cell clones from nasally tolerized rats. Nasal tolerance decreased leukocyte function-associated antigen-1 (LFA-1) expression in CD4+T cells from tolerized rats. There was no difference between nasally tolerized and control rats in expression of intercellular adhesion molecule-1. The levels of transforming growth factor-β (TGF-β) mRNA-expressing cells were upregulated in CD4+T cell clones after tolerance induction. These findings suggest that decreased LFA-1 expression in CD4+T cells contributes to reduction of the infiltration of inflammatory CD4+T cells, while upregulated TGF-β may inhibit lymphocyte functions.
Surface and intracellular immunologic and apoptotic markers and functional lymphocyte assays after stimulation with anti-CD3/anti-CD28 antibodies or phytohemagglutinin (PHA) were studied in 44 patients fulfilling the Oxford criteria for chronic fatigue syndrome (CFS). Results were then correlated to scores for the Short Form-36 health questionnaire (SF-36), which assesses eight aspects of patient's well-being, and for the general health questionnaire (GHQ), which detects current psychiatric disorder. Patients had significantly increased mean fluorescence intensity readings of HLA-DR in CD4 and CD8 cells (P< 0.05). Expression of the costimulatory receptor CD28 in CD8 cells was significantly reduced, and the apoptosis repressor ratio of bcl-2/bax in both CD4 and CD8 was increased in patients (P< 0.05). Patients with increased HLA-DR expression had significantly lower SF-36 total scores, worse body pains, and poorer general health perception and physical functioning scores. Increased spontaneous lymphocyte proliferation was associated with poor general health perception. PHA proliferative responses were lower in patients with poor emotional and mental health scores, and the anti-CD3/anti-CD28 response was low in those with low general health perception scores. Higher spontaneous proliferation and reduced PHA responses correlated with higher GHQ scores. Similarly, GHQ scores were significantly higher, indicating worse mental health, in those with lower total SF-36 scores and worse general and mental health scores in the SF-36 questionnaire. Finally, higher expression of the costimulatory molecule CD28 correlated with higher total SF-36 scores, general health perception and social functioning scores, and with lower role limitation due to physical health. The increased expression of class II antigens and the reduced expression of the costimulatory receptor CD28, which is a marker of terminally differentiated cells, lend further support to the concept of immunoactivation of T-lymphocytes in CFS and may be consistent with the notion of a viral etiopathogenesis in the illness. We report, for the first time, increased expression of the apoptosis repressor protein bcl-2, which may contribute to enhanced survival of activated lymphocytes. Using the SF-36 health assessment questionnaire and the GHQ, we demonstrated changes in different immunological parameters, each of which correlated with particular aspects of disease symptomatology.
Regeneration is generally considered to be a phenomenon restricted to amphibians in which amputated limbs reform and regrow. We have recently noted a strain of mouse, the MRL, which displays a remarkable capacity for cartilagenous wound closure and provides an example of a phenomenon previously considered to be a form of regeneration. Specifically, through-and-through ear punches rapidly attain full closure with normal tissue architecture reminiscent of regeneration seen in amphibians as opposed to scarring, as usually seen in mammals. Histologically, we have demonstrated normal cell growth and microanatomy, including angiogenesis and chondrogenesis, as opposed to control C57BL/6 mice which have ear holes that contract minimally but do not close. Finally, this phenomenon is a genetically definable quantitative trait.
Myasthenia gravis is an organ-specific autoimmune disorder generally thought to be caused by an antibody-mediated attack against the skeletal muscle nicotinic acetylcholine (Ach) receptor (AchR) at the neuromuscular junction. Extraocular muscle weakness and double vision are present in about 90% of patients with myasthenia gravis and are the predominant complaints in about 20% of patients, when the condition is called ocular myasthenia gravis (OMG). While serum antibodies against the AchR are detected in most patients with generalized myasthenia gravis (GMG), they are not found in about one-third of patients with the ocular variety, and epidemiological, clinical, and serological studies suggest that OMG and GMG are two separate diseases. Both forms of myasthenia gravis are sometimes associated with thyroid autoimmunity or thyroid-associated ophthalmopathy (TAO). We have therefore tested the sera of patients with GMG and OMG by Western blotting for antibodies against porcine eye muscle membrane proteins in general, and by enzyme-linked immunosorbent assays (ELISA) specifically for reaction with two skeletal muscle antigens which are prominent marker antigens for TAO, namely, the calcium-binding protein calsequestrin and the so-called "64-kDa protein." The 64-kDa protein has recently been identified as the flavoprotein subunit of mitochondrial succinate dehydrogenase. Patients with ophthalmopathy and myasthenia were excluded. Nine of the patients had associated Graves' hyperthyroidism without evident ophthalmopathy and one had Hashimoto's thyroiditis. Antibodies against porcine eye muscle membrane antigens of M(r) 15-110 kDa were detected in patients with GMG or OMG, one or more antibodies being detected in 100% of patients with GMG and in 88% of those with OMG. The most frequently found antibodies were those targeting eye muscle membrane proteins of 15, 67, and 110 kDa. Antibodies reactive with purified calsequestrin (63 kDa) were detected in 21% of patients with OMG but in no patient with GMG. Antibodies recognizing purified succinate dehydrogenase (67 kDa) were found in 42% of patients with OMG, in 100% (5 of 5) of patients with GMG, and in 48% of all patients with myasthenia gravis not associated with Graves' hyperthyroidism. There was no close correlation between any eye muscle-reactive antibody and antibodies against the AchR in either group of myasthenic patients. The findings support the notion that immunoreactivity against skeletal muscle proteins other than the AchR may play a role in the development of the muscle weakness in AchR antibody-negative patients with OMG and GMG, although it is unlikely that any of the antibodies demonstrated in this study are directly implicated. Similarly, while the demonstration of antibodies reactive with eye muscle antigens associated with TAO in patients with OMG raises the possibility that the link between the ocular lesions of myasthenia gravis and Graves' disease may be autoimmunity against a common antigen(s), it is more likely that both disorders are mediated by cytotoxic T cells recognizing another cell membrane antigen, such as the novel thyroid and eye muscle shared protein G2s, and that serum antibodies reactive with succinate dehydrogenase Fp subunit and calsequestrin are markers of an immune-mediated eye muscle reaction.
Rheumatoid arthritis (RA) is characterized by recruitment of leukocytes from the vasculature into inflamed synovial tissue (ST) and synovial fluid (SF), which depends, in part, upon the continued maintenance of chemotactic stimuli. RANTES is a potent chemoattractant for leukocytes including monocytes and CD45RO+ memory T lymphocytes. The aim of this study was to determine the production, the source, and the function of antigenic RANTES in arthritis. We detected antigenic RANTES in SFs from RA and OA patients (100 +/- 22.7 and 72 +/- 30.7 pg/ml, respectively). CM from RA ST fibroblasts stimulated with interleukin-1beta or tumor necrosis factor-alpha contained significantly more antigenic RANTES than unstimulated CM (452 +/- 181.6 and 581 +/- 200.2 pg/ml, respectively, versus 12 +/- 4.4 pg/ml, P < 0.05). PHA-stimulated RA SF mononuclear cells secreted 5- to 15-fold more antigenic RANTES than did nonstimulated mononuclear cells, while LPS induced secretion up to 4-fold. We immunolocalized antigenic RANTES to sublining macrophages (28 +/- 3.7 and 8 +/- 2.0% immunopositive cells), perivascular macrophages (56 +/- 6.9 and 19 +/- 3.4%), and synovial lining cells (37 +/- 5.8 and 60 +/- 10.4%) in RA and OA tissue, respectively. Anti-RANTES neutralized 20.2 +/- 1.3% of the RA SF chemotactic activity for normal peripheral blood monocytes (P < 0.05). These results demonstrate antigenic RANTES in RA and OA ST and SF and identify RANTES as a chemoattractant for monocytes in the RA joint.
To investigate the molecular mechanism of glandular parenchyma destruction in Sjögren's syndrome (SS), Bcl-2, Bax, Bcl-X, and Bak expression were studied. SS (n= 18) and control salivary glands (n= 6) were examined by immunohistochemistry. Apoptosis was assessed byin situDNA nick end labeling. Infiltrating mononuclear cells in the SS salivary gland showed elevated Bcl-2. These mononuclear cells expressed increased Bax but did not undergo apoptosis. Both SS and control salivary gland ductal epithelial cells expressed Bcl-2, Bax, and Bcl-X. SS, but not normal, salivary gland acinar cells expressed Bax and underwent apoptosis. These results suggest that elevated Bax expression in SS salivary gland acinar cells may play an important role in the apoptotic pathway. In contrast, Bcl-2 expression in SS infiltrating mononuclear cells and ductal cells may contribute to their survival.
We previously reported that intramuscular (i.m.) immunization of DNA vaccine encoding human immunodeficiency virus type 1 (HIV-1)IIIB env and rev genes alone or in combination with appropriate adjuvant induces substantial and enhanced immune response against HIV-1. In the present study, we examined whether a polymer, low-viscosity carboxymethylcellulose sodium salt (CMCS-L), has an adjuvant effect on immune response induced by DNA vaccination. BALB/c mice were immunized with HIV-DNA vaccine formulated with CMCS-L via the intranasal (i.n.) and i.m. routes. The combination with the polymer elicited higher levels of antigen-specific serum IgG and fecal IgA antibodies than DNA vaccine alone. For cell-mediated immunity, HIV-specific delayed-type hypersensitivity response and cytotoxic T lymphocyte activity were measured by the footpad-swelling test and the 51Cr-release assay, respectively. Both were enhanced by the combination with CMCS-L via i.n. and i.m. inoculation. Cytokine analysis in culture media of bulk splenocytes harvested from immunized animals showed higher levels of IL-4 production in i.n. -immunized mice compared with i.m.-immunized mice. Nevertheless, the increased IFN-gamma production resulting from the combination with CMCS-L was observed only in i.n.-immunized mice. These data indicate that i.n. immunization of HIV-DNA vaccine formulated with CMCS-L enhances HIV-specific mucosal antibody (Ab) and systemic Ab and cell-mediated immune response.
Considering an autoimmunity and autism connection, brain autoantibodies to myelin basic protein (anti-MBP) and neuron–axon filament protein (anti-NAFP) have been found in autistic children. In this current study, we examined associations between virus serology and autoantibody by simultaneous analysis of measles virus antibody (measles-IgG), human herpesvirus-6 antibody (HHV-6-IgG), anti-MBP, and anti-NAFP. We found that measles-IgG and HHV-6-IgG titers were moderately higher in autistic children but they did not significantly differ from normal controls. Moreover, we found that a vast majority of virus serology-positive autistic sera was also positive for brain autoantibody: (i) 90% of measles-IgG-positive autistic sera was also positive for anti-MBP; (ii) 73% of measles-IgG-positive autistic sera was also positive for anti-NAFP; (iii) 84% of HHV-6-IgG-positive autistic sera was also positive for anti-MBP; and (iv) 72% of HHV-6-IgG-positive autistic sera was also positive for anti-NAFP. This study is the first to report an association between virus serology and brain autoantibody in autism; it supports the hypothesis that a virus-induced autoimmune response may play a causal role in autism.
The immune response to three cow's milk antigens, beta-lactoglobulin (BLG), alpha-lactalbumin (AL), and casein (CA) was studied in 15 milk-intolerant adult patients and 11 adult controls. IgG, IgE, and IgG subclasses (IgG1, IgG2, IgG3, IgG4) and T cell-derived antigen-binding molecules (TABM) specific for each antigen were measured in both groups. In the patient group, a significant elevation of total IgG and TABM against each of the milk antigens was found as well as raised levels of IgG1 to BLG and CA, IgG4 to BLG, and IgE to CA. TABM specific for BLG were isolated by affinity for BLG and found to be Mr 28,000-46,000 polypeptides functionally and physically associated with TGF-beta1 and TGF-beta2. These results indicate a Th2-type immune response to the milk antigens in milk-intolerant individuals compared with the control group which shows a pattern typical of anergy or deletion.
Anti-thyroid autoimmune responses have been examined in fetal lambs, the immune systems of which had matured in the absence of exposure to thyroid-specific antigens. The lymphocytic infiltrate in self-thyroid tissue reintroduced into autoimmune lambs showed well-differentiated B and T cell domains. However, T cells from these fetuses were not sensitized against ovine thyroglobulin nor did serum antibodies appear against ovine thyroglobulin or thyroid peroxidase. In the light of these observations, it is inferred that the primary abnormality in the immune systems of fetuses deprived of exposure to thyroid autoantigens is likely to be a failure of the development of a normal T cell subpopulation responsible for down-regulation of autoreactivity. It is also concluded that overt autoimmunity develops only when these fetuses are challenged with thyroid tissue and that B cells may undertake an antigen-presentation role in its induction.
Pristane induces a lupus-like syndrome characterized by autoantibody production and glomerulonephritis in nonautoimmune strains of mice. Although it has been suggested that this syndrome results from nonspecific immune activation, there is little evidence so far that B cells are activated nonspecifically by pristane or that this promotes autoimmunity. In this study, we examined whether polyclonal hypergammaglobulinemia occurs in pristane-induced lupus, and its relationship to the production of anti-DNA, nRNP/Sm, and Su autoantibodies. In conventionally housed mice, there was a marked increase in total IgM and IgG3 2 weeks after i.p. pristane injection, followed by increased IgG1, IgG2a, and IgG2b levels. IgM levels were higher in pristane-treated specific pathogen-free (SPF) mice than in conventionally housed mice, whereas IgG and IgA levels were reduced. Pristane induced anti-nRNP/Sm and Su autoantibodies in SPF mice, but their onset was delayed and levels were lower than those in conventionally housed mice. There was no consistent relationship between total IgG1, 2a, and 2b hypergammaglobulinemia and production of anti-nRNP/Sm and Su autoantibodies. Moreover, the total Ig levels were similar in the anti-nRNP/Sm-positive and -negative groups. In contrast, production of IgM anti-ssDNA antibodies paralleled IgM hypergammaglobulinemia in some, but not all, mice. These studies indicate that pristane-induced lupus is associated with marked hypergammaglobulinemia, the magnitude of which is influenced by the microbial environment. However, anti-nRNP/Sm and Su autoantibody production is at least partly independent of polyclonal B cell activation. The data strongly suggest that pristane-induced lupus is not exclusively the consequence of nonspecific immune stimulation. They also point to the importance of microbial stimulation in the development of hypergammaglobulinemia in this inducible lupus model.
Defensins are small cationic cysteine-rich and amphipathic peptides that form of three-dimensional β-strand structure connected by disulfide bonds. Defensins form key elements of the innate immune system of multicellular organisms. They not only possess broad-spectrum antimicrobial activity but also have diverse roles, including cell signaling, ion channel agitation, toxic functions, and enzyme inhibitor activities in various animals. Although the role of β-defensins in immune responses against infectious agents and reproduction could be significant, inadequate genomic information is available to explain the whole β-defensin repertoire in cattle. No domain or motif-based functional analyses have been previously reported. In addition, how do defensins possess this magnitude of functions in the immune system is still not clear. Our present study, therefore, investigated the sequence divergence and evolutionary relations of bovine defensin proteins with those of humans. Our domain-based evolutionary analysis revealed four major clusters with significant domain variation while reserving a main antimicrobial activity. Our study revealed the β-defensin domain as the ancestor domain, and it is preserved in the first group of defensin protein with no α-helix in its structure. Due to natural selection, some domains have evolved independently within clusters II and III, while some proteins have lost their domain characteristics. Cluster IV contains the most recently evolved domains. Some proteins of all but cluster I might have adopted the functional characteristics of α-defensins which is largely absent in cattle. The proteins show different patterns of disulfide bridges and multiple signature patterns which might render them specialized functions in different tissue to combat against various pathogens.
