Clinical immunology and immunopathology最新文献

Innervation of the lacrimal gland of MRL/Mp-Fas-lpr/lpr(MRL/lpr), a murine model for Sjögren's syndrome, is unaltered with the onset or progression of the lymphocytic infiltration. To determine whether lacrimal and submandibular gland cells are able to respond to external stimuli, acini were prepared from MRL/lpr (diseased) and MRL/Mp-+/+ (MRL/+, control) mice at 4, 8, and 12 weeks of age and loaded with the fluorescent dye fura-2 to monitor changes in the intracellular Ca²⁺concentration ([Ca²⁺]_i) in response to cholinergic and α₁-adrenergic stimulation, two major stimuli of lacrimal gland protein secretion. Cholinergic-induced [Ca²⁺]_iincrease was up-regulated 3- and 4-fold in lacrimal gland acini isolated from 8- and 12-week-old MRL/lpr mice, respectively, compared to 4-week-old animals, but was not up-regulated in age-matched MRL/+ control mice. Similarly, α₁-adrenergic-induced [Ca²⁺]_iincrease was up-regulated 7- and 12-fold in acini isolated from 8- and 12-week-old MRL/lpr mice, respectively, compared to 4-week-old animals, but was not up-regulated in MRL/+ mice. Cholinergic-induced [Ca²⁺]_iincrease in submandibular gland acini of MRL/lpr and MRL/+ mice was the same at all ages. In contrast, α₁-adrenergic-induced [Ca²⁺]_iincrease was up-regulated 3-fold in acini from 12-week-old MRL/lpr mice, compared to 4-week-old mice, but was not up-regulated in age-matched MRL/+ mice. We conclude that the Ca²⁺signaling portion of cholinergic and α₁-adrenergic pathway in the lacrimal gland and the Ca²⁺signaling portion of α₁-adrenergic pathway in the submandibular gland is up-regulated with the onset and progression of the lymphocytic infiltration in the MRL/lpr murine model of Sjögren's syndrome.

Lymphotoxin knock-out (KO) mice generate specific immune responses to orally administered immunogens despite having neither gut-associated nor peripheral lymphoid tissues. The spleen, therefore, was expected to play a role in the generation of immune responses in these KO mice. KO and wild-type (wt) mice were splenectomized and orally immunized withSalmonella typhimurium.Splenectomy produced the most profound effects on serum and fecal IgA levels in KO mice. Total and antigen-specific serum and fecal IgA were increased in splenectomized wt mice but decreased in splenectomized KO mice. Antigen-specific serum IgG was decreased in both KO and wt splenectomized mice while total IgG increased in splenectomized wt mice. Both splenectomized wt and KO mice demonstrated a compensatory expansion of the lamina propria compartment characterized by a significant increase in the number of IgA spot-forming cells. KO mice demonstrated further compensation for the loss of the spleen in the accelerated development of ectopic lymphoid tissues. We conclude that the spleen plays a prominent role as a lymphoid organ in KO mice but its removal does not abolish immune responsiveness. Residual immune responsiveness in splenectomized KO mice following oral immunization appears to be due to expansion and/or development of alternate effector compartments.

Systemic lupus erythematosus (SLE) predominantly affects women (9:1 compared to men) of childbearing age and often decreases its intensity in postmenopausal women, suggesting that sex hormones play a role in its pathogenesis. Comparison of steady-state levels of calcineurin mRNA using RNase protection assays revealed increased calcineurin expression in response to estradiol in cultured T cells from nine female lupus patients. Calcineurin mRNA levels did not increase significantly in T cells from eight age-matched normal control female volunteers. Estrogen-dependent calcineurin mRNA increased in a dose-dependent fashion, while progesterone and dexamethasone did not increase calcineurin mRNA in patient cells. Lupus T cell calcineurin mRNA increased in response to estradiol at 6 h but not at 3 h. Calcineurin phosphatase activity increased in lupus T cell extracts after incubation of cells with estradiol, while phosphatase activity in normal T cells was unaffected by estrogen. Calcineurin expression in T cells from patients with vasculitis and rheumatoid arthritis taking medications similar to those taken by the lupus patients was unaffected by estradiol. This study provides the first evidence for a molecular marker of estrogen action in lupus patients and suggests that estrogen-dependent changes in lupus T cell calcineurin could alter proinflammatory cytokine gene regulation and T–B cell interactions.

We have previously shown that apoptosis induced in thymocytes by dexamethasone or teniposide (VM-26) could be inhibited by 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7) and sangivamycin, both relatively specific inhibitors for protein kinase C, but not byN-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA1004), a more specific inhibitor for cAMP-dependent protein kinases. Apoptosis in this model system was not blocked by EGTA and no increase in cytosolic Ca²⁺was observed during apoptosis induced by either dexamethasone or VM-26, suggesting that this kinase was Ca²⁺-independent. In the present study, we demonstrate that addition of 10 μM sangivamycin to thymocyte cultures up to 2 h after addition of either inducer resulted in virtually complete inhibition of apoptosis. Addition of 10 μM sangivamycin at 3 or 4 h after addition of inducer resulted in partial inhibition of apoptosis. Computerized image analysis of two-dimensional PAGE analyses of whole-cell lysates demonstrated that treatment of mouse thymocytes with VM-26 resulted in a limited number ofde novophosphorylation events within 1 h of treatment. The most prominent phosphorylation events associated with VM-26-induced apoptosis were that two intracellular protein species (Protein 1: m.w. = 22.9 kDa, pI, 5.11; and Protein 2: m.w. = 22.9 kDa, pI, 4.98). Similar phosphorylation events were seen in cells treated with dexamethasone. Finally, Western blot analysis suggests thatde novoprotein phosphorylation induced by VM-26 is on serine/threonine residues. These results provide further evidence that the mechanism of VM-26-induced apoptosis of murine thymocytes involves the action of one or more serine/threonine kinases.

The immune response to three cow's milk antigens, β-lactoglobulin (BLG), α-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 beM_r28,000–46,000 polypeptides functionally and physically associated with TGF-β1 and TGF-β2. 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.

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-1β or tumor necrosis factor-α 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.

Transformed B cells making monoclonal IgM-λ anti-PR3 antibody WGH1 from a patient with Wegener's granulomatosis were used to prepare mRNA and synthesize cDNA. PCR primers for human μ and λ chains were then employed to amplify heavy- and light-chain V-regions followed by cloning into pCR2-1 vector and sequencing. Molecular modeling of VH regions employed knowledge-based homology modeling to obtain minimum energy conformation. The VH sequence was subgroup III with marked overall homology to VH1.9III. The VHCDR3 region of WGH1 was unique, consisting of 21 amino acid residues which included seven tyrosines as well as three negatively charged aspartic acid residues. The VL region was subgroup II with a negatively charged glutamic acid at position 100 in CDR3. Molecular modeling of VH revealed a major conformational difference in the shape of CDR3 compared with other antibodies for which three-dimensional structures have been determined. Monoclonal antibody WGH1 reacting with PR3 (a highly positively charged molecule) shows a unique reactive cassette within VHCDR3 with a number of negatively charged aspartic acid residues. WGH1 VHCDR3 contains a loop which shows a major projection not usually recorded in other previously studied antibody molecules.

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.

In a small group of subjects we had identified persistent expansions (range 6–72%) of CD4⁺CD8⁺double-positive (DP) peripheral blood (PB) cells which express the CD8 α/α homodimer. Here, DP cells present in a larger cohort were further investigated and found by FACS analysis to express a single or a dominant TCRBV family. In these subjects, with a mean age of about 64 years, expansions of CD4⁺cells with the same TCRBV family specificity as in the respective DP cells also were consistently detected. TCR heterogeneity of the dominant TCRBV family was specifically evaluated: The amplified CDR3 region was cloned and found to consist of one single or two largely dominant sequence patterns. Furthermore, cloning of the CDR3 region from FACS-sorted DP, CD4⁺, or CD8⁺cells indicates that both DP and CD4⁺, but not CD8⁺cells, isolated from the same individual possess a striking identity of the CDR3 regions. As indicated by FACS analysis, the clonally expanded cells occur in the CD4⁺CD28⁻cells. Taken together, these results suggest that expanded CD4⁺CD28⁻cells might also acquire CD8 α/α expression and become DP and imply that CD4 clonality is a more frequent phenomenon than previously suspected. In conclusion, the persistent expansions described in this report represent a novel group of age-related benign clonal expansions of still undefined significance of a rare CD28⁻T cell subset.