The effect of sex on immune responses to a homocitrullinated peptide in the DR4-transgenic mouse model of Rheumatoid Arthritis.

Rheumatoid Arthritis (RA) is more common and severe in women compared to men. Both women and men with RA express autoantibodies to post-translationally modified antigens, including citrullinated and homocitrullinated proteins or peptides. These autoantibodies are strongly linked with the HLA-DR4 gene. The objective of this study was to determine sex differences in immune responses to homocitrullinated antigens. We used a humanized animal model of RA, DR4-transgenic mice and immunized them with a homocitrullinated peptide called HomoCitJED. Immune responses in these mice were measured for splenocyte proliferation by tritiated thymidine incorporation, serum autoantibody production by ELISA and cytokine levels by multiplex. We found that T cell and antibody responses to homocitrullinated antigens were similar in male and female mice. However, we found sex differences in serum cytokine profiles with female mice having higher ratio of IL-1α to IL-5, suggesting imbalances in immune regulation. This is the first study to report that immune responses to homocitrullinated antigens can be differentiated by sex.

Immune responses to peptides containing homocitrulline or citrulline in the DR4-transgenic mouse model of rheumatoid arthritis.

Antibodies to proteins/peptides containing citrulline are hallmarks of Rheumatoid Arthritis (RA). These antibodies are strongly associated with the expression of the Shared Epitope (SE). RA patients also generate antibodies to homocitrulline-containing proteins/peptides (also referred to as anti-carbamylated protein antibodies (Anti-CarP)). This study was undertaken to investigate the relationship between homocitrulline and citrulline immune responses using an established mouse model of RA: DR4-transgenic (DR4tg) mice that express the human SE. C57BL/6 (B6) and DR4tg (on a B6 background) mice were immunized subcutaneously with a homocitrullinated peptide (HomoCitJED). Splenic T cell proliferation was evaluated by 3H-thymidine incorporation assay. Antibodies to homocitrullinated and citrullinated antigens were screened by enzyme-linked immunosorbent assay (ELISA). Antibody cross-reactivity was examined by inhibition with HomoCitJED and its citrullinated counterpart peptide, CitJED (the number of homocitrullines in HomoCitJED is equal to the number of citrullines in CitJED). HomoCitJED-immunized DR4tg mice developed early T and B cell responses to HomoCitJED and late responses to CitJED. These mice also developed anti-CCP2 antibodies. In some mice, antibodies to HomoCitJED were also reactive to CitJED. B6 mice immunized with HomoCitJED developed late T and B cell responses to HomoCitJED, but did not generate responses to citrullinated antigens. Unlike DR4tg mice, anti-HomoCitJED antibodies from B6 mice did not react to CitJED. In conclusion, DR4tg mice immunized with HomoCitJED developed immune responses to CitJED, indicating cross-reactivity. CitJED immune responses were dependent on the SE. HomoCitJED responses occurred in the absence of the SE (B6 mice); however, they developed earlier in DR4tg SE-expressing mice.

Anti-citrullinated protein antibodies in unaffected first-degree relatives of rheumatoid arthritis patients.

OBJECTIVE: First-degree relatives (FDRs) of rheumatoid arthritis (RA) patients sharing genetic and environmental risk factors for RA may represent a pre-RA state. Since anti-cyclic citrullinated protein/peptide antibodies (ACPAs) appear years before the onset of RA, the purpose of this study was to determine the prevalence of various ACPAs in FDRs of RA patients. METHODS: We evaluated 88 RA patients, 50 unaffected FDRs, and 20 healthy control subjects. Six different types of ACPAs were determined by enzyme-linked immunosorbent assay. Joint and periodontal disease symptoms were self-reported. Patients and FDRs were HLA typed for the shared epitope (SE) and the RA-protective alleles HLA-DRB*1301/1302. RESULTS: FDRs had a high prevalence of ACPAs (48%) as compared to controls (10%). Prevalence of the SE and smoking in FDRs was also high (62% and 49%, respectively). Of all of the ACPAs in the FDRs, 13 of 32 (41%) were of the IgA isotype. The most commonly expressed IgG ACPA targeted citrullinated vimentin, occurring in 20% of FDRs. The FDRs had an average of 1 type of ACPA, whereas the RA patients expressed a median of 5 different ACPAs. The only FDR to later develop RA expressed 4 different ACPAs. Joint and periodontal disease symptoms in the FDRs were significantly associated with smoking (OR 5.714 [95% confidence interval (95% CI) 1.151-28.3] and OR 12.25 [95% CI 2.544-58.99], respectively), but not with ACPAs. CONCLUSION: The rate of ACPA positivity in unaffected FDRs of RA patients with a high prevalence of the SE and smoking was 48%, whereas ACPAs were rare in the healthy controls. ACPAs in the FDRs of RA patients was most commonly of the IgA isotype, but IgG ACPA targeting citrullinated vimentin was also frequently found.

Effect of bacterial vaginosis, Lactobacillus and Premarin estrogen replacement therapy on vaginal gene expression changes.

The aim of the study was to investigate gene expression profiles of post-menopausal women receiving Premarin estrogen replacement therapy (ERT), compared to controls, and to examine any correlations between the bacterial vaginosis (BV) status of the subjects. Based upon an expected finding of a 50-60% difference between gene expression of host antimicrobials with alpha=0.05 (2-sided), beta=0.20 the calculation of 7 subjects per group, led to a sample size of 10 subjects receiving Premarin estrogen replacement therapy and 10 healthy, age-matched controls. Vaginal samples were collected at a single timepoint and processed for RNA recovery and Affymetrix array analysis, as well as Nugent scoring and denaturing gradient gel electrophoresis to identify bacteria. Lactobacillus iners was the most commonly detected species in the normal flora and this was confirmed with L. iners-specific PCR method. Vaginal swabs from 6 Premarin and 8 control vaginal samples provided a non-invasive means to analyze human gene expression. There was no significant up-regulation of cancer-associated gene expression in subject receiving Premarin ERT, but some evidence that the potentially protective innate immunity was reduced in patients with BV. Of those with a normal flora, there was a 2-fold down-regulation of carcinoma associated forkhead box A1 gene expression. BV was associated with 7-fold down-regulation of host antimicrobial colony stimulating factor, -9.83-fold for IL-1alpha, -8.33 for IL-1beta and -3.63 for IL-6. This is the first study to use gene arrays to correlate changes in host expression response to estrogen replacement therapy and BV.

Effect of Lactobacillus challenge on Gardnerella vaginalis biofilms.

Bacterial vaginosis (BV) is the most common infectious condition in women. It is caused primarily by anaerobic bacteria which rapidly form biofilms recalcitrant to antibiotic treatment, elevate vaginal pH, induce inflammatory processes and displace indigenous lactobacilli from the vault. Gardnerella vaginalis is commonly associated with these infections. Microscopy analysis showed that within 72 h, viable G. vaginalis covered a surface area of 567 microm(2), reached a depth of 16 microm and a density of approximately 104 microm(3). They maintained these levels for a further 3 days unless challenged with lactobacilli strains. Lactobacillus reuteri RC-14 produced the biggest displacement of Gardnerella. This was not due to pH, which remained between 4.7 and 5.1 for all experiments, nor by hydrogen peroxide which is produced in low amounts by strain L. reuteri RC-14, high amounts by L. crispatus 33820 and not at all by L. rhamnosus GR-1. Deconvolution microscopy showed changes in structure and viability of the biofilms, with loss of dense Gardnerella biofilm pods. For the first time, a strain of L. iners, the most commonly isolated vaginal Lactobacillus in healthy women, was tested for potential probiotic properties. It was found to disrupt Gardnerella biofilm surface area, density and depth, albeit to a lesser extent than L. reuteri RC-14. These studies help to provide insight into the clinical situation in which probiotic and indigenous vaginal lactobacilli can interfere with Gardnerella's presence and reduce the risk of bacterial vaginosis.