Regulatory B and T cell responses in patients with autoimmune thyroid disease and healthy controls.

Autoimmune diseases occur due to faulty self-tolerance. Graves' disease (GD) and Hashimoto's thyroiditis (HT) are classic examples of organ-specific autoimmune diseases. GD is an auto-antibody-mediated disease where autoantibodies are produced against the thyroid stimulating hormone receptor (TSHR). HT is primarily a T-cell mediated disease, and whether B cells play a pathogenic role in the pathogenesis is still unclear. Both GD and HT are characterized by infiltration of the thyroid gland by self-reactive T cells and B cells. In the first paper of this thesis, the role of regulatory B cells (Bregs) and regulatory T cells (Tregs) were investigated in the context of GD and HT. First, we studied the role of the thyroid self-antigen, thyroglobulin (TG) in healthy donors. The self-antigen TG, but not the foreign recall antigen tetanus toxoid (TT), was able to induce interleukin 10 (IL-10) secretion by B cells and CD4+ T cells. These IL-10 producing B cells (B10 cells) from healthy donors were enriched with the CD5+ and CD24hi phenotype. In addition, TG was able to induce IL-6 production by B cells. In contrast, TT induced production of Th1-type pro-inflammatory cytokines including interferon-gamma (IFN-γ) and IL-2. In the second paper, the frequency and phenotype of B10 was investigated in healthy donors and patients with GD or HT.  The frequencies of B10 cells were similar in the three groups, irrespective of whether IL-10 was induced by a combination of phorbol 12-myristate 13-acetate (PMA) and ionomycin, by CpG oligodeoxynucletodies (ODN) 2006, or by TG. Several phenotypes have been associated with B10 cells such as CD5+, CD25+, TIM-1+, CD24hiCD38hi and CD27+CD43+. We found that larger proportions of B10 cells in patients with GD or HT were CD25+ and TIM-1+ than B10 cells in healthy donors. In healthy donors, B10 cells were CD24hiCD38-, whereas for HT patients these cells were primarily CD24intCD38int. For GD patients, we found lower proportions of B10 cells within the CD27+CD43- and CD27-CD43- fractions than for healthy donors. Our data show that GD and HT are not associated with decreased frequencies of B10 cells. Accordingly, B10 cells may not be confined to one phenotype or subset of B cells. In the third paper, we studied the balance between IL-17-producing CD4+ T cells (Th17 cells) and IL-10-producing CD4+ T cells (Th10 cells) in healthy donors and patients with GD or HT. In HT patients, we found increased proportions of naïve Th17 cells after stimulation with the thyroid self-antigen thyroid peroxidase (TPO) and the Escherichia coli lipopolysaccharide (E. coli LPS).  The proportions of Th10 cells were similar in healthy donors and in HT patients after antigen-specific stimulation. After TG stimulation, an increased Th17:Th10 ratio was found in HT patients within the naïve T cell compartment. Taken together, these data indicate that the thyroid self-antigens TG and TPO induced a skewed Th17:Th10 differentiation in HT patients. IL-6 and TGF-ß have been reported to be important for human Th17 differentiation and, accordingly, HT patients showed higher baseline production of IL-6 and TGF-ß1 than healthy donors. Moreover, the baseline expression of mRNA encoding the transcription factor Forkhead box protein 3 (FOXP3) was similar in HT patients and healthy donors, but HT patients displayed higher constitutive expression of the splice variant FOXP3Δ2, lacking exon 2, than healthy donors. Full-length FOXP3 has been shown to inhibit Th17 differentiation, while FOXP3Δ2 does not. Thus, increased IL-6 and TGF-ß1 in the microenvironment and the increased expression of FOXP3Δ2 may contribute to the skewing of Th17 cells in HT patients. In conclusion, the human thyroid self-antigen TG is able to induce antigen-specific production of IL-10 in CD19+ B cells and CD4+ T cells among healthy donors and patients with GD or HT. Our data indicates that patients with GD or HT were not impaired in producing IL-10 and thus an immunoregulatory response. Additionally, TPO, a thyroid self-antigen, induced a higher frequency of Th17 cells in HT. This indicates that Th17 cells may play an important role in HT pathogenesis.

Characterization of Regulatory B Cells in Graves' Disease and Hashimoto's Thyroiditis.

A hallmark of regulatory B cells is IL-10 production, hence their designation as IL-10+ B cells. Little is known about the ability of self-antigens to induce IL-10+ B cells in Graves' disease (GD), Hashimoto's thyroiditis (HT), or other autoimmune disease. Here we pulsed purified B cells from 12 HT patients, 12 GD patients, and 12 healthy donors with the thyroid self-antigen, thyroglobulin (TG) and added the B cells back to the remaining peripheral blood mononuclear cells (PBMCs). This procedure induced IL-10+ B-cell differentiation in GD. A similar tendency was observed in healthy donors, but not in cells from patients with HT. In GD, B cells primed with TG induced IL-10-producing CD4+ T cells. To assess the maximal frequency of inducible IL-10+ B cells in the three donor groups PBMCs were stimulated with PMA/ionomycin. The resulting IL-10+ B-cell frequency was similar in the three groups and correlated with free T3 levels in GD patients. IL-10+ B cells from both patient groups displayed CD25 or TIM-1 more frequently than did those from healthy donors. B-cell expression of two surface marker combinations previously associated with regulatory B-cell functions, CD24hiCD38hi and CD27+CD43+, did not differ between patients and healthy donors. In conclusion, our findings indicate that autoimmune thyroiditis is not associated with reduced frequency of IL-10+ B cells. These results do not rule out regulatory B-cell dysfunction, however. The observed phenotypic differences between IL-10+ B cells from patients and healthy donors are discussed.

B-cell exposure to self-antigen induces IL-10 producing B cells as well as IL-6- and TNF-α-producing B-cell subsets in healthy humans.

Human B cells are able to secrete IL-10 after stimulation with mitogens, but their ability to produce IL-10 and regulate T-cell responses after stimulation with self-antigens is unclear. We co-cultured thyroglobulin-pulsed B cells from healthy donors with autologous T cells and observed production of IL-10 and TGF-ß, in addition to TNF-α and IL-6. Pulsing with foreign antigen, tetanus toxoid (TT), induced a Th1-response with minimal IL-10 production. After thyroglobulin-pulsing, 1.10±0.50% of B cells and 1.00±0.20% of CD4(+) T cells produced IL-10, compared to 0.29±0.19% of B cells (P=0.01) and 0.13±0.15% of CD4(+) T cells (P=0.006) following TT-pulsing. Thyroglobulin-stimulated, IL-10-secreting B cells were enriched within CD5(+) and CD24(high) cells. While thyroglobulin-pulsed B cells induced only modest proliferation of CD4(+) T cells, B cells pulsed with TT induced vigorous proliferation. Thus, B cells mediate self-antigen-specific IL-10, TNF-α and IL-6 production in co-cultures with T cells and contribute actively to these cytokine secretions.

DNA vaccines encoding proteins from wild-type and attenuated canine distemper virus protect equally well against wild-type virus challenge.

Immunity induced by DNA vaccines containing the hemagglutinin (H) and nucleoprotein (N) genes of wild-type and attenuated canine distemper virus (CDV) was investigated in mink (Mustela vison), a highly susceptible natural host of CDV. All DNA-immunized mink seroconverted, and significant levels of virus-neutralizing (VN) antibodies were present on the day of challenge with wild-type CDV. The DNA vaccines also primed the cell-mediated memory responses, as indicated by an early increase in the number of interferon-gamma (IFN-γ)-producing lymphocytes after challenge. Importantly, the wild-type and attenuated CDV DNA vaccines had a long-term protective effect against wild-type CDV challenge. The vaccine-induced immunity induced by the H and N genes from wild-type CDV and those from attenuated CDV was comparable. Because these two DNA vaccines were shown to protect equally well against wild-type virus challenge, it is suggested that the genetic/antigenic heterogeneity between vaccine strains and contemporary wild-type strains are unlikely to cause vaccine failure.

Room for improvement in the treatment of hip fractures in Denmark.

INTRODUCTION: Treatment of hip fractures has evolved since the introduction of fast-track surgical programs in the late 1990s. The aim of our study was to describe the quality of treatment and care related to fast-track hip fracture surgery in Denmark by external audit of patient records. MATERIAL AND METHODS: This was a national multicenter audit of hospital charts from each hospital treating ≥ 50 hip fracture patients per year (n = 594). RESULTS: The study demonstrated significant variability in treatment and care of patients with hip fractures among the regions of Denmark. Pain management, nutritional screening, ambulation characteristics, training in activities of daily living, and rehabilitation planning were consistently inadequate. Length of stay was 7-11 days. CONCLUSION: Although the principles for fast-track surgery have been adapted to some extent at all departments in Denmark with an annual treatment of at least 50 patients with hip fractures, no single department has implemented the whole package. Hospital stay has been reduced since the introduction of fast-track regimes, and improvements were seen in many of the quality indicators. Implications for future practice include better adherence to clinical guidelines, a more homogeneous documentation system in nursing, promotion of evidence-based standards, and improved treatment and care of the physical and psychological consequences of hospitalization.

Immune response characteristics following emergency vaccination of pigs against foot-and-mouth disease.

Pigs were vaccinated with the emergency inactivated foot-and-mouth disease virus (FMDV) vaccine--water-in-oil-in-water emulsion with Montanide ISA206--known to protect after 3-5 days. Peripheral blood leukocyte (PBL) sub-populations did not differ between vaccinates and controls post-vaccination. There was neither lymphopenia nor inflammatory reaction. FMDV-specific antibody and T lymphocyte activity developed in the vaccinates. Virus-induced Th1-like cytokine protein and mRNA (IFNgamma and IL-2) were identified, particularly IFNgamma. Th2-like cytokine protein and mRNA (IL-4 and IL-6) were also induced in an FMDV-specific manner. IL-10 was induced by both virus and mock antigen. The current emergency FMDV vaccine induces a diverse immune defence network--innate, and both Th1-like and Th2-like responses--without adverse reactions such as lymphopenia or inflammatory responses.

Influence of routes and administration parameters on antibody response of pigs following DNA vaccination.

Using the nucleoprotein of porcine reproductive and respiratory syndrome virus as model antigen, we optimised parameters for gene gun vaccination of pigs, including firing pressure and vaccination site. As criteria for optimisation, we characterised particle penetration and local tissue damage by histology. For selected combinations, vaccination efficiency in terms of antibody response was studied. Gene gun vaccination on ear alone was as efficient as a multi-site (ear, thorax, inguinal area, tongue mucosa) gene gun approach, and more efficient than combined intramuscular (i.m.)/intradermal (i.d.) injection of plasmid DNA. This indicates, that the ear is an attractive site for gene gun vaccination of pigs.