PAR(2) expression in peripheral blood monocytes of patients with rheumatoid arthritis.

OBJECTIVES: Proteinase-activated receptor 2 (PAR(2)) is a G protein-coupled receptor activated by serine proteinases with proinflammatory activity. A study was undertaken to investigate the presence and functional significance of PAR(2) expression on rheumatoid arthritis (RA)-derived leucocyte subsets. METHODS: Venous blood was obtained from patients with RA and osteoarthritis (OA) as well as healthy control subjects. Surface expression of PAR(2) on peripheral blood mononuclear cells (PBMCs) was analysed by flow cytometry and interleukin 6 (IL-6) generation by ELISA. RESULTS: Patients with RA had elevated but variable surface expression of PAR(2) on CD14+ monocytes compared with control subjects (median (1st to 3rd quartiles) 1.76% (0.86-4.10%) vs 0.06% (0.03-0.81%), p<0.0001). CD3+ T cells showed a similar pattern with significantly higher PAR(2) expression in patients with RA compared with controls (3.05% (0.36-11.82%) vs 0.08% (0.02-0.28%), p<0.0001). For both subsets, PAR(2) expression was significantly higher (p<0.00001) in patients with high levels of disease activity: PAR(2) expression for both CD14+ and CD3+ cells correlated to C reactive protein and erythrocyte sedimentation rate. Furthermore, in a cohort of patients with newly diagnosed RA, elevated PAR(2) expression in both CD14+ and CD3+ cells was significantly reduced 3 months after methotrexate or sulfasalazine treatment and this reduction correlated significantly with the reduction in the 28-joint Disease Activity Scale score (p<0.05). PAR(2) expression on cells from patients with OA was low, similar to levels seen in control subjects. Generation of IL-6 by monocytes in response to a selective PAR(2) agonist was significantly greater in patients with RA than in patients with OA and control subjects (p<0.05). CONCLUSIONS: These findings are consistent with a pathogenic role for PAR(2) in RA.

T cell requirements for the rejection of renal allografts bearing an isolated class I MHC disparity.

This study has examined the cellular and humoral responses underlying the rejection of rat renal allografts bearing an isolated RT1Aa class I MHC disparity. RT1Aa disparate kidneys were rejected promptly by high responder RT1u but not by low responder RT1c recipients (median survival time 10 d and greater than 100 d, respectively). The magnitude and phenotype of the cellular infiltrate were similar in rejecting and nonrejecting RT1Aa disparate kidneys. Paradoxically, graft infiltrating cells and spleen cells from RT1u recipients showed minimal ability to lyse donor strain lymphoblasts in vitro, whereas effector cells from RT1c recipients showed modest levels of cytotoxicity. Injection of RT1u rats with MRC OX8 mAb was highly effective at selectively depleting CD8+ cells from graft recipients but had no effect in prolonging the survival of RT1Aa disparate grafts despite the complete absence of CD8+ cells from the graft infiltrate, which included numerous CD4+ T cells and macrophages. RT1u, but not RT1c, recipients mounted a strong alloantibody response against RT1Aa disparate kidneys. Immune serum obtained from RT1u recipients that had rejected a RT1Aa disparate graft was able, when injected into cyclosporin-treated RT1u recipients, to restore their ability to reject a RT1Aa, but not a third-party RT1c, kidney. These results suggest that CD8+ cells in general and CD8+ cytotoxic effector cells in particular are unnecessary for the rapid rejection of RT1Aa class I disparate kidney grafts by high responder RT1u recipients. By implication, CD4+ T cells alone are sufficient to cause prompt rejection of such grafts and they may do so by providing T cell help for the generation of alloantibody.

Cellular requirements for renal allograft rejection in the athymic nude rat.

This study has examined the ability of adoptively transferred CD4+ and CD8+ T cells to mediate rejection of a fully allogeneic DA renal graft in the PVG nude rat. Transfer, at the time of transplantation, of naive CD4+ T cells caused rapid graft rejection and primed CD4+ cells were several times more potent. In contrast, naive or specifically sensitized CD8+ cells were entirely ineffective at mediating renal allograft rejection. Whereas nonrejecting grafts showed only a mild cellular infiltrate, rejecting grafts in CD4+ reconstituted animals showed a substantial infiltrate and many of the infiltrating cells had a phenotype (MRC OX8+, MRC OX19-), consistent with NK cells. Experiments using a mAb (HIS 41) against an allotypic determinant of the leukocyte common antigen confirmed that the majority (greater than 80%) of the cellular infiltrate in rejecting grafts derived from the host rather than from the CD4+ inoculum. Infiltrating mononuclear cells, obtained from rejecting allografts 7 d after transplantation in CD4+-injected PVG nude hosts, showed high levels of in vitro cytotoxicity against not only kidney donor strain Con A blasts but also third-party allogeneic Con A blasts, as well as against both NK and LAK susceptible targets. When splenocytes from nontransplanted nude PVG rats were tested in vitro they also demonstrated high levels of lytic activity against both NK and LAK susceptible targets as well as allogeneic Con A blasts, which were not susceptible to lysis by spleen cells from euthymic rats. These findings suggest that injected CD4+ cells may cause renal allograft rejection by the recruitment of extrathymically derived, widely alloreactive cells into the kidney in this model of graft rejection.

Radiology teaching improves Anatomy scores for medical students.

OBJECTIVE: The aim of this study was to evaluate if small group teaching in Radiology impacted Anatomy scores in the summative end of year examination. METHODS: Small group teaching in Radiology was incorporated into Anatomy of year one medical students during the academic years 2016/17 and 2017/18. Examination outcome for 2 years before and 1 year after the study period were compared.Question papers for end of year summative examinations were retrieved; questions relating to Anatomy were identified and anonymised scores for students were obtained. RESULTS: Student numbers ranged 238 to 290/year. Mean Anatomy scores ranged 62-74%, this compared with mean total exam score of 62-65%. No significant difference in Anatomy and Total examination scores for 2015, 2016 and 2019. Mean (SD) Anatomy scores were significantly higher than the Total examination scores for the study period of 2017 and 2018 [68.97 (17.32) vs 63.12 (11.51) and 73.77 (17.85) vs 64.99 (10.31) (p < 0.001)]. Combined Anatomy scores 2017 and 2018 were significantly higher than 2015 and 2016, difference of 5.50 (95% C.I. 3.31-7.70; p < 0.001). CONCLUSION: This is the first study to objectively demonstrate Radiology small group teaching significantly improved Anatomy scores for medical students in the summative end of year examination. ADVANCES IN KNOWLEDGE: No evidence in the literature that Radiology teaching improves examination outcomes for medical students.This is the first study to directly link Radiology teaching with improved Anatomy examination result.Small group teaching in Radiology is a feasible way to teach Anatomy.

The phosphorycholine moiety of the filarial nematode immunomodulator ES-62 is responsible for its anti-inflammatory action in arthritis.

OBJECTIVE: In countries where parasitic infections are endemic, autoimmune disease is relatively rare, leading to the hypothesis that parasite-derived immunomodulators may protect against its development. Consistent with this, we have previously demonstrated that ES-62, a 62 kDa phosphorylcholine (PC)-containing glycoprotein that is secreted by filarial nematodes, can exert anti-inflammatory action in the murine collagen-induced arthritis (CIA) model and human rheumatoid arthritis-derived synovial tissue cultures. As a first step to developing ES-62-based drugs, the aim of this study was to determine whether the PC-moiety of ES-62 was responsible for its anti-inflammatory actions. METHODS: We compared the anti-inflammatory activity of a PC-free form of recombinant ES-62 (rES-62) and a synthetic PC-ovalbumin conjugate (OVA-PC) with that of native ES-62 in the CIA model and synovial tissues from patients with rheumatoid arthritis. RESULTS: The anti-inflammatory actions of ES-62 in CIA appear to be dependent on the PC moiety as indicated by the reduction in severity of disease and also suppression of collagen-specific T helper 1 cytokine production observed when testing OVA-PC, but not rES-62. Interestingly, the anti-inflammatory activity of PC did not correlate with a reduction in anti-collagen IgG2a levels. Also, the ES-62-mediated suppression of interferon-gamma from human patient tissues could be mimicked by OVA-PC but not rES-62 or ovalbumin. CONCLUSIONS: In countries where filariasis is endemic the reduced detection of inflammatory diseases, such as rheumatoid arthritis may be because of the anti-inflammatory action of the PC moieties of ES-62. PC may thus provide the starting point for the development of novel, safe immunomodulatory therapies.

Inflammatory cytokines in juvenile idiopathic arthritis: effects on physical growth and the insulin-like-growth factor axis.

OBJECTIVE: To investigate the relationship between markers of inflammation with physical growth and systemic markers of GH secretion in JIA. DESIGN: This is a cross sectional prospective study of patients with JIA recruited during therapeutic arthrocentesis of 17 children with JIA (F,10): 8 oligoarticular (OJIA) and 9 polyarticular (PJIA). RESULTS: Median adjusted height (AHt) SDS was -0.3 (-2.2 to 1.6). Serum ALS SDS (median -1.3, range -2.7 to -0.6) was reduced compared with serum IGFBP-3 SDS (median -0.5, range -7.7 to 2.3) and IGF-1 SDS (median -0.2, range -0.5 to 0.5). Log serum IL5 (95% CI -3.25, -0.81) and log serum IL15 (95% CI -9.58, -4.10) were independent factors associated with AHt SDS. Inflammatory cytokines individually showed no association with IGF-1, IGFBP-1, -2, -3 and ALS. CONCLUSION: Children with JIA and mild degree of growth retardation show decreased ALS and IGFBP-3. Cytokines did not show an association to systemic markers of GH secretion. However, this study reports the novel, preliminary association between serum levels of IL5 and IL15 and the extent of short stature.

A proinflammatory role for IL-18 in rheumatoid arthritis.

IL-18 is a novel cytokine with pleiotropic activities critical to the development of T-helper 1 (Th1) responses. We detected IL-18 mRNA and protein within rheumatoid arthritis (RA) synovial tissues in significantly higher levels than in osteoarthritis controls. Similarly, IL-18 receptor expression was detected on synovial lymphocytes and macrophages. Together with IL-12 or IL-15, IL-18 induced significant IFN-gamma production by synovial tissues in vitro. IL-18 independently promoted GM-CSF and nitric oxide production, and it induced significant TNF-alpha synthesis by CD14(+) macrophages in synovial cultures; the latter effect was potentiated by IL-12 or IL-15. TNF-alpha and IFN-gamma synthesis was suppressed by IL-10 and TGF-beta. IL-18 production in primary synovial cultures and purified synovial fibroblasts was, in turn, upregulated by TNF-alpha and IL-1beta, suggesting that monokine expression can feed back to promote Th1 cell development in synovial membrane. Finally, IL-18 administration to collagen/incomplete Freund's adjuvant-immunized DBA/1 mice facilitated the development of an erosive, inflammatory arthritis, suggesting that IL-18 can be proinflammatory in vivo. Together, these data indicate that synergistic combinations of IL-18, IL-12, and IL-15 may be of importance in sustaining both Th1 responses and monokine production in RA.

Use of monoclonal antibodies to quantitate T lymphocyte subpopulations in human cardiac allografts.

T cell subsets have been quantitated in 40 human cardiac biopsies to characterize the surface phenotype of T lymphocytes involved in acute rejection. Seventeen biopsies were from patients receiving conventional immunosuppression, and 18 were from patients receiving cyclosporine (Cys) as the major immunosuppressive drug. Five biopsies were from nontransplanted hearts. Frozen sections were treated with monoclonal antibodies of the Leu series and an immunoperoxidase technique to determine numbers of Leu 4 (T3 or pan-T-cell), Leu 2a (T8 or suppressor/cytotoxic), and Leu 3a (T4 or helper/inducer) positive cells. Biopsies from patients on conventional immunosuppression during rejection contained 68.3 +/- 10 infiltrating leukocytes/0.048 mm2 of the biopsy, of which 47 +/- 7.4% were T cells. Most of the T cells (86%) were of the 2a/T8 phenotype. In contrast, nonrejecting hearts contained substantially fewer infiltrating leukocytes (43.3 +/- 18.8/0.048 mm2), of which only 12.6% were T cells. There was no predominance of the 2a/T8 subset in nonrejecting biopsies. It is concluded that in patients receiving conventional immunosuppression, rejection is associated with an influx of T cells, most of which are 2a/T8-positive, into the cardiac allograft. In contrast, biopsies from patients on Cys contained large numbers of infiltrating leukocytes that did not appear to correlate with the histological assessment of rejection. However, in these patients, rejection was associated with an increase in the number of T cells with no consistent pattern of subset distribution. Biopsies from nontransplanted hearts contained few infiltrating leukocytes (31.5 +/- 9.4/0.048 mm2), and none of them were T cells.

Skin allograft rejection in mice lacking inducible nitric oxide synthase.

BACKGROUND: During allograft rejection, up-regulation of cytokine-inducible nitric oxide synthase (iNOS) leads to the production of large amounts of nitric oxide (NO). The net effect of NO on the alloimmune response is, however, difficult to predict because of its diverse biological effects, which include potentially opposing roles as an effector and immunoregulatory molecule. METHODS: In this study, the role of iNOS on the in vitro and in vivo alloimmune response was defined using mutant mice that lack a functional iNOS gene. The ability of spleen cells obtained from iNOS-deficient mutants to proliferate and to produce cytokines in response to irradiated BALB/c stimulator cells was determined, and the rate at which iNOS-deficient mice were able to reject BALB/c skin allografts was observed. RESULTS: Spleen cells from homozygous iNOS-deficient (129xMF1)F1 mice, when compared with cells from heterozygous control mice, showed an increased in vitro proliferative response and produced substantially higher levels of interferon-gamma, and also more interleukin-2 and interleukin-12, in response to allogeneic stimulation. The kinetics of BALB/c skin graft rejection were comparable in heterozygous control animals and iNOS-deficient mice. Moreover, no net effect of iNOS on skin allograft rejection was apparent in mice treated with depleting monoclonal antibodies (mAb) to CD4 or CD8 T cells, either alone or in combination, or in mice treated with both anti-CD8 mAb and a neutralizing anti-tumor necrosis factor mAb. CONCLUSIONS: These results show that iNOS has an immunomodulatory effect on the in vitro alloimmune response but lack of iNOS has no net influence on the kinetics of murine skin allograft rejection in either unmodified recipients or recipients in which the early contribution of T-cell subsets and tumor necrosis factor-alpha to graft rejection has been abrogated.

Renal allograft rejection in CD4+ T cell-reconstituted athymic nude rats. The origin of CD4+ and CD8+ graft-infiltrating cells.

PVG-rnu/rnu nude rats reject a fully allogeneic DA renal allograft after the adoptive transfer of naive CD4+ T cells alone, but rejection is accompanied by the accumulation of many CD8+ leukocytes within the graft. In order to clearly establish the provenance of these CD8+ cells infiltrating rejecting kidney allografts, nude recipients (PVG-RT7a) were injected with CD4+ T cells from the PVG-RT7b congenic strain bearing an allotypic variant of the leukocyte-common antigen. Dual fluorescence and immunohistochemistry demonstrated that approximately 75% of the total infiltrate was host-derived; the donor-derived RT7b population was almost entirely (92-99%) CD4+, CD5+, CD3+, and alpha beta TCR+. At least 97% of the CD8+ cells were of nude origin. There was no evidence of donor-derived CD8+ cells or of a CD4+8+ double-staining population. Unexpectedly, nearly half of the alpha beta TCR+ cells from the grafts were of nude origin.

B cell dysfunction in haemophilia in the absence and presence of HIV-1 infection.

56 haemophiliacs selected on the basis of HIV-1 antibody status, liver disease grade and mean annual dose of clotting factor concentrate used were studied. Spontaneous and stimulated IgG and IgM production in vitro were measured. HIV-1 infection was associated with increased spontaneous immunoglobulin production and an impaired response to pokeweed mitogen and Staph Aureus protein A. Implying a shift in the proportions of partially and fully activated B cells. In the absence of HIV-1 infection there was a shift to a greater proportion of partially activated B cells in patients with severe liver disease. The remainder had in vitro immunoglobulin production comparable to controls. B cell abnormalities occur early in the course of HIV-1 infection. Liver disease and not clotting factor concentrate treatment cause B cell abnormalities in the absence of HIV-1 infection in haemophilia.

Normal values for the different classes of venous blood mononuclear cells defined by monoclonal antibodies.

We present the data obtained from routine quantitation of normal venous blood mononuclear cells using 19 monoclonal antibodies against defined mononuclear cell surface antigens. The results indicate different values for percentage and absolute numbers of T lymphocytes and of T lymphocyte subsets depending on the monoclonal antibodies used to quantify these populations. In most instances the total number of identified cells was significantly less than the total number of recovered blood mononuclear cells, which suggests the existence in blood of a null cell population. Evidence is advanced to support previous observations that at least a proportion of this population is of B cell lineage, expressing cytoplasmic immunoglobulin but lacking other class or lineage specific markers. The value of a diverse monoclonal panel in routine quantitation of peripheral blood mononuclear cells is discussed.