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Background The functional relevance of OX40 ligand (OX40L) in the effector phase of crescentic glomerulonephritis (GN) is unknown. These studies defined the role of endogenous OX40L during the effector stage of murine crescentic GN. Methods GN was induced by immunization with sheep globulin/adjuvant on Day 0 and injection of sheep anti-mouse glomerular basement membrane immunoglobulin (Ig) on Day 10. Rat IgG or neutralizing anti-OX40L antibody was administered on Days 10-18 and immune responses and renal injury assessed on Day 20. Results Compared with naive animals, OX40L was upregulated in the lymph nodes (LNs) and on leucocytes and resident non-immune cells in the kidneys of mice with GN. Inhibition of OX40L in GN augmented renal injury, as indicated by increased crescent formation, proteinuria and glomerular leucocyte accumulation. In line with increased injury, anti-OX40L treatment increased proliferation and decreased apoptosis of CD4 T cells in the LNs, without affecting LN CD4 cytokine production and CD8 T-cell responses. Blockade of OX40L decreased LN regulatory T-cell (Treg) proliferation, transforming growth factor β production and foxp3 expression. OX40L inhibition did not affect B cell expansion or circulating antibody levels. In the kidney, neutralization of OX40L augmented interferon γ (IFNγ) expression by CD4 and CD8 T cells and shifted macrophage polarization towards the pro-inflammatory M1 phenotype. Conclusions OX40L is protective during the effector phase of murine crescentic GN by reducing the expansion of CD4 T cells and enhancing Treg responses in the LNs, and by locally inhibiting T-cell IFNγ production and pro-inflammatory macrophage phenotype in the kidney.

Summary Non-typeable Haemophilus influenzae (NTHi) is a major cause of respiratory but rarely systemic infection. The host defence to this bacterium has not been well defined in patients with chronic airway infection. The aim of this study was to assess the effect of humoral immunity in host defence to NTHi. Responses were measured in control and bronchiectasis subjects who had recurrent bronchial infection. Antibody and complement-mediated killing was assessed by incubating NTHi with serum and the role of the membrane–attack complex and classical/alternate pathways of complement activation measured. The effect of one strain to induce protective immunity against other strains was assessed. The effect of antibody on granulocyte intracellular killing of NTHi was also measured. The results showed that both healthy control subjects and bronchiectasis patients all had detectable antibody to NTHi of similar titre. Both groups demonstrated effective antibody/complement-mediated killing of different strains of NTHi. This killing was mediated through the membrane–attack complex and the classical pathway of complement activation. Immunization of rabbits with one strain of NTHi resulted in protection from other strains in vitro. Antibody activated granulocytes to kill intracellular bacteria. These findings may explain why NTHi rarely causes systemic disease in patients with chronic respiratory mucosal infection and emphasize the potential importance of cellular immunity against this bacterium.

Summary Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells have a key role in host defence against infectious pathogens, but their response to bacteria is not well characterized. Non-typeable Haemophilus influenzae is a major cause of respiratory tract infection including otitis media, sinusitis, tonsillitis and chronic bronchitis (especially in chronic obstructive pulmonary disease and bronchiectasis). This bacterium is also present in the pharynx of most healthy adults. The primary factor that may determine whether clinical disease occurs or not is the nature of the lymphocyte response. Here we examined the CTL cell and NK cell responses to nontypeable H. influenzae in healthy control subjects and in subjects who had bronchiectasis and recurrent bronchial infection with this bacterium. Cells were stimulated with live H. influenzae and intracellular cytokine production and release of cytotoxic granules measured. Control subjects had significantly higher levels of interferon gamma production by both CTL and NK cells, while levels of cytotoxic granule release were similar in both groups. The main lymphocyte subsets that proliferated in response to H. influenzae stimulation were the CTL and NK cells. The results suggest that CTL and NK cell responses may be important in preventing disease from nontypeable H. influenzae infection.

BACKGROUND : The degree to which transplant recipients are immunosuppressed influences their risks of rejection, infection and cancer. Current measures of immune suppression are crude (clinical events) or indirect (drug exposure). We assessed a direct measure of immune status, leukocyte phenotype and function (LPF, a composite measure of five aspects of peripheral blood leukocyte phenotype and function), as a predictor of infection. METHODS : A double-blind, prospective, cohort study was conducted, to determine the burden of infection in stable renal transplant recipients with moderate-severe (Group I, n = 34) or minimal (Group II, n = 36) impairment of LPF, a composite score of: (i) CD4 count; (ii) lymphocyte proliferation in response to phytohaemagglutinin A (PHA); (iii) serum Ig concentrations; (iv) neutrophil phagocytic function; and (v) reactive oxygen species generation. Subjects completed a 6 month diary and each recorded infection was scored 1-4: 1, minor undefined infection (e.g. URTI); 2, minor, microbiologically defined infection (e.g. UTI); 3, major defined infection (requiring hospitalization); 4, opportunistic infection (e.g. Herpes zoster). Final infection score was the sum of all infective episodes. Subjects were then followed-up for 5 years for outcome measures. RESULTS : Groups were well matched for age, sex, diabetes, serum creatinine, rejection and trough cyclosporin concentrations. Group I (moderate to severe impairment of LPF) recorded a higher infection score, 2.4+/-2.8 vs 1.2+/-1.2 for Group II, P = 0.02, due to a higher incidence of moderate to severe infection. This relationship was confirmed by multivariate analysis (OR 1.83, CI 1.08, 3.11, P = 0.03 per unit increase in infection score). During the 5 year follow-up period they had significantly more episodes of admission to hospital, and twice as many admissions due to infections, but no difference in malignancy, graft or patient outcome. CONCLUSION : LPF testing prospectively identified a cohort who incurred a higher burden of infection. Further studies are required to determine the predictive value of LPF for acute rejection, infection and cancer, and to determine whether adjustments to therapy on the basis of LPF can lead to improved outcomes.

Background Advances in immunosuppression have made renal transplantation an effective therapy for end stage renal failure; with low rejection rates and long graft survival times. However, the major adverse consequences, infection and malignancy have not diminished. To predict this risk a score of immune competence has been developed from the simultaneous laboratory assessment of multiple parameters of immune function. Methods The immune status of 152 transplant recipients (138 renal and 14 pancreas/renal) was assessed by measurement of lymphocyte subsets, mitogen-induced T-cell proliferative responses, neutrophil phagocytic capacity and reactive oxygen species (ROS) generation. A scoring system was devised based on the average number of these parameters below 10th percentile of normal. Results The most common abnormality was B-cell lymphopenia (85%) followed by reduced neutrophil ROS production (63% of patients), NK cell lymphopenia (50%), lymphocyte mitogen response (49%) and CD4 number (23%). The abnormalities were unrelated to the duration of immunosuppression (up to 15 years), and variable combinations of cyclosporine A, azathioprine, prednisolone and mycophenolate mofetil (MMF) (except for a consistent reduction in lymphocyte mitogen response in MMF treated patients). Retrospective comparison of infective episodes showed a significantly greater index of infections in patients with the worst score compared with a normal score. Conclusions The data suggests that this quantification of immune function may allow assessment of the level of host immune defence reflecting the level of drug-induced immunosuppression and thus risks of immunosuppressive complications.

SUMMARYThe effect of cytotoxic T-lymphocyte-associated molecule 4-immunoglobulin fusion protein (CTLA4-Fc) on humorally-mediated glomerulonephritis was studied in accelerated anti-glomerular basement membrane (anti-GBM) glomerulonephritis induced in BALB/c mice. This strain of mice develops antibody and complement dependent glomerulonephritis under this protocol. Sensitized BALB/c mice developed high levels of circulating autologous antibody titres, intense glomerular deposition of mouse immunoglobulin and complement, significant proteinuria, renal impairment, significant glomerular necrosis and a minor component of crescent formation 10 days after challenge with a nephritogenic antigen (sheep anti-GBM globulin). Early treatment during the primary immune response, or continuous treatment throughout the disease with CTLA4-Fc, significantly suppressed mouse anti-sheep globulin antibody titres in serum, and immunoglobulin and complement deposition in glomeruli. The degree of glomerular necrosis was improved and proteinuria was reduced, particularly in the earlier stages of disease. Late treatment by CTLA4-Fc starting one day after challenge with sheep anti-mouse GBM did not affect antibody production and did not attenuate glomerulonephritis. The low level of crescent formation found in BALB/c mice developing glomerulonephritis was not prevented by the administration of CTLA4-Fc. These results demonstrate that CTLA4-Fc is of benefit in this model of glomerulonephritis by its capacity to attenuate antibody production, without affecting the minor degree of cell-mediated glomerular injury.

SUMMARY The influence of endogenous glucocorticoids (GC) on glomerular injury was studied in a rat model of heterologous anti-glomerular basement membrane (GBM) glomerulonephritis (GN). Sprague-Dawley rats underwent adrenalectomy (ADX) or sham-operation 3 days prior to i.v. administration of both nephritogenic (100 μg/g) and subnephritogenic (50 μg/g) doses of sheep anti-rat GBM globulin. Administration of a subnephritogenic dose of anti-GBM globulin resulted in GN in adrenalectomized animals only. Similarly, ADX performed prior to administration of anti-GBM in the nephritogenic dose range resulted in exacerbation of GN compared with sham-operated animals (24 h protein excretion: 190.8 ± 32.8 versus 42.5 ± 2.6 mg/24 h; P

The introduction of recombinant human erythropoietin (EPO) into routine clinical practice in the late 1980s heralded a new dawn for the treatment of anaemia in chronic kidney disease (CKD). Apart from the financial burden, limiting its use in the developing world, the early sideeffect profile was mild. It has been shown that full correction of anaemia, requiring high doses of EPO, does not confer mortality benefit to patients suffering from CKD [1,2], and is potentially detrimental to survival [1] even in haemodialysis patients with cardiac disease; the administration of EPO to raise haematocrit to 42% is not recommended [3]. Pure red cell aplasia (PRCA), secondary to EPO, will form the basis of this review. This idiosyncratic immunological reaction involves the generation of anti-EPO antibodies (Abs) resulting in significant anaemia. The mechanisms governing this immunological response remain only partially understood. Whilst PRCA is infrequent, the disease is severe, and the aggressive treatment requirements present a significant complication to patients and a therapeutic conundrum to the treating clinician.

SUMMARYPharmacologic glucocorticoids are powerful inhibitors of the inflammatory response at many levels, including leucocyte trafficking and function. The adhesion molecule P-selectin is a key participant in polymorphonuclear neutrophil (PMN) migration to sites of inflammation. The extent to which endogenous glucocorticoids influence PMN migration and activation is not clear. We used the glucocorticoid receptor antagonist RU486 to examine the effect of endogenous glucocorticoid blockade on PMN migration and function in carrageenan monoarthritis in the rat. Arthritis was induced by intra-articular injection of carrageenan and disease severity measured by PMN count in synovial lavage fluid. Decalcified frozen sections of injected joints were analysed for expression of P-selectin by immunohistochemistry. Adrenal glucocorticoid action was blocked in vivo with RU486 20 mg/kg. PMN phagocytosis and reactive oxygen species synthesis were measured by flow cytometry. Carrageenan injection was associated with severe arthritis (synovial lavage PMN 5.9 ± 0.7 × 106, P

SUMMARYThe aim of this study was to investigate the role of P-selectin in the accumulation of neutrophils in the direct passive Arthus reaction in rat skin. Direct passive Arthus dermal reaction was induced in male Sprague-Dawley (SD) rats by a single i.v. injection of rat anti-sheep globulin (SG) 1 h before i.d. injection of SG antigen. Anti-P-selectin or irrelevant control antibody was given 1 h before rat anti-SG injection. Complement depletion was also performed in a separate group by pretreatment with cobra venom factor (CVF). In all groups dermal swelling was assessed 4 h after antigen challenge. Four hours after antigen challenge, rats treated with control antibody developed skin swelling (2·29 ± 0·47 mm), prominent complement deposition and neutrophil accumulation. This response was associated with local up-regulation of endothelial P-selectin. Pre-treatment with anti-P-selectin antibody 1 h before passive Arthus induction prevented skin swelling (0·29 ± 0·06 mm, P

SUMMARY The aim of this study was to determine the contribution of neutrophils to adjuvant arthritis (AA) by in vivo depletion of peripheral blood neutrophils. Specific anti-neutrophil MoAb, RP3 (10 mg), or a control antibody was given twice daily on days 8–11 after injection of Mycobacterium tuberculosis in inbred male Sprague-Dawley rats. RP3 treatment inhibited the neutrophil leukocytosis associated with AA (3.3 ± 0.6 × 103/mm3versus 21.2 ± 6.9 × 103/mm3; P

Four commercially available assays were compared with a 14C DNA Farr assay for their ability to detect anti-DNA antibodies in 119 sera from 109 patients with systemic lupus erythematosus (SLE) and 25 control sera. The 14C DNA Farr assay was the most sensitive and specific assay (SLE, 57% positive, controls, 0%). A commercial 125I DNA Farr assay was significantly less sensitive (SLE, 39% positive). The fluorescence human chromosomal preparation assay was as sensitive as the 14C DNA Farr assay (SLE, 58% positive) but less specific (controls, 8% positive). The immunofluorescence Crithidia luciliae assay was specific, but less sensitive (SLE, 37% positive) than the 14C DNA Farr assay. Adaptation of Crithidia to immunoperoxidase did not alter its sensitivity or performance. These results confirm that the 14C DNA Farr assay, locally refined and performed by experienced hands, is the most sensitive and specific assay for anti-DNA antibodies. The 125I DNA Farr was no more sensitive than the Crithidia assay but considerably more laborious. The human chromosomal preparation may be suitable as a rapid screening test for anti-DNA antibodies.

The aetiology, clinical features and outcome of 40 patients presenting with Goodpasture's syndrome (glomerulonephritis with haemoptysis and pulmonary infiltrates) are reviewed. The diseases of the patients studied could be divided into three groups: antiglomerular basement membrane (anti-GBM) antibody-induced disease (7/40); systemic vasculitis (22/40) and idiopathic Goodpasture's syndrome (i.e. no systemic disease or anti-GBM antibody detected) (11/40). Overall mortality was 57.5 per cent (anti-GBM disease 4/7; systemic vasculitis 15/22; and idiopathic Goodpasture's syndrome 4/11). Most patients died of disease progression or infection. End-stage renal failure developed in 26 patients (anti-GBM (7), vasculitis (14) and idiopathic Goodpasture's syndrome (5). End-stage renal failure developed in 23 of 24 patients presenting with a creatinine of greater than 600 microM/l regardless of the aetiology of Goodpasture's syndrome or treatment used. Review of renal histology showed that all had proliferative nephritis, with 80 per cent of patients having more than 30 per cent crescents. Thus Goodpasture's syndrome was associated with a wide variety of underlying disease. It had a poor prognosis, with the degree of renal impairment at presentation, the extent of crescent formation and the nature of the underlying disease being the major determinants of outcome.