Mass oseltamivir prophylaxis halts pandemic influenza A H1N1 2009 outbreak in a secondary school in Ashanti Region, Ghana.

OBJECTIVES: To describe the epidemiology and public health response to H1N1 outbreak and make recommendations to prevent future outbreaks. DESIGN: A descriptive study of an outbreak investigation. SETTING: A secondary school in Asante Akim South District. METHODS: Influenza A H1N1 2009 infection was laboratory-confirmed by Polymerase Chain Reaction (PCR) in clinically ill students after collecting throat swabs. Sixty students of the school had presented with fever, cough, headache and sore throat. The students' dormitories were also inspected to assess degree of ventilation and general level of cleanliness in the rooms. RESULTS: The outbreak followed a propagated transmission lasting 10 days with two peaks on 22(nd) and 24(th) June, 2010.The clinical attack rate was 9.9%. Secondary attack rates at the highly congested female dormitory were 28%, 31.3% and 17.8% for Rooms 1, 2 and 3 respectively. The generation time for the Influenza H1N1 a 2009 outbreak in the school was about two days. CONCLUSION: A mild form of Influenza A H1N1 2009 was confirmed in a secondary school affecting mainly those in the boarding house. Cases identified were treated, but post-exposure prophylaxis with oseltamivir administered to the remaining school population actually halted the outbreak, after social distancing interventions had not succeeded.

European consensus statement on the terminology used in the management of lupus glomerulonephritis.

Systemic lupus erythematosus (SLE) is a complex, multisystem autoimmune disorder, which often involves referral to multiple medical specialists. Lupus nephritis (LN) occurs in ~35% of adults with SLE and predicts poor survival. There is currently no consensus on how to manage patients with SLE or LN across specialties and across different European countries. The Lupus Nephritis Terminology Advisory Group was formed to address this issue as it impacts upon LN treatment. It has developed consensus statements based on opinions from expert panel meetings with nephrologists, nephropathologists, rheumatologists, clinical immunologists and internal medicine specialists from many European countries, after reviewing current guidelines from the European League Against Rheumatism, the American College of Rheumatology and the participants' experience. In this article, we report consensus statements that were developed in six important areas: classification of patients with LN, how classification affects the selection of treatment options and definitions of induction, response, flare and maintenance. We have also proposed a consensus for the terminology involved in the management of LN that is consistent with clinical opinion gathered from multidisciplinary expert meetings and with existing guidelines. We believe this consensus approach provides agreed expert opinion to clinicians and will form the basis for optimising LN treatment.

Meta-analysis of myeloperoxidase G-463/A polymorphism in anti-neutrophil cytoplasmic autoantibody-positive vasculitis.

Wegener's granulomatosis, microscopic polyangiitis and Churg Strauss syndrome are small-vessel vasculitides associated with anti-neutrophil cytoplasmic antibodies (ANCA) directed against proteinase 3 (PR3) and myeloperoxidase (MPO). A G to A polymorphism at position 463 in the promoter region of the MPO gene, which leads to the loss of a SP1 transcription binding site in an Alu hormone responsive element, reduces MPO expression. We hypothesized that MPO alleles may play a role in determining disease susceptibility or severity in ANCA-associated vasculitis (AASV). MPO genotypes were determined by restriction fragment length polymorphism polymerase chain reaction (RFLP/PCR) in 134 Caucasian patients (Wegener's granulomatosis, n = 69; microscopic polyangiitis, n = 65; PR3-ANCA n = 91; MPO-ANCA, n = 43) and 150 matched healthy controls. There was no difference in survival to renal failure or death in patients with the different MPO alleles (chi(2) = 0.904, P = 0.6362) or in presenting serum creatinine concentration based on MPO genotype (chi(2) = 0.389, P = 0.8232). There was no significant difference in genotype frequencies between controls (13AA, 102GG, 35GA) and patients (14AA, 97GG, 23GA: chi(2) = 1.75, P = 0.417), patients with Wegener's granulomatosis (5AA, 53GG, 11GA: chi(2) = 1.864, P = 0.3938) or patients with microscopic polyangiitis (9AA, 44GG, 12GA: chi(2) = 1.682, P = 0.4317). A meta-analysis of our study and two previous studies showed that there was no association between the myeloperoxidase G-463/A polymorphism and the risk of developing ANCA-associated vasculitis; GG versus GA plus AA (odds ratio 1.14; 95% confidence interval 0.86-1.50). The MPO G-463/A polymorphism is not a risk factor for the development or severity of AASV.

Treatment of proliferative lupus nephritis: a changing landscape.

Grootscholten et al. report a randomized controlled trial comparing azathioprine plus intravenous methylprednisolone and oral prednisolone (AZA group) with intermittent intravenous cyclophosphamide and oral prednisolone (CY group) in patients with proliferative lupus nephritis. AZA-treated patients were more likely to develop non-sustained doubling of their serum creatinine, although not significantly so, and significantly more likely to have a relapse of their nephritis than CY-treated patients.

The relationship between albuminuria, MCP-1/CCL2, and interstitial macrophages in chronic kidney disease.

Glomerular-derived proteins may activate tubular cells to express the macrophage-directed chemokine monocyte chemoattractant protein-1 (MCP-1/CCL2). Macrophages at interstitial sites have a central role in directing renal scarring. We have prospectively assessed the relationship between albuminuria, urinary MCP-1/CCL2, interstitial macrophage infiltration, in situ damage, and clinical outcomes in a large group of patients with chronic kidney disease. We studied 215 patients and quantified albumin-creatinine ratio (ACR), urinary MCP-1/CCL2, interstitial macrophage numbers, and in situ damage. ACR correlated with urinary MCP-1/CCL2 (correlation 0.499; P<0.001), interstitial macrophage numbers (correlation 0.481; P<0.001), and index of chronic damage (correlation 0.363; P<0.001). Macrophage numbers closely correlated with in situ damage (correlation 0.755; P<0.001). By multivariate analysis ACR, urinary MCP-1/CCL2, and interstitial macrophage numbers were interdependent. By Kaplan-Meier survival analysis albuminuria, urinary MCP-1/CCL2, interstitial macrophages, and chronic damage predict the outcome. ACR, macrophage numbers, chronic damage, and creatinine independently predicted renal survival. The association of ACR with other variables was strongest in patients with less advanced disease states. There is a close association between albuminuria, urinary MCP-1/CCL2, and interstitial macrophage infiltration with in situ damage and clinical outcomes. These findings support the hypothesis that albuminuria triggers tubular MCP-1/CCL2 expression with subsequent macrophage infiltration. These processes may represent the dominant pathway for the progression of renal injury before the establishment of advanced renal scarring.

Treatment and outcome of adult patients with primary focal segmental glomerulosclerosis in five UK renal units.

BACKGROUND: Focal segmental glomerulosclerosis (FSGS) is the least studied of the causes of idiopathic nephrotic syndrome, and there are few specific guidelines for treatment. AIM: To review data from five UK renal units to investigate whether adult patients with FSGS were treated uniformly, and to examine the effect of treatment on proteinuria and survival. DESIGN: Retrospective record review. METHODS: We examined electronic records of patients with idiopathic FSGS for information on baseline clinical parameters, treatment regimens and outcomes. RESULTS: Of 136 patients with primary FSGS and nephrotic range proteinuria, 76 (56%) were treated with prednisolone and of this group, 59% were treated with additional immunosuppression. Among the treated patients, the total remission rate (complete and partial) was 67%, and one hospital achieved a remission rate of 80%. Treated patients had a significantly higher remission rate than those who were not treated. Remission was associated with a 5-year survival off dialysis of 94%, compared with 53% if remission was not achieved. Baseline serum creatinine and remission were independently associated with survival off dialysis in a multivariate Cox proportional hazards model. DISCUSSION: Patients with primary FSGS and nephrotic range proteinuria, who are treated with corticosteroids, are more likely to enter remission than those who are not treated. Remission rates of up to 80% can be achieved with prolonged treatment, and remission is an independent predictor of survival off dialysis. Patients who do not achieve remission have a poor prognosis. Further clarification of optimal treatment regimens requires additional, prospective studies.

Anti-neutrophil cytoplasm antibody IgG subclasses in Wegener's granulomatosis: a possible pathogenic role for the IgG4 subclass.

A characteristic feature of Wegener's granulomatosis is the presence of antineutrophil cytoplasm antibodies (ANCA) to proteinase 3 (PR3). In vitro, ANCA activate neutrophils by co-ligating PR3 and FcgammaRIIa/IIIb receptors. ANCA are predominantly of the IgG isotype, and IgG1, IgG3 and IgG4 subclasses are particularly represented. To address the pathogenic role of individual ANCA-IgG subclass antibodies, patients' sera were screened using indirect immunofluorescence, enzyme-linked immunosorbent assay (ELISA) and subclass PR3-ELISA to identify patients with high titres of PR3-ANCA within the IgG1, IgG3 or IgG4 subclasses. Unfractionated ANCA-IgG and subclass fractions were isolated by affinity chromatography and compared for their capacities to stimulate superoxide production by primed human neutrophils. Donor neutrophils were analysed for constitutive and induced FcgammaRI expression by flow cytometry. The IgG1, IgG3 and IgG4 subclass fractions, isolated from three different ANCA sera, each stimulated superoxide production from neutrophils derived from multiple donors. Subsequently, IgG4 subclass fractions isolated from a further four ANCA positive sera demonstrated varying abilities to stimulate release of superoxide; unrelated to PR3-ANCA titre, neutrophil donor, or neutrophil FcgammaRI expression. The stimulation of superoxide release by IgG1- and IgG3-ANCA subclass fractions is consistent with the proposed mechanism of co-ligation of PR3 antigen and FcgammaRIIa/IIIb receptors. However, the demonstration of similar activity for the IgG4-ANCA subclass fractions isolated from some sera was unexpected. This activity was independent of neutrophil donor and expression of FcgammaRI, suggesting it was capable of activating neutrophils via constitutively expressed FcgammaRIIa/IIIb or co-ligation of other, unidentified, cell surface molecules.

Improving prescribing using a rule based prescribing system.

OBJECTIVE: To test the hypothesis that the prescribing behaviour of doctors would improve after having experience with a computerised rule based prescribing system. DESIGN: A prospective observational study of changes in prescribing habits resulting from the use of a computerised prescribing system in (1) a cohort of experienced users compared with a new cohort, and (2) a single cohort at the beginning and after 3 weeks of computer aided prescribing. SETTING: 64 bed renal unit in a teaching hospital. INTERVENTION: Routine use of a computerised prescribing system by doctors and nurses on a renal unit from 1 July to 31 August 2001. MAIN OUTCOME MEASURES: Number of warning messages generated by the system; proportion of warning messages overridden; comparison between doctors of different grades; comparison by doctors' familiarity with the system. RESULTS: A total of 51,612 records relating to 5995 prescriptions made by 103 users, of whom 42 were doctors, were analysed. The prescriptions generated 15,853 messages, of which 6592 were warning messages indicating prescribing errors or problems. Doctors new to the system generated fewer warning messages after using the system for 3 weeks (0.81 warning messages per prescription v 0.42 after 3 weeks, p = 0.03). Doctors with more experience of the system were less likely to generate a warning message (Spearman's rho = -0.90, p = 0.04) but were more likely to disregard one (Spearman's rho = -1, p<0.01). Senior doctors were more likely than junior doctors to ignore a warning message. CONCLUSIONS: Doctors are influenced by the experience of using a computerised prescribing system. When judged by the number of warning messages generated per prescription, their prescribing improves with time and number of prescriptions written. Consultants and registrars are more likely to use their clinical judgement to override warning messages regarding prescribed drugs.

EULAR randomised controlled trial of pulse cyclophosphamide and methylprednisolone versus continuous cyclophosphamide and prednisolone followed by azathioprine and prednisolone in lupus nephritis.

OBJECTIVE: To compare the efficacy and side effects of intermittent pulse cyclophosphamide plus methylprednisolone with continuous oral cyclophosphamide plus prednisolone, followed by azathioprine, in patients with proliferative glomerulonephritis caused by systemic lupus erythematosus (SLE). METHODS: A multicentre randomised controlled trial was conducted between June 1992 and May 1996 involving eight European centres. All patients satisfied the American College of Rheumatology criteria for SLE and had biopsy proven proliferative lupus nephritis. All received corticosteroids in addition to cytotoxic drugs, as defined in the protocol, for two years. The trial was terminated after four years as recruitment was disappointing. RESULTS: 32 SLE patients with lupus nephritis were recruited: 16 were randomised to intermittent pulse cyclophosphamide and 16 to continuous cyclophosphamide plus azathioprine. Mean duration of follow up was 3.7 years in the continuous group (range 0 to 5.6) and 3.3 years in the pulse group (range 0.25 to 6). Three patients were excluded from the pulse therapy group as they were later found to have pure mesangial glomerulonephritis. Two patients in the continuous therapy group developed end stage renal failure requiring dialysis, but none in the intermittent pulse therapy (p = 0.488; NS). There were similar numbers of side effects and withdrawals from treatment in both groups. There were three deaths: two in the intermittent pulse therapy group and one in the continuous therapy group. CONCLUSIONS: There was no statistically significant difference in efficacy and side effects between the two regimens. Infectious complications occurred commonly, so careful monitoring is required during treatment.

Powerful morphometric indicator of prognosis in lupus nephritis.

BACKGROUND: Changes in renal biopsies in lupus nephritis have been analysed in many ways, but few have had prognostic value. AIM: To see whether a morphometric measure of chronic renal damage that was a prognostic indicator in other conditions had similar value in lupus nephritis. DESIGN: Retrospective analysis of biopsies and study of outcome. METHODS: On sections of 260 biopsies from 182 consecutive patients with systemic lupus erythematosus, an image analysis system measured chronic damage as a proportion of cortical area, to give the index of chronic damage. This was related to survival (until death or onset of dialysis). Patients were followed for up to 20 years. RESULTS: The index of chronic damage ranged from 0 to 93%. Twenty-three patients (13%) died before dialysis, many from infection or myocardial infarction, and 40 (22%) went onto permanent dialysis. There were strong correlations between the index and time until death or dialysis (log rank test: chi(2) = 51.08, three degrees of freedom [df], p < 0.001) and time to dialysis (log rank test: chi(2) = 72.88, 3df, p < 0.001), but there was no correlation with time until death before dialysis (log rank test: chi(2) = 0.36, 3df, p > 0.9). WHO class of nephritis had no major relation to outcome after the index was taken into account and after appropriate treatment of the different classes. DISCUSSION: The index was a strong indicator of risk of progression to renal failure in lupus nephritis, but not of risk of death before dialysis. This will be useful in clinical management and treatment trials.

Hypogalactosylation of serum IgG in patients with ANCA-associated systemic vasculitis.

The triad of small vessel vasculitides (SVV) comprise Wegener's granulomatosis (WG), microscopic polyangiitis (MPA) and Churg-Strauss syndrome (CS). All three are associated with presence of circulating IgG antineutrophil cytoplasm antibodies (ANCA) which target autoantigens contained, primarily, within neutrophil azurophilic granules. The widely accepted model of pathogenesis suggests that ANCA activate cytokine-primed neutrophils within the microvasculature, leading to by-stander damage to endothelial cells, and rapid escalation of inflammation with recruitment of mononuclear cells. Activation may be initiated, in vitro, by the coligation of the PR3 or MPO antigen, translocated to the cell surface, and FcgammaRIIa/FcgammaRIIIb receptors. This suggests that the IgG subclass profile of ANCA and, possibly, its glycosylation status could influence the inflammatory mechanisms activated. The glycosylation status of total IgG isolated from the sera of patients with WG (13), MPA (6) and CSS (1) was determined by analysis of the released oligosaccharides. A deficit in IgG galactosylation is demonstrated for all patient samples, compared to controls. The mean percentage values for the agalactosylated (G0) oligosaccharides were 57% (SD +/- 9.71), 47% (SD +/- 4.25) and 28% (SD +/- 4.09) for WG, MPO and control samples, respectively. The G0 levels for polyclonal IgG isolated from the sera of both WG and MPA patients were significantly increased compared to controls (P < 0.0001). The major glycoform present therefore is agalactosylated (G0) IgG. In previous studies the G0 glycoform of IgG has been shown to bind and activate mannan binding lectin, and hence to activate the complement cascade, and to facilitate mannose receptor binding and the uptake of IgG complexes by macrophages and dendritic cells. Both of these activities could impact on the processing and presentation of self-antigens in autoimmune disease.

The value of pulse cyclophosphamide in ANCA-associated vasculitis: meta-analysis and critical review.

BACKGROUND: The study aimed at studying efficacy and adverse effects of pulse cyclophosphamide (pCyc) treatment and to compare it to continuous cyclophosphamide (cCyc) for induction of remission in ANCA-associated vasculitides from data in the published literature. METHODS: A Medline search identified 14 studies, containing more than five patients. From the 11 non-randomized studies, data on outcome following pCyc treatment were extracted. Results were given as fraction of the number of evaluable patients. A meta-analysis was performed on the three prospective, randomized controlled trials to compare outcomes concerning remission, relapses, infection, leucopenia, death and renal failure in patients treated with pCyc as opposed to cCyc. RESULTS: The 11 non-randomized studies comprised 202 patients receiving pCyc. Cyc pulses of 375-1000 mg/sqm/pulse were applied at weekly to monthly intervals with different concomitant prednisolone regimens and variable adjunctive therapy. Complete remission was achieved in 112/191, partial remission in 23/191 evaluable patients. Relapses occurred in 68/135 patients, 40/115 patients were non-responders. Leucopenia, infections, haemorrhagic cystitis, and deaths were rare. The meta-analysis, comprising 143 patients, showed that pCyc compared with cCyc treatment was significantly less likely to fail to induce remission (OR 0.29; 95% CI 0.12-0.73) and had a significantly lower risk of infection (OR 0.45; 95% CI 0.23-0.89) and leucopenia (OR 0.36; 95% CI 0.17-0.78). Relapses occurred slightly, although not statistically significantly, more often under pCyc treatment (OR 1.79; 95% CI 0.85-3.75). There were no differences in end-stage renal failure or deaths between the two regimens. CONCLUSIONS: The currently available, rather sparse data show that pCyc is less toxic than cCyc therapy and is an at least equally potent inductor of remission, but possibly at the expense of a higher relapse rate. The existing data do not give sufficient information on outcomes as time to remission and relapse, irreversible damage or quality of life without which a treatment regimen cannot satisfactorily be evaluated today. A large prospective randomized controlled trial is needed to address these issues and their relative importance.

Prognostic value of simple measurement of chronic damage in renal biopsy specimens.

BACKGROUND: A simple method of measurement of chronic damage in renal biopsy specimens would be useful in clinical management, prognosis, comparisons between different centres and trials. METHODS: An interactive image analysis system was used to outline and measure areas of chronic damage in 247 renal biopsy specimens to give an index of chronic damage, expressed as a percentage of cortical cross-sectional area. Prognostic value was analysed by the Kaplan-Meier method to study time between biopsy and onset of permanent dialysis. RESULTS: There was no significant bias between measurements by the same observer or different observers. The index of chronic damage ranged from 0 to 90%. Increasing severity of chronic damage was associated with shortened renal survival. Each increase of 10% in the index increased the hazard ratio of risk of permanent dialysis by 1.5 times (95% confidence interval 1.4-1.7, P<0.001). CONCLUSIONS: A simple measure of chronic damage was a powerful indicator of prognosis. This is likely to be clinically useful in routine practice and trials.

Treatment of systemic lupus erythematosus with mycophenolate mofetil.

Mycophenolate mofetil is an immunosuppressive drug that is of established efficacy in renal transplantation. It inhibits the de novo pathway of purine synthesis and therefore lymphocyte proliferation. Mycophenylate mofetil has been shown to ameliorate the severity of renal injury in murine models of lupus nephritis. Recent studies suggest that it may also be effective in the treatment of patients with lupus nephritis when used in conjunction with steroids. These observations need to be confirmed in adequately sized randomised-controlled studies.

IgG from myeloperoxidase-antineutrophil cytoplasmic antibody-positive patients stimulates greater activation of primed neutrophils than IgG from proteinase 3-antineutrophil cytosplasmic antibody-positive patients.

OBJECTIVE: Wegener's granulomatosis (WG) and microscopic polyangiitis (MPA) have been reported to be pathologically and clinically different. The aim of this study was to assess whether these differences could be explained by differing abilities of proteinase 3-antineutrophil cytoplasmic antibody (PR3-ANCA)-positive IgG preparations or myeloperoxidase-ANCA (MPO-ANCA)-positive IgG preparations to activate neutrophils (polymorphonuclear cells [PMN]) in vitro. METHODS: Using Percoll density gradients, PMN were isolated (concentration 2 x 10(6)/ml) and primed with cytochalasin B (1 ng/ml) and tumor necrosis factor alpha (TNFalpha; 2 ng/ml). The PMN were activated with 200 microg/ml of normal IgG or ANCA. Activation was determined by 1) superoxide anion generation as determined by the superoxide dismutase-inhibitable reduction of ferricytochrome c, 2) monitoring fluxes in Ca2+ concentration using Fura 2-AM-loaded PMN, and 3) degranulation using an MPO assay. Surface expression of PR3 and MPO was determined by fluorescence-activated cell sorter analysis. ANCA isotypes were investigated by enzyme-linked immunosorbent assay. RESULTS: Activation of PMN by MPO-ANCA-positive IgG preparations compared with PR3-ANCA-positive IgG preparations resulted in greater generation of superoxide anions (MPO-ANCA-positive IgG preparations 9.13 +/- 0.39 nmoles [mean +/- SEM], PR3-ANCA-positive IgG preparations 6.32 +/- 0.35 nmoles; P < 0.001), Ca2+ fluxes (MPO-ANCA-positive IgG preparations 0.735 +/- 0.10, PR3-ANCA-positive IgG preparations 0.33 +/- 0.098; P < 0.01), and MPO degranulation (MPO-ANCA-positive IgG preparations 251.98 +/- 26.7 ng, PR3-ANCA-positive IgG preparations 145.19 +/- 19.4 ng; P < 0.001). The increased activation seen with MPO-ANCA-positive IgG preparations was not due to increased expression of MPO on the cell surface, because following TNFalpha priming PR3 was expressed on significantly more cells than was MPO (PR3 expression 54.2 +/- 5.18%, MPO 31.6 +/- 3.55%; P < 0.001). IgG1 and IgG4 were the predominant isotypes in both MPO-ANCA-positive IgG preparations and PR3-ANCA. MPO-ANCA contained significantly more IgG1 than did PR3-ANCA, and PR3-ANCA-positive IgG preparations contained significantly more IgG3. CONCLUSION: In vitro MPO-ANCA-positive IgG preparations are more activating than PR3-ANCA-positive IgG preparations. The increased activation cannot be explained by increased MPO expression on the cell surface or greater IgG3 present in MPO-ANCA-positive IgG preparations. Differences in activation of PMN by these antibodies may determine some differences between WG and MPA.

Neutrophil priming and apoptosis in anti-neutrophil cytoplasmic autoantibody-associated vasculitis.

BACKGROUND: Interactions between anti-neutrophil cytoplasmic autoantibody (ANCA) and primed neutrophils (PMNs) may be central to the pathogenesis of primary small vessel vasculitis. PMNs from patients are primed, expressing proteinase 3 (PR3) on the cell surface, which permits interaction with ANCA. In vitro ANCA activates primed PMN to degranulate and generate a respiratory burst. Resultant reactive oxygen species are important in triggering apoptosis, but the fate of PMN in ANCA-associated vasculitis is unknown. Failure to remove apoptotic PMN in a nonphlogistic manner may sustain the inflammatory response. METHODS: PMNs from patients or controls were isolated, and the basal production of superoxide was measured by the superoxide dismutase-inhibitable reduction of ferricytochrome C. ANCA antigen expression on apoptotic PMN was assessed at 0, 12, and 18 hours by flow cytometry using dual staining with FITC-conjugated annexin V and PE-conjugated anti-murine IgG against monoclonal ANCA. Apoptosis was also assessed by morphology. In further studies, apoptotic PMNs were opsonized with monoclonal anti-myeloperoxidase (MPO) or anti-proteinase-3 (PR3) or irrelevant isotype-matched IgG (N IgG) and phagocytosis by macrophages was measured using interaction assays. Cytokines interleukin-8 (IL-8) and interleukin-1 were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: Proteinase-3 expression (active 63.04 +/- 5.6% of total number of cells, remission 51.47 +/- 7.9% of total number of cells, control 17.7 +/- 4.7% of total number of cells, P < 0.05) and basal superoxide production (active 6.9 +/- 0.8 nmol/L x 10(6) cells, remission 5.15 +/- 0.4 nmol/L/10(6) cells, control 3.63 +/- 0.3 nmol/L/10(6) cells, P < 0.001) were significantly greater with freshly isolated PMN from patients than controls. PR3 expression and superoxide generation were positively correlated. PMN from patients with active disease became apoptotic at a greater rate than those of controls (at 18 hours, patients 72.3 +/- 3.9% apoptosis, controls 53.2 +/- 2.7% apoptosis, P < 0.05). PR3 and MPO expression were significantly greater on PMN isolated from patients at 12 and 18 hours. Opsonization of apoptotic PMN with ANCA significantly enhanced recognition and phagocytosis by scavenger macrophages (anti-MPO 88.95 +/- 6.27, anti-PR3 93.98 +/- 4.90, N IgG 44.89 +/- 3.44, P < 0.01) with increased secretion of IL-1 (anti-PR3 34.73 +/- 6.8 pg/mL, anti-MPO 42.01 +/- 12.3 pg/mL, N IgG 8.04 +/- 6.3 pg/mL, P < 0.05) and IL-8 (anti-PR3 8.97 +/- 0.93 ng/mL, anti-MPO 8.45 +/- 1.46 ng/mL, N IgG 0.96 +/- 0.15 ng/mL, P < 0.01). CONCLUSION: In vivo circulating PMNs are primed as assessed by PR3 expression and basal superoxide production, thereby enhancing their inflammatory potential. These PMNs undergo apoptosis more readily, at which times they express PR3 and MPO on their surface. These antigens may then provide targets for ANCA. Opsonization of apoptotic PMN will enhance clearance by macrophages but will also trigger the release of pro-inflammatory cytokines that may contribute to chronic inflammation.

Antineutrophil cytoplasm antibody-induced neutrophil nitric oxide production is nitric oxide synthase independent.

BACKGROUND: Antineutrophil cytoplasm antibodies (ANCAs) are implicated in the pathogenesis of systemic vasculitis. We asked whether ANCA could induce nitric oxide (NO) release from human neutrophils and, if so, whether this NO production was dependent on NO synthase (NOS) activity. METHODS: Neutrophil NO production was measured using a chemiluminescence assay, and NOS activity was determined by the conversion of [(14)C] L-arginine to [(14)C] L-citrulline and NOS mRNA expression by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Human neutrophils isolated from healthy donors were incubated at 37 degrees C with human ANCA, normal human IgG, murine monoclonal myeloperoxidase ANCA, murine proteinase-3 ANCA, or their respective isotypic controls for 6 to 12 hours in RPMI. Both human and monoclonal ANCA led to a dose-dependent increase of NO compared with control IgG. Neutrophils, either freshly isolated or incubated for seven hours with murine monoclonal myeloperoxidase ANCA, proteinase-3 ANCA, or a mixture of interleukin-1 beta, tumor necrosis factor-alpha, interferon-gamma plus lipopolysaccharide showed no NOS activity with low conversion rates of [(14)C] L-arginine to [(14)C] L-citrulline, which could not be inhibited by N(G)-monomethyl-L-arginine (NOS inhibitor). To detect NOS mRNA expression, RT-PCR was performed using oligonucleotide primers derived from mRNA sequences of either human constitutive endothelial NOS (eNOS), constitutive neuroneal NOS (nNOS), or human hepatocyte inducible NOS (iNOS). There was no expression of either eNOS, nNOS, or iNOS in untreated, human and murine monoclonal ANCA-treated, or cytokine-treated neutrophils. CONCLUSION: These data suggest that human neutrophils produce NO in response to ANCA but in a NOS-independent way. NO can be generated from a nonenzymatic interaction between hydrogen peroxide and arginine. We postulate that this is the predominant pathway of NO synthesis in neutrophils, since ANCAs are capable of inducing reactive oxygen species production from neutrophils.