Sandro F. Perazzio

Increased neutrophil oxidative burst metabolism in systemic lupus erythematosus

Introduction: There is increased frequency of discoid lesions (2.7%) and SLE (0.5%) in patients with chronic granulomatosus disease, but the literature is still controversial about phagocyte oxidative burst in SLE patients. Materials and methods: 300 SLE patients and 301 blood donors were evaluated for quantitation of the oxidative burst in phagocytes by flow cytometry based on the oxidation of 2,7-dichlorofluorescein-diacetate after stimuli with Staphylococcus aureus and Pseudomonas aeruginosa. Results: Neutrophils from SLE patients displayed higher basal reactive oxygen species (ROS) production than healthy controls [Mean of fluorescence intensity (MFI) = 53.77 ± 11.38 vs 15.08 ± 2.63, p < 0.001] and after stimulation with S. aureus (MFI = 355.46 ± 58.55 vs 151.92 ± 28.25, p < 0.001) or P. aeruginosa (MFI = 82.53 ± 10.1 vs 48.99 ± 6.74, p < 0.001). There was stronger neutrophil response after bacterial stimuli (ΔMFI) in SLE patients than in healthy controls (S. aureus = 301.69 ± 54.42 vs 118.38 ± 26.03, p < 0.001; P. aeruginosa = 28.76 ± 12.3 vs 15.45 ± 5.15, p < 0.001), but no difference with respect to the oxidative burst profile according to disease activity (SLEDAI ≥ 6) or severity (SLICC-DI ≥2). Patients with kidney involvement presented higher basal and stimulated ROS production in neutrophils. Discussion: The present findings corroborate the important role of innate immunity in SLE and implicate neutrophils in the pathophysiology of the disease.

Primary immunodeficiency association with systemic lupus erythematosus: review of literature and lessons learned by the Rheumatology Division of a tertiary university hospital at São Paulo, Brazil

Primary immunodeficiency disorders (PID) represent a heterogeneous group of diseases resulting from inherited defects in the development, maturation and normal function of immune cells; thus, turning individuals susceptible to recurrent infections, allergy, autoimmunity, and malignancies. In this retrospective study, autoimmune diseases (AIDs), in special systemic lupus erythematosus (SLE) which arose associated to the course of PID, are described. Classically, the literature describes three groups of PID associated with SLE: (1) deficiency of Complement pathway components, (2) defects in immunoglobulin synthesis, and (3) chronic granulomatous disease (CGD). Currently, other PID have been described with clinical manifestation of SLE, such as Wiskott-Aldrich syndrome (WAS), autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED), autoimmune lymphoproliferative syndrome (ALPS) and idiopathic CD4(+) lymphocytopenia. Also we present findings from an adult cohort from the outpatient clinic of the Rheumatology Division of Universidade Federal de São Paulo. The PID manifestations found by our study group were considered mild in terms of severity of infections and mortality in early life. Thus, it is possible that some immunodeficiency states are compatible with survival regarding infectious susceptibility; however these states might represent a strong predisposing factor for the development of immune disorders like those observed in SLE.

Differential capacity of therapeutic drugs to induce Rods/Rings structures in vitro and in vivo and generation of anti-Rods/Rings autoantibodies

Some HCV patients using ribavirin and interferon alpha (IFN-α) develop anti-rods and rings (RR) autoantibodies, the main target of which is inosine monophosphate dehydrogenase (IMPDH), the rate-determining enzyme in de novo GTP biosynthesis. In vitro inhibition of IMPDH by ribavirin induces RR formation. Here we investigate whether other commonly used drugs that interfere with GTP biosynthesis can induce RR structures in vitro and vivo and elicit generation of autoantibodies. HEp-2 cells treated for 24h with ribavirin, mycophenolic acid (MPA), azathioprine, methotrexate or acyclovir were positive for RR structures. However, adefovir, entecavir, tenofovir and lamivudine did not induce RR structures in these cells. Structures induced by ribavirin in HEp-2 cells are stable after 24h drug-washout, while structures induced by other drugs are relatively labile, disappearing within 2h. Looking at patients treated with these drugs, HCV patients treated with ribavirin (n=17) showed higher average percentage of RR-positive peripheral mononuclear cells than autoimmune patients treated with RR-inducing immunosuppressant drugs (n=21). Serum from 173 autoimmune patients who had been treated with MPA, azathioprine or methotrexate was tested for presence of anti-RR autoantibodies, and only one sample was found to be positive. Conversely, of 48 anti-RR autoantibody positive samples identified at Fleury Laboratories over 30months, 94% were from HCV patients treated with ribavirin plus IFN-α. These data indicate that RR structures can be induced by a variety of drugs in vitro and in vivo, but anti-RR autoantibody production is mostly restricted to HCV patients under ribavirin+IFN-α treatment.

Quantification of basal digital blood flow and after cold stimulus by laser doppler imaging in patients with systemic sclerosis

OBJECTIVES The objective of this study was to investigate the dynamic behavior of the blood flow of the microvascular circulation of the fingertips before and after two cold stimuli (CS), using Laser Doppler Imaging with different intensities in patients with systemic sclerosis (SSc) and in healthy individuals. PATIENTS AND METHODS Fourteen SSc patients (51.2 ± 5.5 years) with Raynauds phenomenon and 12 healthy controls (44.8 ± 9.0 years) were included in this study. Two CS protocols (submersion of the hands in water at 10 ºC or 15 ºC for 1 minute) were performed on the same day. Mean fingertip blood flow (FBF) of four digits of the left hand was measured using LDI (Moor LDI-VR, Moor Instruments) at baseline and at 1, 4, 10, 25, and 40 minutes after CS. RESULTS Baseline blood flow was significantly lower in both CS protocols in SSc patients when compared to controls (312.9 ± 102.7 vs 465.4 ± 135.4 PU, P = 0.006 at 15 ºC; 305.2 ± 121.0 vs 437.9 ± 119.8 PU; P = 0.01 at 10 ºC). In the control group, a significant decrease in FBF after CS, when compared to baseline, was observed 1 minute (P = 0.001) after CS at 15 ºC and at 1 (P = 0.005) and 25 minutes (P = 0.001) after CS at 10 ºC. In SSc patients, a significant decrease in FBF was observed in both CS protocols at 1, 4, and 10 minutes (P < 0.000; P = 0.002; P = 0.014, after CS at 15 ºC; P < 0.000; P = 0.004; P = 0.001, after CS at 10 ºC). CONCLUSIONS Laser Doppler Imaging showed lower baseline fingertip perfusion and further reduction after CS in SSc patients compared to controls. Quantification of fingertip blood flow by LDI may be useful in the longitudinal monitoring of the disease status and therapeutic interventions in SSc.

CD4(+) T helper cells and regulatory T cells in active lupus nephritis: an imbalance towards a predominant Th1 response?

The objective of this study was to evaluate the frequency of CD4+ T cell subsets in peripheral blood mononuclear cells (PBMC), urine and renal tissue from patients with lupus nephritis (LN). PBMC and urinary cells were collected from 17 patients with active LN, 20 disease controls (DC) with primary glomerulonephritis and 10 healthy controls (HC) and were analysed by flow cytometry with markers for T helper type 1 (Th1), Th2, Th17 and regulatory T cells (Treg) cells. T cell subsets were assessed by immunohistochemistry from LN biopsy specimens from 12 LN patients. T cell subtypes in PBMC were re‐evaluated at 6 months of therapy. CD4+ T cells were decreased in PBMC in LN compared with DC and HC (P = 0·0001). No differences were observed in urinary CD4+ T cell subsets between LN and DC. The frequency of urinary Th17 cells was higher in patients with non‐proliferative than in proliferative LN (P = 0·041). CD3+ and T‐box 21 ( Tbet+ ) cells were found in glomeruli and interstitium of LN patients, while forkhead box protein 3 (FoxP3), retinoid‐related orphan receptor gamma (ROR‐γ) and GATA binding protein 3 (GATA‐3) were present only in glomeruli. Th1 cells in PBMC were correlated negatively with urinary Th1 cells (Rho = –0·531; P = 0·028) and with Tbet in renal interstitium (Rho = –0·782; P = 0·004). At 6 months, LN patients showed an increase in Th17 cells in PBMC. In conclusion, the inverse association between Th1 cells from PBMC and urinary/renal tissue indicate a role for Th1 in LN pathophysiology. Urinary Th17 cells were associated with less severe LN, and Th17 increased in PBMC during therapy. Urinary CD4+ T cells were not different between LN and DC.

High frequency of immunodeficiency-like states in systemic lupus erythematosus: a cross-sectional study in 300 consecutive patients

OBJECTIVE To determine the frequency of immunodeficiency-like states in SLE and related clinical features. METHODS Three hundred and fifteen SLE patients and 301 controls were evaluated for C4A and C4B gene copy number, immunoglobulin isotypes, IgG subclasses, total haemolytic complement (CH50), C2, C3 and neutrophil oxidative burst. C2 and C3 genes were sequenced in cases of low C2 or C3 levels. Those presenting abnormal CH50 with normal C2 and C3 underwent C1q-C9 determination. Patients with active SLE and abnormal results were re-tested after the flare or were excluded if no remission was attained. Fifteen patients were excluded on this basis. Persistent abnormal results characterized an immunodeficiency-like state. RESULTS A significantly higher percentage of SLE patients presented an immunodeficiency-like state compared with controls (28.7% vs 3.3%; P < 0.001), especially low immunoglobulin serum levels. Rigorous testing confirmed only two cases of C2 deficiency carriers among the SLE patients. There were significantly more SLE patients with less than two C4A copies compared with controls. SLE patients had higher frequency of low IgG2, IgG3, IgG4 and IgM levels compared with controls. Patients with low IgG3 or IgG4 presented higher frequency of lupus nephropathy. Patients with low IgM had longer disease duration, older age at SLE onset and lower frequency of oral ulcers. CONCLUSION An immunodeficiency-like state is present in a sizable fraction of SLE patients. Further studies are warranted to determine the impact of these immunodeficiency states and whether they are a primary condition or are secondary to confounding factors, including SLE itself.

High mobility group box 1 serum levels are increased in Behçet’s disease, but not associated with disease activity or disease manifestations

OBJECTIVES High mobility group box 1 (HMGB1) is a nuclear protein that acts as an alarmin when released into the extracellular milieu. HMGB1 is a biomarker of active disease in several systemic autoimmune diseases. Behçets disease (BD) is a systemic inflammatory disorder with a waxing/waning course. The objective of this study is to evaluate serum HMGB1 levels as a possible biomarker for disease activity in BD. METHODS A cross-sectional study measuring serum HMGB1 levels was performed in 26 BD patients and 20 healthy controls. The Brazilian version of the simplified BD Current Activity Form (BR-BDCAFs) was used to measure disease activity. RESULTS Serum HMGB1 levels were higher in patients with active disease [3.82 (2.54-6.11) ng/ml], in patients with BD without active disease but still on therapy [2.76 (1.89-5.78) ng/ml] and in patients in remission without treatment [2.66 (1.86-4.70) ng/ml] than in healthy controls [0.96 (0.59-1.39) ng/ml], P < 0.001. Levels were comparable between BD patients with active disease, BD without active disease but still on therapy and those in remission without treatment (P = 0.432). There was no correlation between serum HMGB1 levels and BR-BDCAF(s) (ρ = 0.195; P = 0.339). No association could be found between serum HMGB1 levels and specific disease involvement or therapy. So serum HMGB1 levels cannot be used as a biomarker in BD. CONCLUSION Serum HMGB1 levels are increased in patients with BD as compared with healthy controls. However, no association was found with disease activity, specific organ involvement or therapy in BD.

Síndrome de Werner associada a quadro esclerodermiforme: relato de caso e revisão da literatura

A sindrome de Werner e uma doenca autossomica recessiva rara associada a envelhecimento precoce, cujo quadro cutâneo deve ser distinguido daquele encontrado na esclerose sistemica (ES). Descrevemos aqui o caso de uma paciente de 39 anos de idade, portadora de sindrome de Werner, encaminhada ao nosso servico com hipotese diagnostica inicial de ES. A paciente apresentava varias manifestacoes associadas a sindrome de Werner, incluindo cabelos precocemente grisalhos, voz estridente, baixa estatura, alteracoes cutâneas esclerodermiformes, diabetes melito, catarata, hipogonadismo, hipotireoidismo e hiperlipidemia. Nao apresentava fenomeno de Raynaud, manifestacoes viscerais tipicas da ES, alteracoes capilaroscopicas periungueais ou auto-anticorpos. O diagnostico de sindrome de Werner, apesar de raro, deve ser lembrado no diagnostico diferencial de ES, principalmente na presenca de manifestacoes atipicas e na ausencia de alteracoes tipicas da ES.