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Aims: To validate an SPE-ultra-HPLC-MS/MS method for thalidomide (THD) measurement in dried plasma spot (DPS). Methods: Extraction included acetonitrile/water clean-up and online SPE. The LOD, LLOQ, linearity, precision, accuracy, recovery, matrix effect, process efficiency, carryover, stability, drug interference and dilution integrity were assessed. Results: The method was linear from 50 to 2000 ng/ml with a LOD of 20 ng/ml and LLOQ of 50 ng/ml. The coefficient of variation for precision was 0.4–7.9% for intra-assay and 1.3–8.9% for interassay, and accuracy was 81.4–97.1%. Adequate matrix effect (100.6–107.0%), recovery (88.7–105.0%) and process efficiency (91.3–109.3%) were registered. DPS was stable for 14 days at room temperature and 45°C ,and for 4 months at -80°C. The method was applied to quantify THD in both wet plasma and DPS from patients with cutaneous lupus receiving THD treatment. The difference between THD wet plasma and DPS concentration was

Background Few prospective studies in cutaneous and systemic lupus erythematosus (CLE/SLE) assessed thalidomide-induced peripheral neuropathy (TiPN) incidence/reversibility, and most have not excluded confounding causes neither monitored thalidomide plasma levels. Objectives To evaluate TiPN incidence/reversibility, coasting effect and its association with thalidomide plasma levels in CLE/SLE. Methods One-year prospective study of thalidomide in 20 CLE/SLE patients without pregnancy potential, with normal nerve conduction study (NCS), and excluded other PN causes. Thalidomide levels were determined by high-performance liquid chromatography/tandem mass spectrometry. Results Twelve patients (60%) developed TiPN: 33.3% were symptomatic and 66.6% asymptomatic. Half of this latter group developed coasting effect (TiPN symptoms 1-3 months after drug withdrawal). The main predictive factors for TiPN were treatment duration ≥6 months (p = 0.025) and cumulative dose (p = 0.023). No difference in plasma thalidomide levels between patients with/without TiPN was observed (p = 0.464). After drug withdrawal, 75% symptomatic TiPN patients improved their symptoms. Seven TiPN patients underwent an additional NCS after drug withdrawal: 42.8% worsened NCS, 14.2% was stable, and 42.8% had improved NCS. Conclusion Our data provides novel evidence of coasting effect in half of asymptomatic patients with TiPN. The irreversible nature of this lesion in 25% of TiPN patients reinforces the relevance of early NCS monitoring, and suggests thalidomide use solely as a bridge for other effective therapy for refractory cutaneous lupus patients.

The American Academy of Ophthalmology (2016-AAO) recommended hydroxychloroquine (HCQ) dose not to exceed 5 mg/kg/day (real body weight). Recently, it was reported that prescribed 2016-AAO dose provided adequate HCQ levels for most lupus nephritis (LN) patients, with low flare risk. However, the minimum HCQ dose required to keep adequate levels is unknown. To evaluate if a further reduction in 2016-AAO dose (2–3 mg/kg/day) would sustain 12-month HCQ levels in LN patients with stable inactive disease. Seventy-three stable LN patients under prescribed full HCQ 2016-AAO dose for ≥6 months and adequate baseline HCQ levels (≥613.5 ng/mL) were divided in two groups: reduced 2016-AAO dose (2–3 mg/kg/day), n = 32, and full 2016-AAO dose (5 mg/kg/day), n = 41. All patients were assessed at baseline, 3, 6, and 12 months. HCQ levels were measured by liquid chromatography-tandem mass spectrometry. Flare was defined as augment ≥ 3 in SLE Disease Activity Index-2000 and/or change in treatment. Rigorous clinical/laboratorial surveillance was performed. Prospective evaluation revealed for reduced 2016-AAO dose group a decrease of HCQ levels from baseline to 3 months (1,404.9 ± 492.0 vs. 731.6 ± 385.0 ng/mL, p 0.05). Prescribed HCQ low-dose regimen (2–3 mg/kg/day) does not sustain, for most patients, 6- and 12-month adequate HCQ levels. Full 2016-AAO dose maintained HCQ levels way above this limit. ClinicalTrials.gov : NCT03122431, registered on April 20, 2017

ObjectivesTo evaluate the effect on immunogenicity and safety of 2-week methotrexate(MTX) discontinuation after each dose of the Sinovac-CoronaVac vaccine versus MTX maintenance in rheumatoid arthritis(RA) patients.MethodsThis was a single-center, prospective, randomized, investigator-blinded, intervention study (#NCT04754698, CoronavRheum), including adult RA patients(stable CDAI10 at D28).ResultsRandomization included 138 patients with 9 exclusions(5 COVID-19, 4 protocol violations). Safety evaluation included 60(MTX-hold) and 69(MTX-maintain) patients. Further exclusions consisted of 27 patients[13(21.7%) vs. 14(20.3%),p=0.848] with positive baseline IgG/NAb and 10 patients(21.3%) in MTX-hold with CDAI>10 at D28. At D69, a higher increase in SC[29(78.4%) vs 30(54.5%),p=0.019] was observed in MTX-hold(n=37) in comparison to MTX-maintain(n=55), with parallel augmentation in GMT[34.2(25.2-46.4) vs 16.8(11.9-23.6),p=0.006]. No differences were observed for NAb positivity[23(62.2%) vs 27(49.1%),p=0.217]. Longitudinal variations in disease activity scores were alike in both groups(CDAI,p=0.144; DAS28-CRP,p=0.718).ConclusionWe provided novel data that 2-week MTX withdrawal after each vaccine dose improves anti-SARS-CoV-2 immunogenicity. The comparable longitudinal variations of disease activity in both groups suggest that discontinuation is a feasible and efficient strategy in well-controlled RA patients, and may be even safer for vaccines with longer interval between doses or single dose schedules.FundingFAPESP/CNPq/B3-Bolsa de Valores-Brasil.

ObjectivesTo evaluate the distinct impact of disease modifying antirheumatic drugs (DMARD) combination and monotherapy in immune response to an inactivated SARS-CoV-2 vaccine in patients with rheumatoid arthritis (RA).MethodsThis phase 4 prospective study analysed seroconversion (SC) of anti-SARS-CoV-2 immunoglobulin G (IgG) and neutralising antibodies (NAb) induced by the inactivated vaccine (CoronaVac) in patients with RA in comparison to controls (CG). Disease activity and treatment were also assessed. Only participants with baseline negative IgG/NAb were included.ResultsPatients with RA (N=260) and CG (N=104) had comparable median ages (59 years (50–65 years) vs 58 years (49.8–64 years), p=0.483). Patients with RA had moderate but lower SC (61.8% vs 94.2%, p0.05). After multivariate analyses, factors independently related to reduced SC were: older age (OR=0.79 (0.70–0.89) for each 5-year interval, p7.5 mg/day (OR=0.38 (0.19–0.74), p=0.004) were negatively related to the presence of NAb. Further comparison of SC/NAb positivity among RA treatment subgroups and CG revealed that methotrexate/tofacitinib/abatacept/tocilizumab use, in monotherapy or in combination, resulted in lower responses (pConclusionsPatients with RA under DMARD have a moderate immunogenicity to CoronaVac. We identified that nearly all DMARD combinations have a deleterious effect in immunogenicity, whereas a more restricted number of drugs (methotrexate/tofacitinib/abatacept/tocilizumab) also hampered this response as monotherapy. These findings reinforce the need of a broader approach, not limited to specific drugs, to improve vaccine response for this population.Trial registration detailsNCT04754698.

There is no study specifically focused on SARS-CoV-2 vaccine in primary Sjögren's syndrome (pSS).To assess the immunogenicity, safety, possible effects on disease activity, and autoantibody profile of the Sinovac-CoronaVac vaccine in pSS.Fifty-one pSS patients and 102 sex- and age-balanced controls without autoimmune diseases were included in a prospective phase 4 trial of the Sinovac-CoronaVac vaccine (two doses 28 days apart, D0/D28). Participants were assessed in three face-to-face visits (D0/D28 and six weeks after the 2nd dose (D69)) regarding adverse effects; clinical EULAR Sjögren's Syndrome Disease Activity Index (clinESSDAI); anti-SARS-CoV-2 S1/S2 IgG (seroconversion (SC) and geometric mean titers (GMT)); neutralizing antibodies (NAb); and pSS autoantibody profile.Patients and controls had comparable female sex frequency (98.0% vs. 98.0%, p = 1.000) and mean age (53.5 ± 11.7 vs. 53.4 ± 11.4 years, p = 0.924), respectively. On D69, pSS patients presented moderate SC (67.5% vs. 93.0%, p 0.001) and GMT (22.5 (95% CI 14.6-34.5) vs. 59.6 (95% CI 51.1-69.4) AU/mL, p 0.001) of anti-SARS-CoV-2 S1/S2 IgG but lower than controls, and also, moderate NAb frequency (52.5% vs. 73.3%, p = 0.021) but lower than controls. Median neutralizing activity on D69 was comparable in pSS (58.6% (IQR 43.7-63.6)) and controls (64% (IQR 46.4-81.1)) (p = 0.219). Adverse events were mild. clinESSDAI and anti-Ro(SS-A)/anti-La(SS-B) levels were stable throughout the study (p 0.05).Sinovac-CoronaVac vaccine is safe in pSS, without a deleterious impact on disease activity, and has a moderate short-term humoral response, though lower than controls. Thus, a booster dose needs to be studied in these patients.ClinicalTrials.gov Identifier: NCT04754698. Key Points • Sinovac-CoronaVac vaccine is safe in pSS, without a detrimental effect on systemic disease activity, and has a moderate short-term humoral response • A booster dose should be considered in these patients.

Background: Coronavirus disease 19 (COVID-19) has an increased risk of coagulopathy with high frequency of antiphospholipid antibodies (aPL). The recent reports of thrombosis associated with the adenovirus-based vaccines raises concern that SARS-CoV-2 immunization in primary antiphospholipid syndrome (PAPS) patients may trigger a dysregulated immune response with possible clotting complications. Therefore, the objectives of this study were to assess immunogenicity, aPL production and safety of Sinovac-Coronavac in PAPS patients.Methods: This prospective controlled phase 4 study of SARS-CoV-2-naïve PAPS patients and a control group (CG) consisted of a two-dose Sinovac-CoronaVac (D0/D28) and blood collection before vaccination (D0), at D28 and 6 weeks after second dose (D69) for immunogenicity/aPL levels. Outcomes were seroconversion (SC) rates of anti-SARS-CoV-2 S1/S2 IgG and/or neutralizing antibodies (NAb) at D28/D69. Safety and aPL production were also assessed. Results: Forty-four PAPS patients and 132 CG had comparable age (p=0.982) and sex (p>0.999). At D69, both groups had high and comparable SC (83.9% vs. 93.5%, p=0.092) and GMT [50.2(95%CI 34.5-73.2) vs. 61.7 (95%CI 52.8-72.3), p=0.249] as well as NAb positivity (77.4% vs. 78.7%, p=0.440), and NAb-activity [64.3% (49.0-77.0) vs. 60.9% (45.6-81.3), p=0.689]. Of note, for D28, the antibody response was very low and also similar in both groups SC (25.8% vs. 30.6% p=0.609). Antiphospholipid levels remained stable throughout the study at D0 vs D28 vs D69 (IgG aCL, p=0.058; IgM aCL, p=0.091; IgG aβ2GPI, p=0.513 and IgM aβ2GPI, p=0.468). Thrombotic event up to 6 months or other moderate/severe side effects were not observed.Conclusions: We provide novel evidence that Sinovac-CoronaVac vaccine has a high immunogenicity and excellent safety profile in PAPS. We further demonstrated that this vaccine did not trigger thrombosis or induced changes in aPL-related antibodies production. Our findings strongly support the recommendation of SARS-CoV-2 vaccination for PAPS patients.Trial registration: ClinicalTrials.gov - Identifier: NCT04754698 first registered on February 8th, 2021.

Objectives To evaluate immunogenicity and safety of an inactivated SARS-CoV-2 vaccine in systemic autoimmune myopathies (SAMs) and the possible influence of baseline disease parameters, comorbidities and therapy on immune response. Methods This prospective controlled study included 53 patients with SAMs and 106 non-immunocompromised control group (CTRL). All participants received two doses of the Sinovac-CoronaVac vaccine (28-day interval). Immunogenicity was assessed by anti-SARS-CoV-2 S1/S2 IgG seroconversion (SC), anti-S1/S2 IgG geometric mean titre (GMT), factor increase GMT (FI-GMT), neutralizing antibodies (NAb) positivity, and median neutralizing activity after each vaccine dose (D0 and D28) and six weeks after the second dose (D69). Participants with pre-vaccination positive IgG serology and/or NAb and those with RT-PCR confirmed COVID-19 during the protocol were excluded from immunogenicity analysis. Results Patients and CTRL had comparable sex (P>0.99) and age (P=0.90). Immunogenicity of 37 patients and 79 CTRL-naïve participants revealed at D69, a moderate but significantly lower SC (64.9% vs 91.1%, P0.05). Conclusion Sinovac-CoronaVac is safe and has a moderate short-term immunogenicity in SAMs, but reduced compared with CTRL. We further identified that immunosuppression is associated with diminished NAb positivity. Trial registration COVID-19 CoronaVac in Patients With Autoimmune Rheumatic Diseases and HIV/AIDS (CoronavRheum), http://clinicaltrials.gov/ct2/show/NCT04754698

Introduction In 2016 the American Academy of Ophthalmology(2016-AAO) recommended a maximum daily HCQ use of 5.0 mg/kg real body weight(RBW) taking into consideration minimizing eye toxicity. Retinopathy in systemic lupus erythematosus(SLE) patients was recently associated with obesity and this condition is progressively more common in these patients. However, the impact of obesity in HCQ blood levels remains controversial. Objective To determine if the 2016-AAO recommendation based on RBW with and without maximum daily dose restriction results in adequate and safe blood levels in obese lupus nephritis(LN) patients. Methods A cross-sectional study was performed with 108 LN patients under the prescribed 2016-AAO dose for at least 3 months. LN patients were assessed for demographic characteristics, body mass index(BMI), disease parameters, HCQ dose, concomitant treatment and HCQ blood levels measured by liquid chromatography-tandem mass spectrometry. Obesity was defined as BMI ≥30kg/m2. Results Obesity was identified in 35/108(32%) LN patients. The calculation of HCQ daily dosage revealed that obese patients were under a lower prescribed daily dose according to the real body weight (RBW) [4.4(2.9-5.4) vs. 4.9(4-5.5)mg/Kg/day, p Conclusion Obese patients under the 2016-AAO prescribed dose of HCQ based on RBW with and without maximum daily dose restriction have a very high HCQ blood levels compared to non-obese patients, with a potential increased risk of ocular toxicity. The use of 2016-AAO dose of HCQ according to the ideal body weight for this group of patients should be considered.Clinicaltrials.gov #NCT0312243.

Background: Limited information is available on response to Covid-19 vaccines in autoimmune rheumatic disease patients(ARD) previously exposed to the SARS-CoV-2. We compared the dynamics of vaccine induced antibody production after immunization with CoronaVac in SARS-CoV-2 - seropositive ARD patients(ARD+) with two age/sex balanced groups: SARS-CoV-2 naive ARD patients(NAIVE-ARD) and SARS-CoV-2-seropositive control group(CTRL+). Methods: Participants of this phase 4 prospective controlled study were vaccinated with two doses of CoronaVac(28-days interval). Primary objective was immunogenicity dynamics evaluated by median neutralizing activity(NAb-activity)/anti-SARS-Cov-2 ln(IgG) titers[ln(IgG)] from D0-D28 and from D28-D69. Secondary objectives included safety and other immunogenicity parameters. Findings: Disease and therapy were similar in ARD+ and NAIVE-ARD groups(p>0·05). A comparable dynamics was observed for ARD+ and CTRL+ with a plateau increase occurring from D0-D28[ARD+, NAb-activity:59·1% to 81·8%, mean difference -12·1%,p=0·002 and anti-S1/S2-GMT:52·3 to 128·9, ln(IgG) mean difference -0·9,p 0·999) and anti-S1/S2-GMT(p 0·999 and D69:-12·3%,p=0·167/0·32%,p=0·258 with minor difference at D28:-13·6%, p=0·067/-0·45,p=0·006. Interpretation: ARD+ patients mount a robust plateau response after a single dose of inactivated SARS-CoV-2 vaccine, independent of pre-existing ARD/therapy, whereas NAIVE-ARD patients require the second dose to ensure a moderate antibody production. Our findings raise the possibility of a single dose regimen in ARD patients previously exposed to SARS-CoV-2.[clinicaltrials.gov#NCT04754698] Funding: FAPESP/CNPq/B3-Bolsa de Valores-Brasil. Declaration of Interest: The authors declare no competing interests. Ethical Approval: The protocol was approved by the National and Institutional Ethical Committee (CAAE: 42566621.0.0000.0068)

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BackgroundHydroxychloroquine (HCQ) blood levels are used to monitor efficacy, safety, and patient adherence during treatment. Oral fluid has emerged as an alternative noninvasive, easily accessible, and low-complexity matrix for drug monitoring. However, there is no analytical method to measure HCQ in oral fluid. Therefore, we developed and validated an ultra-high-performance liquid chromatography-tandem mass (UHPLC-MS/MS) method for the measurement of HCQ and its main metabolites in oral fluid and compared to whole blood.MethodsTen microliters of matrices were used for sample preparation by protein precipitation with acetonitrile followed by online solid phase extraction. The validation process included assessment of lower limit of quantification, linearity, precision, recovery, matrix effect, interferences assessment, carryover, and sample dilution validation.ResultsThe lower limit of quantification was 50 ng/mL for HCQ and metabolites in both oral fluid and whole blood. The calibration curve was linear from 50 to 2000 ng/mL (r2 = 0.999). The coefficient of variation for precision assay was 1.2% to 9.7% for intraday and 1.1% to 14.2% for interday for both HCQ and metabolites in oral fluid and whole blood samples at 150, 750, and 1250 ng/mL. The recovery was 85.3% to 118.5% for 150, 750, and 1250 ng/mL of HCQ and metabolites in both oral fluid and whole blood. Dilution factor up to 5-fold was validated for concentrations higher than the upper limit of quantification.ConclusionsThe validated method is specific, precise, and accurate to determine the analytical range for therapeutic monitoring of HCQ and its main metabolites in oral fluid and blood.

Robust correlation between the severity of rheumatoid arthritis (RA) and cigarette smoking has been clinically demonstrated. Nevertheless, cigarette compounds responsible for this toxic effect and their mechanisms have not been described. Considering that hydroquinone (HQ) is an abundant, pro-oxidative compound of the matter particle phase of cigarette smoke, we investigated whether HQ exposure during the initial phase of collagen-induced arthritis (CIA) could aggravate the disease. For this purpose, male Wistar rats were exposed to aerosolized HQ (25 ppm), saline or 5% ethanol solution (HQ vehicle) for 1 h per day during 14 days. CIA was induced through s.c. injection of bovine collagen Type II (0.4 mg/100 μL) at days seven and 14 of exposure. Clinical signs of disease and the cell profile and chemical mediators in the synovial fluid and membrane were analysed at day 35 after the beginning of exposure. HQ exposure aggravated CIA-related paw edema and increased the cell infiltrate and interleukin-6 (IL-6) levels in the synovial fluid, promoted intense tissue collagen deposition and enhanced synoviocyte proliferation and higher frequency of aryl hydrocarbon receptor (AhR+) and interleukin (IL-17+) neutrophils in the synovial membrane. in vitro data also highlighted that neutrophils expressed increased levels of AhR, IL-17 and reactive oxygen species (ROS) generation. However, only AhR expression and ROS generation were blocked by in vitro treatment with AhR antagonist. Therefore, we conclude that in vivo HQ exposure at the early phase of AR onset worsens RA, leading to high frequency of AhR/IL-17+ neutrophils into the joint.

The genesis of rheumatoid arthritis (RA) is complex and dependent on genetic background and exposure to environmental xenobiotic. Indeed, smoking is associated to developing and worsening pre-existing RA. Nevertheless, the mechanisms and cigarette compounds involved in the harmful processes have not been elucidated. Here, we investigated if the exposure to hydroquinone (HQ), an abundant pro-oxidative compound of cigarette and benzene metabolite, could worsen the ongoing RA. Hence, collagen-induced arthritis (CIA) was induced in male Wistar rats by s.c. injection of 400 μg (200 μL) of bovine collagen type II emulsified in complete Freund's adjuvant on day 1, and a booster injection was performed on day 7. Exposures to nebulized HQ (25 ppm), saline solution or HQ vehicle solution (5% ethanol in saline) were carried out for 1 h, once a day, on days 21–27 after CIA induction. On day 27, animals were euthanized and samples were collected for further analyses. Exposure to HQ caused loss of weight, intensified paw edema, enhanced levels of tumor necrosis factor-α (TNF-α) and anti-citrullinated protein antibody (ACPA) in the serum; augmented synoviocyte proliferation and influx of aril hydrocarbon receptor (AhR) positive cells into the synovial membrane, altered collagen fibre rearrangement in the synovia, and synoviocytes isolated from HQ exposed rats secreted higher levels of pro-inflammatory cytokines, TNF-α and interleukin-1β. Associated, we point out HQ as an environmental pollutant that aggravates RA, suggesting its participation on worsening RA in smoking patients.

Neutrophils are the first line of defence during inflammatory processes; nevertheless, exacerbated influx and actions of neutrophils in terms of uncontrolled inflammation are harmful to the host. Hence, neutrophil activity is the target of drugs seeking to address undesired inflammation. Here, we investigated the mechanisms of action of a ligand of the three isoforms of peroxisome proliferator-activated receptors (PPAR; (5 Z )-5-[(5-bromo-1H-indole-3-yl)methylene]-3-(4-chlorobenzyl)-thiazolidine-2,4-dione), dubbed LYSO-7, on neutrophils activated by N-formyl-l-methionyl-l-leucyl-l-phenylalanine (fMLP), an agonist of G-protein coupled receptors (GPCRs) that binds to membrane-formylated peptide and activates intracellular inflammation pathways. Neutrophils were collected from the peritoneal cavity of male Wistar rats four hours after oyster glycogen injection. Afterwards, the neutrophils were incubated with saline or LYSO-7 (1 or 10 μM, 30 min), washed and stimulated with fMLP (10 −7 μM, 1 h). LYSO-7 treatment inhibited gene and protein expression of adhesion molecules, CD62 L and CD18, abolished adhesion of neutrophils to endothelial cells, impaired chemotaxis, blocked the enhancement of intracellular calcium levels, induced the expression of PPARγ as well as PPARβδ and reduced nuclear translocation of nuclear factor κB (NF-κB). Moreover, topical application of LYSO-7 (10 mM) prior to local application of fMLP (10 −7 μM) diminished the in vivo leukocyte-endothelial interactions in the mesentery microcirculation of rats. Together, our data highlight the effectiveness of anti-inflammatory actions of LYSO-7 on neutrophils activated by GPCRs, depending, at least in part, on impaired of NF-κB activation and induction of PPAR expression.

Novel N-triazolyl maleimide derivatives were synthesized by azide-alkyne Huisgen cycloaddition (1,3-dipolar cycloaddition) and tested for cytotoxicity against a cell line derived from human melanomas SK-Mel-28 and SK-Mel-103, and human umbilical vein endothelial cell lines (HUVEC). The 4l was chose to be biologically tested due to incorporation of benzyl triazolic to the nitrogen of maleimide has not been tested before, and due the satisfactory yield. The analysis of cell metabolism, using the MTT method, showed that the compound 4l impaired cell metabolism in HUVEC only in high concentration (100µM). A lower concentration of compound 4l, whether in association or not with paclitaxel, was required to cause toxicity in both SK-Mel-28 and SK-Mel-103 cells in comparison with HUVEC cells. Moreover, the ability of 4l to cause cell death was evaluated by flow cytometry, and the data obtained highlighted the apoptotic action of 4l and paclitaxel co-treatment on Sk-Mel-28 cells only, which corroborated the greater efficacy of maleimide compounds against cancer cells. Together, our data provide promising data on the selectivity of maleimide compounds to cancer cells, and suggest that novel maleimide-substituted compounds may be synthesized and tested on different cancer cell lines, as primary or co-adjuvant agents of cancer cell toxicity.

We report a method to obtain biaryl dipeptide tyrosine via Suzuki-Miyaura and alkynyl dipeptide tyrosine by Sonogashira cross-coupling reactions. Analysis of the biological action of biaryl dipeptide tyrosine 4d compound showed its ability to impair the metabolism and proliferation of SK-Mel-28 human melanoma lineage cells, independently of mitochondrial membrane depolarization, apoptosis and necrosis. Moreover, 4d compound did not cause toxicity to human umbilical vein endothelial cells (HUVEC), suggesting its toxic specificity to cancer cells.

O TSPO (Translocator protein 18 KDa) é uma proteína intracelular localizada na membrana mitocondrial externa, mas também na membrana citoplasmática, e no núcleo. O TSPO está envolvido na biossíntese de esteroides, proliferação celular, apoptose, estresse oxidativo, e na modulação da inflamação, principalmente no sistema nervoso central, onde a proteína é considerada um marcador da neuroinflamação. Os neutrófilos representam células-chave no processo inflamatório sendo as primeiras células a chegarem no foco inflamatório onde exercem atividades fagocíticas, secretórias e microbicidas. O presente trabalho investigou os efeitos de diferentes ligantes de TSPO Diazepam, Ro5-4864 (agonistas parciais) e PK-11195 (antagonista) na ativação dos neutrófilos in vitro focando na via de ativação do Toll-like receptor (TLR) e de receptores transmembranas ligados a proteína G (GPCR). Neutrófilos obtidos da cavidade peritoneal de camundongos BalbC machos quatro horas após injeção do glicogênio de ostra (1%), foram tratados in vitro com meio de cultura, veículo, Diazepam, Ro5-4864, PK-11195 (10, 100, e 1000 nM), e estimulados ou não com Lipopolissacarídeo (LPS) ou Leucotrieno B4 (LTB4). Foram avaliados em condições basais e após estímulo: a expressão de TSPO e de moléculas de adesão por citometria de fluxo; a migração pelo ensaio de quimiotaxia em placa; a produção de citocinas e do óxido nítrico por ELISA e pela reação de Griess, respectivamente; e finalmente, a geração de espécies reativas de oxigênio por espectrofotômetro de fluorescência. Os resultados obtidos mostram que o TSPO é expresso em neutrófilos em condições basais, e que os estímulos inflamatórios com LPS ou LTB4 não alteram essa expressão. Os ligantes de TSPO não afetam as funções de neutrófilos ativados pelo LPS, salvo a acentuação da geração de espécies reativas (ROS) observada com Ro5-4864 em células estimuladas com LPS. Os neutrófilos estimulados pelo LTB4, quando pré-tratados com os ligantes de TSPO, apresentaram redução na clivagem da L-selectina, redução de quimiotaxia, e indução da geração de ROS. Baseado nestes resultados e nos dados da literatura, concluímos que os efeitos dos ligantes de TSPO sobre as funções neutrofílicas concentram-se na expressão de moléculas de adesão, no estresse oxidativo e na migração. Estes efeitos dependem da via de ativação e do tipo celular. TSPO (Translocator protein 18 kDa) is an intracellular protein located on the out mitochondrial membrane, but also on the cytoplasmatic membrane and in the nucleus. TSPO is involved in endogen steroids substances biosynthesis, cellular proliferation, apoptosis, oxidative stress and in the modulation of inflammatory process, principally in the central nervous system where the protein is a marker of neuroinflammation. Neutrophils are key-cells in the inflammatory process, being the first cell line that reach the inflammatory focus, where they realize their phagocytic, secretory and microbicidal activities. This study assessed the effects of TSPO ligands Diazepam, Ro5-4864 (partial agonists) and PK-11195 (antagonist) on in vitro neutrophils activation, focusing on the Toll-like receptor (TLR) and G protein coupled receptors (GPCRs) pathways. Neutrophils obtained from de peritoneal cavity of male BalbC mouse after four hours of Oyster glycogen injection (1%), were treated in vitro with culture medium, vehicle, Diazepam, Ro5-4864, PK-11195 (10, 100, e 1000 nM) and stimulated or not with lipopolysaccharide (LPS) or Leukotriene B4 (LTB4). We assessed in basal conditions and after stimulus:The TSPO and adhesion molecules proteic expression by flow cytometry; the migration by a plate chemotaxis assay; Nitric oxide and cytokines production by ELISA and the Griess reaction, respectively; and finally the reactive oxygen species generation by a fluorescence spectrophotometer. The results show that TSPO is expressed in neutrophils in basal conditions, and that inflammatory stimulus with LPS and LTB4 did not alter this expression. We also show that TSPO ligands did not affect neutrophil function activated by LPS. However, neutrophils stimulated by LTB4, when pre-treated with TSPO ligands shown a reduced L-selectina cleavage, chemotaxis reduction and induction of ROS generation. Based on these data and in literature data, we concluded that the effects of TSPO ligands in neutrophilic functions is concentrated on adhesion molecules expression, on oxidative stress and on the migration. These effects depend to the activation pathways and to the cellular type.