A GPCR-neuropeptide axis dampens hyperactive neutrophils by promoting an alternative-like polarization during bacterial infection.

The notion that neutrophils exist as a homogeneous population is being replaced with the knowledge that neutrophils adopt different functional states. Neutrophils can have a pro-inflammatory phenotype or an anti-inflammatory state, but how these states are regulated remains unclear. Here, we demonstrated that the neutrophil-expressed G-protein-coupled receptor (GPCR) Mrgpra1 is a negative regulator of neutrophil bactericidal functions. Mrgpra1-mediated signaling was driven by its ligand, neuropeptide FF (NPFF), which dictated the balance between pro- and anti-inflammatory programming. Specifically, the Mrgpra1-NPFF axis counter-regulated interferon (IFN) γ-mediated neutrophil polarization during acute lung infection by favoring an alternative-like polarization, suggesting that it may act to balance overzealous neutrophilic responses. Distinct, cross-regulated populations of neutrophils were the primary source of NPFF and IFNγ during infection. As a subset of neutrophils at steady state expressed NPFF, these findings could have broad implications in various infectious and inflammatory diseases. Therefore, a neutrophil-intrinsic pathway determines their cellular fate, function, and magnitude of infection.

The latency to awake from induced-obstructive sleep apnea is reduced in rats with chronic epilepsy.

OSA is known to increase the risk for SUDEP in persons with epilepsy, but the relationship between these two factors is not clear. Also, there is no study showing the acute responses to obstructive apnea in a chronic epilepsy model. Therefore, this study aimed to characterize cardiorespiratory responses to obstructive apnea and chemoreceptor stimulation in rats. In addition, we analyzed respiratory centers in the brain stem by immunohistochemistry. Epilepsy was induced with pilocarpine. About 30-60 days after the first spontaneous seizure, tracheal and thoracic balloons, and electrodes for recording the electroencephalogram, electromyogram, and electrocardiogram were implanted. Intermittent apneas were made by inflation of the tracheal balloon during wakefulness, NREM sleep, and REM sleep. During apnea, respiratory effort increased, and heart rate fell, especially with apneas made during wakefulness, both in control rats and rats with epilepsy. Latency to awake from apnea was longer with apneas made during REM than NREM, but rats with epilepsy awoke more rapidly than controls with apneas made during REM sleep. Rats with epilepsy also had less REM sleep. Cardiorespiratory responses to stimulation of carotid chemoreceptors with cyanide were similar in rats with epilepsy and controls. Immunohistochemical analysis of Phox2b, tryptophan hydroxylase, and NK1 in brain stem nuclei involved in breathing and sleep (retrotrapezoid nucleus, pre-Bötzinger complex, Bötzinger complex, and caudal raphe nuclei) revealed no differences between control rats and rats with epilepsy. In conclusion, our study showed that rats with epilepsy had a decrease in the latency to awaken from apneas during REM sleep, which may be related to neuroplasticity in some other brain regions related to respiratory control, awakening mechanisms, and autonomic modulation.

Insights into the study and origin of the citrullinome in rheumatoid arthritis.

The presence of autoantibodies and autoreactive T cells to citrullinated proteins and citrullinating enzymes in patients with rheumatoid arthritis (RA), together with the accumulation of citrullinated proteins in rheumatoid joints, provides substantial evidence that dysregulated citrullination is a hallmark feature of RA. However, understanding mechanisms that dysregulate citrullination in RA has important challenges. Citrullination is a normal process in immune and non-immune cells, which is likely activated by different conditions (eg, inflammation) with no pathogenic consequences. In a complex inflammatory environment such as the RA joint, unique strategies are therefore required to dissect specific mechanisms involved in the abnormal production of citrullinated proteins. Here, we will review current models of citrullination in RA and discuss critical components that, in our view, are relevant to understanding the accumulation of citrullinated proteins in the RA joint, collectively referred to as the RA citrullinome. In particular, we will focus on potential caveats in the study of citrullination in RA and will highlight methods to precisely detect citrullinated proteins in complex biological samples, which is a confirmatory approach to mechanistically link the RA citrullinome with unique pathogenic pathways in RA.

An update on autoantibodies in systemic lupus erythematosus.

PURPOSE OF REVIEW: Autoantibodies are cornerstone biomarkers in systemic lupus erythematosus (SLE), an autoimmune disease characterized by autoantibody-mediated tissue damage. Autoantibodies can inform about disease susceptibility, clinical course, outcomes and the cause of SLE. Identifying pathogenic autoantibodies in SLE, however, remains a significant challenge. This review summarizes recent advances in the field of autoantibodies in SLE. RECENT FINDINGS: High-throughput technologies and innovative hypothesis have been applied to identify autoantibodies linked to pathogenic pathways in SLE. This work has led to the discovery of functional autoantibodies targeting key components is SLE pathogenesis (e.g. DNase1L3, cytokines, extracellular immunoregulatory receptors), as well as the identification of endogenous retroelements and interferon-induced proteins as sources of autoantigens in SLE. Others have reinvigorated the study of mitochondria, which has antigenic parallels with bacteria, as a trigger of autoantibodies in SLE, and identified faecal IgA to nuclear antigens as potential biomarkers linking gut permeability and microbial translocation in SLE pathogenesis. Recent studies showed that levels of autoantibodies against dsDNA, C1q, chromatin, Sm and ribosomal P may serve as biomarkers of proliferative lupus nephritis, and identified novel autoantibodies to several unique species of Ro52 overexpressed by SLE neutrophils. SUMMARY: Autoantibodies hold promise as biomarkers of pathogenic mechanisms in SLE.

Granzyme B Induces IRF-3 Phosphorylation through a Perforin-Independent Proteolysis-Dependent Signaling Cascade without Inducing Cell Death.

Granzyme B (GrB) is an immune protease implicated in the pathogenesis of several human diseases. In the current model of GrB activity, perforin determines whether the downstream actions of GrB occur intracellularly or extracellularly, producing apoptotic cytotoxicity or nonapoptotic effects, respectively. In the current study, we demonstrate the existence of a broad range of GrB-dependent signaling activities that 1) do not require perforin, 2) occur intracellularly, and 3) for which cell death is not the dominant outcome. In the absence of perforin, we show that GrB enzymatic activity still induces substoichiometric activation of caspases, which through nonlethal DNA damage response signals then leads to activity-associated phosphorylation of IFN regulatory factor-3. These findings illustrate an unexpected potential interface between GrB and innate immunity separate from the traditional role of GrB in perforin-dependent GrB-mediated apoptosis that could have mechanistic implications for human disease.

The Brazilian Toxoplasma gondii strain BRI caused greater inflammation and impairment in anxiogenic behavior in mice, which was reverted by rosuvastatin treatment.

This study aimed to investigate the effect of rosuvastatin treatment on anxiety-related behavior and short- and long-term memory impairment in mice infected with acute RH and BRI strains of Toxoplasma gondii. Balb/C mice were infected intraperitoneally and after 2 h, oral treatment with rosuvastatin (40 mg/kg/day) was initiated for 4 days. Behaviors related to anxiety and locomotion were evaluated in the open field (OF), and short- and long-term memory through the novel object recognition test (NOR). At the end of the experiments, peritoneal fluid, brain, liver, and lung were collected for T. gondii DNA quantification and histopathological analysis. Infection with BRI strain reduced the dwell time and central locomotion in the OF (p < 0.05), indicating anxiogenic type behavior, while treatment with rosuvastatin reversed this response (p < 0.05). RH strain infection did not alter any behavior in the OF (p > 0.05) and both strains impaired short- and long-term memory (NOR test), but with no significant treatment effect (p > 0.05). The BRI strain was shown to be more damaging in relation to anxiogenic type behavior when compared to the RH strain (p < 0.05), whereas rosuvastatin reduced this damaging effect in BRI. The treatment reduced the parasite load in the peritoneal lavage, liver, and lung of animals infected with both acute strains; however, it significantly (p < 0.05) attenuated the inflammatory process only in BRI-infected and treated animals, showing that non-archetypal genotypes are more damaging in rodents. This suggests that rosuvastatin may be a drug with great therapeutic potential against T. gondii mainly to reduce damage from virulent strains.

Management of early-stage triple-negative breast cancer: recommendations of a panel of experts from the Brazilian Society of Mastology.

BACKGROUND: Triple-negative breast cancer (TNBC) is a heterogenous subtype involving different patterns of behavior and clinical course, demanding a complex, individualized sequence of treatment. The knowledge and attitudes of the affiliated members of the Brazilian Society of Mastology regarding TNBC were evaluated and a consensus regarding management and treatment was reached. METHODS: Affiliates completed a survey involving 44 objective questions. In addition, a specialist meeting was held with 27 experts and 3 ad hoc consultants. The panelists completed the survey before and after brainstorming. Answers achieving 70% of agreement were considered consensual. The chi-square test was used to compare answers between panelists and affiliates and the Kappa coefficient to calculate agreement. RESULTS: Consensus among the panelists increased from 26 (59.1%) to 32 questions (72.7%) following brainstorming (p = 0.17), including 7/10 questions on systemic treatment. Among the affiliates, consensus was achieved for 24 questions (54.5%), resulting in moderate agreement (κ = 0.445). Neoadjuvant chemotherapy should be indicated for almost all cases (except cT1a-b N0) and should include platinum agents. When indicated, immunotherapy is part of the standard of care. The panel reaffirmed the concept of no ink on tumor as indicative of adequate margins and the possibility of sentinel lymph node biopsy for cN1 patients who become cN0 following neoadjuvant therapy. Controversies remain on combining immunotherapy with capecitabine/olaparib in pertinent cases. CONCLUSION: Expert consensus was achieved for > 70% of the questions, with moderate agreement between panelists and affiliates. Educational interventions on systemic breast cancer treatment affected decision-making in 60% of the questions.

Autoantibodies targeting LINE-1-encoded ORF1p are associated with systemic lupus erythematosus diagnosis but not disease activity.

OBJECTIVES: Long Interspersed Element 1 (LINE-1) is an endogenous retroelement that constitutes a significant portion of the human genome and has been implicated in the pathogenesis of systemic lupus erythematosus (SLE). The LINE-1 RNA chaperone protein ORF1p was recently identified as an SLE autoantigen. Here we analyse ORF1p for qualities underlying SLE autoantigen status, compared anti-ORF1p antibodies to markers of SLE disease activity, and performed screening for antibodies against LINE-1 reverse transcriptase ORF2p. METHODS: ORF1p was examined in epithelial cell lines treated with cytotoxic lymphocyte granules and UV irradiation. Anti-ORF1p and anti-ORF2p antibodies were assayed by ELISA and analysed in two SLE cohorts. RESULTS: We found that ORF1p localises to cytoplasmic RNA-containing blebs in apoptotic cells, and is a substrate of the cytotoxic protease granzyme B (GrB). Anti-ORF1p antibodies were present in 4.2% of healthy controls, compared to 15.8% (p=0.0157) and 15.5% (p=0.036) of subjects in the two SLE cohorts. Anti-ORF1p antibodies were not associated with SLE disease activity nor peripheral blood markers of interferon (IFN) activation. Anti-ORF1p titres demonstrated stability over serial time points. Anti-ORF1p antibodies were not associated with anti-DNA, anti-RNP, or other SLE autoantibodies. There was no difference in anti-ORF2p ELISA results in controls versus SLE patients. CONCLUSIONS: LINE-1 ORF1p is a component of apoptotic blebs and a substrate for GrB. Anti-ORF1p antibodies are enriched in SLE subjects but are not associated with dynamic markers of disease activity. These data support a potential role for LINE-1 dysregulation in SLE pathogenesis.

Molecular characterization of HTLV-1 genomic region hbz from patients with different clinical conditions.

The human T-cell lymphotropic virus type-1 (HTLV-1) is associated with severe pathologies, such as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), adult T-cell leukemia-lymphoma (ATLL), and infective dermatitis associated with the HTLV-1 (IDH). Interestingly, HTLV-1 infection does not necessarily imply the development of pathological processes and it is unknown why some patients remain asymptomatic carriers (AC). Despite some mutations in the HTLV-1 genome appear to influence the outcome of HTLV-1, there are few studies that characterize molecularly the hbz region. This study aimed to perform the molecular characterization of hbz gene isolated from patients with different clinical outcomes. A total of 15 sequences were generated and analyzed with 571 sequences previously published. The analises showed that the R119Q mutation seems to be related to HTLV-1 clinical conditions since the frequency of this HBZ mutation is significantly different in comparison between AC with HAM/TSP and ATLL. The R119Q mutation is possibly a protective factor as the frequency is higher in AC sequences.

An overview of sequencing technology platforms applied to HTLV-1 studies: a systematic review.

Human T-lymphotropic virus type 1 (HTLV-1) was the first human retrovirus described. The viral factors involved in the different clinical manifestations of infected individuals are still unknown, and in this sense, sequencing technologies can support viral genome studies, contributing to a better understanding of infection outcome. Currently, several sequencing technologies are available with different approaches. To understand the methodological advances in the HTLV-1 field, it is necessary to organize a synthesis by a rigorous review. This systematic literature review describes different technologies used to generate HTLV-1 sequences. The review follows the PRISMA guidelines, and the search for articles was performed in PubMed, Lilacs, Embase, and SciELO databases. From the 574 articles found in search, 62 were selected. The articles showed that, even with the emergence of new sequencing technologies, the traditional Sanger method continues to be the most commonly used methodology for generating HTLV-1 genome sequences. There are many questions that remain unanswered in the field of HTLV-1 research, and this reflects on the small number of studies using next-generation sequencing technologies, which could help address these gaps. The data compiled and analyzed here can help research on HTLV-1, assisting in the choice of sequencing technologies.

Pulse oximetry curves in healthy children living at moderate altitude: a cross-sectional study from the Ecuadorian Andes.

BACKGROUND: In populations above 3,000 meters above sea level (m.a.s.l.) normal values of oxygen saturation (SpO2) above 90% have been reported. Few studies have been conducted in cities of moderate altitude (between 2,500 and 3,000 m a.s.l). We set out to describe the range of SpO2 values measured with a pulse oximeter in healthy children between 1 month and 12 years of age living in an Ecuadorian Andean city. METHODS: A cross-sectional study was carried out in Quito, Ecuador, located at 2,810 m a.s.l. SpO2 measurement in healthy children of ages ranging from 1 month to 12 years of age residents in the city were recorded by pulse oximetry. Age and gender were recorded, and median and 2.5th and 5th percentile were drawn. Non parametric tests were used to compare differences in SpO2 values by age and gender. RESULTS: 1,378 healthy children were included for the study, 719 (52.2%) males. The median SpO2 for the entire population was 94.5%. No differences were observed between SpO2 median values by age and gender. The 2.5th percentile for global SpO2 measurements was 90%, in children under 5 years of age was 91% and it was 90% in children older than 7. CONCLUSIONS: Our results provide SpO2 values for healthy children from 1 to 12 years old residents in Quito, a city of moderate altitude. The SpO2 percentile curve could contribute as a healthy range for the clinical evaluation of children residing at this altitude.

Accelerated partial breast irradiation: Current status with a focus on clinical practice.

Accelerated partial breast irradiation (APBI), a radiation technique in which only the tumor bed is treated, has now become an acceptable radiation modality for selected early-stage breast cancer patients. Compared to conventional whole breast irradiation (WBI), APBI has some benefits with regard to the reduced total irradiated breast volume and the shorter treatment time. The role of APBI, which can be delivered using diverse techniques, has been evaluated in several prospective randomized phase III trials. These clinical trials demonstrate diverging outcomes relating to local recurrence, while establishing comparable effect in terms of survival between APBI with WBI. The aim of this study was to review the current status of APBI with a focus on clinical practice.

Effects of 12 and 17 cmH2O positive end-expiratory pressure applied after alveolar recruitment maneuver on pulmonary gas exchange and compliance in isoflurane-anesthetized horses.

OBJECTIVE: To compare static compliance (Cst) and alveolar-arterial oxygen tension difference [P(a-a)O2] between positive end-expiratory pressures (PEEP) of 7, 12 and 17 cmH2O applied after an alveolar recruitment maneuver (RM) in isoflurane-anesthetized horses. STUDY DESIGN: Prospective, randomized, clinical study. ANIMALS: A group of 30 healthy adult horses undergoing arthroscopic surgery. METHODS: Animals in dorsal recumbency and mechanically ventilated with a tidal volume of 14 mL kg-1 and 7 cmH2O PEEP (control; n = 6) were subjected to an RM by increasing PEEP from 7 to 22 cmH2O in 5 cmH2O increments at 5 minute intervals, and then decreased similarly to PEEP of 17 (RM17; n = 8), 12 (RM12; n = 8) or 7 cmH2O (RM7; n = 8). Cst and P(a-a)O2 were assessed prior to (baseline) and after the RM at 5, 10, 15, 20, 40, 60 and 80 minutes after achieving each target PEEP, and during recovery from anesthesia. RESULTS: Post-RM improvements on P(a-a)O2 were maintained (baseline versus 80 minutes) in RM12 [216 ± 77 mmHg (28.8 ± 10.3 kPa) versus 194 ± 39 mmHg (25.9 ± 5.2 kPa)] and RM17 [180 ± 86 mmHg (24.0 ± 11.6 kPa) versus 136 ± 75 mmHg [18.2 ± 10.0 kPa]). The improvements on Cst were maintained only in RM12 (0.80 ± 0.13 versus 0.98 ± 0.13 mL cmH2O-1 kg-1). No such improvements were observed in RM7 and control. No significant differences were observed between groups during recovery from anesthesia. CONCLUSIONS: and clinical relevance The 12 and 17 cmH2O PEEP can be used to maintain the improvements on P(a-a)O2 obtained after an RM. Only 12 cmH2O PEEP maintained the post-RM increase on Cst. Such variables were not influenced by the 7 cmH2O PEEP.

Rheumatoid arthritis and citrullination.

PURPOSE OF REVIEW: Dysregulated citrullination is a key element that drives the production and maintenance of antibodies to citrullinated proteins, a hallmark in rheumatoid arthritis (RA). This article reviews recent literature on the origin of citrullinated antigens in RA. RECENT FINDINGS: The study of synovial fluid from patients with RA has provided important insights into the identity of citrullinated proteins that accumulate in the RA joint (the RA citrullinome) and mechanisms that control their generation. SUMMARY: Citrullinating enzymes (peptidylarginine deiminases, PADs) are tightly controlled to limit their hyperactivation. Calcium and redox conditions are important regulators of PAD activity. Studies suggest that citrullination is dysregulated both intra- and extracellularly in RA. In neutrophils, host (i.e., perforin and the membrane attack complex) and bacterial (i.e., toxins) pore-forming proteins induce prominent calcium influx, cytolysis, and hyperactivation of PADs. These factors likely drive hypercitrullination in the RA joint and at extraarticular sites of disease initiation, respectively. As oxidizing conditions present in the extracellular environment are known to inactivate PADs, extracellular citrullination in RA probably requires the constant release of active enzymes from dying cells and may be accelerated by autoantibodies that activate PADs.

Affinity maturation shapes the function of agonistic antibodies to peptidylarginine deiminase type 4 in rheumatoid arthritis.

OBJECTIVES: The citrullinating enzyme peptidylarginine deiminase type 4 (PAD4) is the target of a polyclonal group of autoantibodies in patients with rheumatoid arthritis (RA). A subgroup of such antibodies, initially identified by cross-reactivity with peptidylarginine deiminase type 3 (PAD3), is strongly associated with progression of radiographic joint damage and interstitial lung disease and has the unique ability to activate PAD4. The features of these antibodies in terms of their T cell-dependent origin, genetic characteristics and effect of individual antibody specificities on PAD4 function remain to be defined. METHODS: We used PAD4 tagged with the monomeric fluorescent protein mWasabi to isolate PAD4-specific memory B cells from anti-PAD4 positive patients with RA and applied single cell cloning technologies to obtain monoclonal antibodies. RESULTS: Among 44 single B cells, we cloned five antibodies with PAD4-activating properties. Sequence analysis, germline reversion experiments and antigen specificity assays suggested that autoantibodies to PAD4 are not polyreactive and arise from PAD4-reactive precursors. Somatic mutations increase the agonistic activity of these antibodies at low calcium concentrations by facilitating their interaction with structural epitopes that modulate calcium-binding site 5 in PAD4. CONCLUSIONS: PAD4-activating antibodies directly amplify a key process in disease pathogenesis, making them unique among other autoantibodies in RA. Understanding the molecular basis for their functionality may inform the design of future PAD4 inhibitors.

Can short-term pasture management increase C balance in the Atlantic Rainforest?

Few studies have shown the importance of different pasture management practices on C storage and the reduction of CO2-C emissions in tropical conditions. The objective of the present study was to determine short-term changes in C pools and C balance from different pasture management practices established in the Atlantic Rainforest. A field study was carried out in Alegre, ES, Brazil from September 2013 to August 2014 to investigate the first-year effect of pasture management practices on a Udult clayey soil. The different pasture management practices studied included the following: control (CON), chiseled (CHI), fertilized (FER), burned (BUR), integrated with crop-livestock (iCL) systems, and plowed and harrowed (PH). Monthly disturbed and undisturbed soil samples were taken at two different layers (0.00-0.05 and 0.05-0.20 m) for chemical, physical, and organic matter characterization. C inputs monitored in aboveground pools included plant aerial parts and litter, and belowground pools included roots and soil C stocks. C outputs monitored were CO2-C emissions, erosion water, and sediment. C balance was considered the difference between inputs and outputs in each treatment during four seasons. The spring and summer seasons had a strong influence on C inputs and outputs where there is significant difference between spring and summer, while the autumn and winter seasons had less influence. All pasture management practices exhibited positive C balance after 1 year. High values of C balance were verified in pastures fertilized (FER) (53.04 Mg ha-1 year-1. Intermediate C balance was found in the burned (BUR) (40.84 Mg ha-1 year-1), traditional control (CON) (40.31 Mg ha-1 year-1), and in the plowing and harrowing (PH) (40.02 Mg ha-1 year-1) management practices. The practices of chiseled (40.00 Mg ha-1 year-1) and integrated crop-livestock systems (iCL) (59.06 Mg ha-1 year-1) resulted in low C balance.

Ventilation distribution assessed with electrical impedance tomography and the influence of tidal volume, recruitment and positive end-expiratory pressure in isoflurane-anesthetized dogs.

OBJECTIVE: To examine the intrapulmonary gas distribution of low and high tidal volumes (VT) and to investigate whether this is altered by an alveolar recruitment maneuver (ARM) and 5 cmH2O positive end-expiratory pressure (PEEP) during anesthesia. STUDY DESIGN: Prospective randomized clinical study. ANIMALS: Fourteen client-owned bitches weighing 26 ± 7 kg undergoing elective ovariohysterectomy. METHODS: Isoflurane-anesthetized dogs in dorsal recumbency were ventilated with 0 cmH2O PEEP and pressure-controlled ventilation by adjusting the peak inspiratory pressure (PIP) to achieve a low (7 mL kg-1; n = 7) or a high (12 mL kg-1; n = 7) VT. Ninety minutes after induction (T90), an ARM (PIP 20 cmH2O for 10 seconds, twice with a 10 second interval) was performed followed by the application of 5 cmH2O PEEP for 35 minutes (RM35). The vertical (ventral=0%; dorsal=100%) and horizontal (right=0%; left=100%) center of ventilation (CoV), four regions of interest (ROI) (ventral, central-ventral, central-dorsal, dorsal) identified in electrical impedance tomography images, and cardiopulmonary data were analyzed using two-way repeated measures anova. RESULTS: The low VT was centered in more ventral (nondependent) areas compared with high VT at T90 (CoV: 38.8 ± 2.5% versus 44.6 ± 7.2%; p = 0.0325). The ARM and PEEP shifted the CoV towards dorsal (dependent) areas only during high VT (50.5 ± 7.9% versus 41.1 ± 2.8% during low VT, p = 0.0108), which was more distributed to the central-dorsal ROI compared with low VT (p = 0.0046). The horizontal CoV was centrally distributed and cardiovascular variables remained unchanged throughout regardless of the VT, ARM, and PEEP. CONCLUSIONS AND CLINICAL RELEVANCE: Both low and high VT were poorly distributed to dorsal dependent regions, where ventilation was improved following the current ARM and PEEP only during high VT. Studies on the role of high VT on pulmonary complications are required.

Antibodies to native and citrullinated RA33 (hnRNP A2/B1) challenge citrullination as the inciting principle underlying loss of tolerance in rheumatoid arthritis.

BACKGROUND: Anti-citrullinated protein antibodies (ACPAs) are the hallmark of rheumatoid arthritis (RA). Protein citrullination is believed to drive autoantigen selection in RA. Nonetheless, several autoantigens in RA are targeted as native (unmodified) proteins. Here, the study of hnRNP A2/B1 (RA33) provides a framework to understand the humoral response to native and citrullinated autoantigens in RA. METHODS: RA synovial fluid (SF) cells were analysed by immunoblotting and mass spectrometry. RA33 was cloned from RASF cells and splice variants expressed as recombinant proteins. Antibodies against native and citrullinated RA33 were characterised by ELISA, immunoblotting and immunoprecipitation. RESULTS: RA33 is citrullinated in the rheumatoid joint and targeted either as a citrullinated or native protein in distinct patient subsets with RA. A novel splice variant (hnRNP B1b) previously associated with disease initiation in experimental arthritis was identified in the RA joint and acts as the major target of the anti-RA33 response. Antibodies exclusively targeting citrullinated RA33 were positively associated with disease duration and erosive disease. In contrast, anti-(native) RA33 antibodies were detected almost exclusively in early RA and identified patients with low radiographic erosion scores. Finally, a unique subset of double-reactive patients demonstrated intermediate severity, but rapid disease progression, suggesting a transitional disease phase in the evolution of an anti-native protein antibody to ACPA response in RA. CONCLUSIONS: These data suggest that native and citrullinated proteins targeted by autoantibodies in RA may be part of a single antibody system and challenge the paradigm of citrullination as the unifying principle underlying loss of tolerance in RA.

Determination of trace levels of triazines in corn matrices by bar adsorptive microextraction with a molecularly imprinted polymer.

This manuscript addresses the determination of triazines (ametryn, atrazine, simazine, and terbutryn) in corn matrices using bar adsorptive microextraction coated with a selective molecularly imprinted polymer phase following microliquid desorption and high-performance liquid chromatography with diode array detection. The molecularly imprinted polymer was synthesized using atrazine as a template and methacrylic acid as a functional monomer. Assays performed in 25 mL of ultrapure water samples spiked at 8.0 µg/L yielded 80-120 % recoveries under the evaluated experimental conditions. The method showed an accuracy (0.2 < bias < 17.9%), precision (relative standard deviation <17.4%), convenient detection (0.2 µg/L), and quantification (0.7 µg/L) limits, as well as linear dynamic ranges (0.8-24.0 µg/L) with remarkable determination coefficients (R(2) > 0.9926). The proposed analytical method was applied to monitor triazines in three types of corn matrices using the standard addition methodology. Experiments performed in corn samples spiked with triazines at the trace level (8.0 µg/kg of each analyte) gave rise to recoveries (81.0-119.4%) with good reproducibility and robustness. The proposed methodology is also easy to implement and showed to be a good analytical alternative to monitor triazines in complex matrices, when compared with other sorption-based microextraction techniques.

Determination of pesticides in sugarcane juice employing microextraction by packed sorbent followed by gas chromatography and mass spectrometry.

This paper describes the development of a method for the determination of six pesticides (tebuthiuron, carbofuran, atrazine, metribuzine, ametryn, and bifenthrin) in sugarcane juice using microextraction by packed sorbent as the extraction technique. The extraction steps were optimized by factorial design, being the variables pH, ionic strength, desorption solvent and solvent volume optimized for comparisons among sorbent materials. Among the evaluated materials C18-Chromabond(®) showed better extraction efficiency. A factorial design 2(3) with central point was used for the extraction cycles optimization. Draw/eject and washes cycles showed significant improvements in the extraction efficiency when the number of cycles increased. The method was validated and showed a limit of quantification in the range of 2.0-10.0 µg.L(-1) . The calibration curves were constructed by weighting models that reduced the sum of absolute residues values and improved determination coefficient. The matrix factor and extraction efficiency were 97.3-77.3% and 27.1-64.8%, respectively. The accuracy was 71.7-106.9%; precision evaluated as the coefficient of variance obtained in intra and inter day analysis was 4.5-15.9%. The method was applied to the determination of pesticide residues in four sugarcane juice samples commercially available in markets from different cities from São Paulo state, Brazil.