Explore the vast range of titles available.

Background Autophagy has emerged as a key mechanism in the survival and function of T and B lymphocytes, and its activation was involved in apoptosis resistance in rheumatoid arthritis (RA). To investigate whether the relationship between autophagy and apoptosis may impact the response to the therapy, we analyzed ex vivo spontaneous autophagy and apoptosis in patients with RA subjected to treatment with anti-tumor necrosis factor (TNF) drugs and in vitro the effects of TNFα and anti-TNF drugs on cell fate. Methods Peripheral blood mononuclear cells (PBMCs) from 25 RA patients treated with anti-TNF drugs were analyzed for levels of autophagy marker LC3-II by western blot and for the percentage of annexin V-positive apoptotic cells by flow cytometry. The same techniques were used to assess autophagy and apoptosis after in vitro treatment with TNFα and etanercept in both PBMCs and fibroblast-like synoviocytes (FLS) from patients with RA. Results PBMCs from patients with RA responsive to treatment showed a significant reduction in LC3-II levels, associated with an increased apoptotic activation after 4 months of therapy with anti-TNF drugs. Additionally, the expression of LC3-II correlated with DAS28. TNFα was able to induce autophagy in a dose-dependent manner after 24 h of culture in RA PBMCs and FLS. Moreover, etanercept caused a significant reduction of autophagy and of levels of citrullinated proteins. Conclusions Our results show how the crosstalk between autophagy and apoptosis can sustain the survival of immune cells, thus influencing RA progression. This suggests that inhibition of autophagy represents a possible therapeutic target in RA.

Psoriatic arthritis (PsA) is a chronic inflammatory arthritis associated with psoriasis. A third of psoriatic patients develop PsA unknown mechanisms. No reliable diagnostic markers are available for PsA, or prognostic biomarkers for PsA development in psoriasis. We previously uncovered a pro-inflammatory role for cathelicidin LL37 in lesional psoriasis skin. LL37 binds nucleic acids and stimulates plasmacytoid/myeloid dendritic cells (pDC, mDCs) to secrete type I interferon (IFN-I) and pro-inflammatory factors. LL37 becomes an autoantigen for psoriatic Th1-Th17/CD8 T cells. Anti-LL37 antibodies were detected in systemic lupus erythematosus, an autoimmune disease characterized by neutrophil-extracellular-traps release (NETosis) in target organs. LL37 can be substrate of irreversible post-translational modifications, citrullination or carbamylation, linked to neutrophil activity. Here we analyzed inflammatory factors, included LL37, in PsA and psoriasis plasma and PsA synovial fluids (SF)/biopsies. We show that LL37 (as a product of infiltrating neutrophils) and autoantibodies to LL37 are elevated in PsA, but not OA SF. Anti-LL37 antibodies correlate with clinical inflammatory markers. Anti-carbamylated/citrullinated-LL37 antibodies are present in PsA SF/plasma and, at lower extent, in psoriasis plasma, but not in controls. Plasma anti-carbamylated-LL37 antibodies correlate with PsA (DAS44) but not psoriasis (PASI) disease activity. Ectopic lymphoid structures, and deposition of immunoglobulin-(Ig)G-complexes (IC) co-localizing with infiltrating neutrophils, are observed in PsA and not OA synovial tissues (ST). Activated complement (C5a, C9), GM-CSF and IFN-I are up-regulated in PsA and not OA synovia and in PsA and psoriasis plasma but not in HD. C9 and GM-CSF levels in PsA SF correlate with clinical inflammatory markers and DAS44 (C9) and with anti-carbamylated/citrullinated-LL37 antibodies (GM-CSF and IFN-I). Thus, we uncover a role for LL37 as a novel PsA autoantibody target and correlation studies suggest participation of anti-LL37 antibodies to PsA pathogenesis. Notably, plasma antibodies to carbamylated-LL37, which correlate with DAS44, suggest their use as new disease activity markers. GM-CSF and complement C5a and C9 elevation may be responsible for autoantigens release by neutrophils and their modification, fueling inflammation and autoreactivity establishment. Finally, targeting GM-CSF, C5a, C9 can be beneficial in PsA.

Most plant species depend upon insect pollination services, including many cash and subsistence crops. Plants compete to attract those insects using visual cues and floral odor which pollinators associate with a reward. The cacao tree, Theobroma cacao, has a highly specialized floral morphology permitting pollination primarily by Ceratopogonid midges. However, these insects do not depend upon cacao flowers for their life cycle, and can use other sugar sources. To understand how floral cues mediate pollination in cacao we developed a method for rearing Ceratopogonidae through several complete lifecycles to provide material for bioassays. We carried out collection and analysis of cacao floral volatiles, and identified a bouquet made up exclusively of saturated and unsaturated, straight-chain hydrocarbons, which is unusual among floral odors. The most abundant components were tridecane, pentadecane, (Z)-7-pentadecene and (Z)-8-heptadecene with a heptadecadiene and heptadecatriene as minor components. We presented adult midges, Forcipomyia sp. (subgen. Forcipomyia), Culicoides paraensis and Dasyhelea borgmeieri, with natural and synthetic cacao flower odors in choice assays. Midges showed weak attraction to the complete natural floral odor in the assay, with no significant evidence of interspecific differences. This suggests that cacao floral volatiles play a role in pollinator behavior. Midges were not attracted to a synthetic blend of the above four major components of cacao flower odor, indicating that a more complete blend is required for attraction. Our findings indicate that cacao pollination is likely facilitated by the volatile blend released by flowers, and that the system involves a generalized odor response common to different species of Ceratopogonidae.

The pathway of Janus tyrosine kinases (JAKs) has a central role in the pathogenesis of Rheumatoid Arthritis (RA) by regulating multiple immune functions and cytokine production. The JAK inhibitor tofacitinib is effective in RA patients not responding to methotrexate or TNF-inhibitors. Since hyperactive autophagy has been associated with impaired apoptosis of RA fibroblast-like synoviocytes (FLS), we aimed to investigate the role of tofacitinib in modulating autophagy and apoptosis in these cells. FLS isolated from RA biopsies were cultured with tofacitinib in presence of autophagy inducer rapamycin and in serum deprivation condition. Levels of autophagy, apoptosis, and citrullinated proteins were analyzed by western blot, flow cytometry, immunocytofluorescence, and Real-Time PCR. Rapamycin induced an increase in RA-FLS autophagy while the levels of autophagy marker LC3-II were reduced after in vitro treatment with tofacitinib. The analysis of autophagic flux by specific fluorescence dye confirmed the reduction of autophagy in RA FLS. The treatment with tofacitinib did not influence apoptosis of RA FLS. Modulation of the autophagic process by tofacitinib did not significantly change citrullination. The results of this study demonstrate that tofacitinib is able to modulate autophagy of FLS contributing to its effectiveness in RA patients.

Background/Objectives: The genomic bases of the adipose tissue abnormalities induced by chronic positive calorie excess have been only partially elucidated. We adopted a genome-wide approach to directly test whether long-term high-fat diet (HFD) exposure affects the DNA methylation profile of the mouse adipose tissue and to identify the functional consequences of these changes. Subjects/Methods: We have used epididymal fat of mice fed either high-fat (HFD) or regular chow (STD) diet for 5 months and performed genome-wide DNA methylation analyses by methylated DNA immunoprecipitation sequencing (MeDIP-seq). Mouse Homeobox ( Hox ) Gene DNA Methylation PCR, RT-qPCR and bisulphite sequencing analyses were then performed. Results: Mice fed the HFD progressively expanded their adipose mass accompanied by a significant decrease in glucose tolerance ( P <0.001) and insulin sensitivity ( P <0.05). MeDIP-seq data analysis revealed a uniform distribution of differentially methylated regions (DMR) through the entire adipocyte genome, with a higher number of hypermethylated regions in HFD mice ( P <0.005). This different methylation profile was accompanied by increased expression of the Dnmt3a DNA methyltransferase (Dnmt; P <0.05) and the methyl-CpG-binding domain protein Mbd3 ( P <0.05) genes in HFD mice. Gene ontology analysis revealed that, in the HFD-treated mice, the Hox family of development genes was highly enriched in differentially methylated genes ( P =0.008). To validate this finding, Hoxa5 , which is implicated in fat tissue differentiation and remodeling, has been selected and analyzed by bisulphite sequencing, confirming hypermethylation in the adipose tissue from the HFD mice. Hoxa5 hypermethylation was associated with downregulation of Hoxa5 mRNA and protein expression. Feeding animals previously exposed to the HFD with a standard chow diet for two further months improved the metabolic phenotype of the animals, accompanied by return of Hoxa5 methylation and expression levels ( P <0.05) to values similar to those of the control mice maintained under standard chow. Conclusions: HFD induces adipose tissue abnormalities accompanied by epigenetic changes at the Hoxa5 adipose tissue remodeling gene.

Background We assessed the impact of two sand fly insecticide interventions (insecticide spraying and insecticide-impregnated dog collars) on the peridomestic abundance and distribution of mosquitoes (Culicidae) and biting midges (Ceratopogonidae) in western São Paulo (Brazil) in a long-term (42-month) evaluation. Both of these dipteran groups are vectors of diseases of medical and veterinary relevance to humans and domestic animals in Brazil. Methods The interventions in the 3-arm stratified randomised control trial were: pheromone + insecticide (PI) (chicken roosts were sprayed with microencapsulated lambda-cyhalothrin; pheromone lure has no effect on the Diptera pests studied here); dog-collars (DC) (dogs fitted with deltamethrin-impregnated collars); and control (C) (unexposed to pyrethroids) were extended by 12 months. During that time, adult mosquitoes and midges were sampled along 280 households at three household locations (inside human dwellings, dog sleeping sites and chicken roosts). Results We collected 3145 culicids (9 genera, 87.6% Culex spp.) distributed relatively uniformly across all 3 arms: 41.9% at chicken roosts; 37.7% inside houses; and 20.3% at dog sleeping sites. We collected 11,464 Culicoides (15 species) found mostly at chicken roosting sites (84.7%) compared with dog sleeping sites (12.9%) or houses (2.4%). Mosquitoes and Culicoides were most abundant during the hot and rainy season. Increased daytime temperature was marginally associated with increased mosquito abundance ( Z  = 1.97, P  = 0.049) and Culicoides abundance ( Z  = 1.71, P  = 0.087). There was no significant association with daily average rainfall for either group. Household-level mosquito and midge numbers were both significantly reduced by the PI intervention 56% [incidence rate ratio, IRR = 0.54 (95% CI: 0.30–0.97), P  ≤ 0.05] and 53% [IRR = 0.47 (95% CI: 0.26–0.85), P  ≤ 0.05], respectively, compared to the control intervention. The abundance of both dipteran groups at dog sleeping sites was largely unaffected by the PI and DC interventions. The PI intervention significantly reduced abundance of mosquitoes inside houses (41%) and at chicken roosting sites (48%) and reduced midge abundance by 51% in chicken roosting sites. Conclusions Sprayed insecticide at chicken roosting sites reduced the abundance of mosquitoes and midges at the peridomestic level while dog collars had no effect on numbers for any group.

Short rotation poplar plantations grow on flat and even terrain, and the interrow spacing is wide enough for easy machine access. If the terrain is firm enough, one may consider moving the classic roadside chipping operation directly into the field (i.e., terrain chipping), thus saving on wood extraction cost. This study compared the efficiency and cost of roadside and terrain chipping conducted with exactly the same equipment, to assess the benefits offered by the versatile deployment of a standard chipping operation, whereby the operation can be moved inside the stand whenever terrain conditions are suitable. The study was conducted at 12 sample plots, containing about one truck load of chips each (i.e., approximately 11 bone-dry tons or BDT). Plots were arranged as alternate windrows on a 8.5 ha field. Data was collected for the whole supply chain, from field to factory. The factory was located 14 km from the field. Delivered cost was 53 € BDT-1 and 70 € BDT-1 for roadside and terrain chipping, respectively, i.e., terrain chipping was 1/3 more expensive than roadside chipping, even if the latter included the additional cost of forwarding the residues to the roadside. The chipper-truck used for the test could not cope with small scattered residue piles (32 BDT ha-1), and the cumbersome reposition maneuver became the main hurdle to efficient operation. Further improvements might be achieved by pre-bunching the residues, introducing a dedicated terrain chipper or bundling the residues and taking the bundles to the factory for centralized chipping.

Background Thrombocytopenia is a manifestation associated with primary antiphospholipid syndrome (PAPS), and many studies have stressed the leading role played by platelets in the pathogenesis of antiphospholipid syndrome (APS). Platelets are highly specialized cells, and their activation involves a series of rapid rearrangements of the actin cytoskeleton. Recently, we described the presence of autoantibodies against D4GDI (Rho GDP dissociation inhibitor beta, ARHGDIB) in the serum of a large subset of SLE patients, and we observed that anti-D4GDI antibodies activated the cytoskeleton remodeling of lymphocytes by inhibiting D4GDI and allowing the upregulation of Rho GTPases, such as Rac1. Proteomic and transcriptomic studies indicate that D4GDI is very abundant in platelets, and small GTPases of the RHO family are critical regulators of actin dynamics in platelets. Methods We enrolled 38 PAPS patients, 15 patients carrying only antiphospholipid antibodies without clinical criteria of APS (aPL carriers) and 20 normal healthy subjects. Sera were stored at − 20 °C to perform an ELISA test to evaluate the presence of anti-D4GDI antibodies. Then, we purified autoantibodies anti-D4GDI from patient sera. These antibodies were used to conduct in vitro studies on platelet activation. Results We identified anti-D4GDI antibodies in sera from 18/38 (47%) patients with PAPS, in sera from 2/15(13%) aPL carriers, but in no sera from normal healthy subjects. Our in vitro results showed a significant 30% increase in the activation of integrin αIIbβ3 upon stimulation of platelets from healthy donors preincubated with the antibody anti-D4GDI purified from the serum of APS patients. Conclusions In conclusion, we show here that antibodies anti-D4GDI are present in the sera of PAPS patients and can prime platelet activation, explaining, at least in part, the pro-thrombotic state and the thrombocytopenia of PAPS patients. These findings may lead to improved diagnosis and treatment of APS.

Background:It is reported that up to 90.4% of individuals diagnosed with Rheumatoid Arthritis (RA) seek medical assistance due to severe pain. The underlying causes of this pain are multifaceted, involving factors such as inflammation, secondary osteoarthritis, as well as central and peripheral sensitization. CGRP (calcitonin gene-related peptide) is a peptide exerting nociceptive and vasodilatory effects and a debated immunomodulatory effect; inflammatory arthropathies have been associated a local increase of CGRP release and CGRP seems to increase IL6 and IL8 secretion from fibroblast-like synoviocytes isolated from RA patients.Objectives:The aim of this prospective pilot study was to determine whether patients with RA have detectable levels of circulating CGRP, to investigate the correlation between CGRP and with pain levels reported by the patients and to assess the effect of baricitinib on pain and CGRP levels.Methods:We enrolled RA patients starting treatment with baricitinib for high-to-moderate active RA. At baseline and after 4 and 12 weeks of treatment we collected clinical data (number of tender and swollen joints), Erytrhosedimentation Rate (ESR), C Reactive Protein (CRP) levels, and patients reported outcomes [Patients Global Assessment (PGA) and pain on a 0-10 cm Visual Analogic Scale (VAS)]. CGRP serum levels were assessed in serum from RA patients at baseline and after 4 and 12 weeks of treatment with baricitinib using an ELISA kit. Data were expressed as mean ± standard deviation or median (IQR) according to distribution. Mann-Whitney and Spearman tests were performed for comparisons and p values < 0.05 were considered statistically significant.Results:We enrolled 43 patients (F:M =36:7, median age=58, IQR 11 years, median disease duration =144, IQR 150) starting baricitinib. We defined responders (R) patients those who achieved at least a EULAR moderate response (1.2 point reduction) of DAS28_CRP from baseline value and not-responders (NR) those who did not. At baseline pain VAS score did not differ significantly between R and NR. Already after 4 weeks of treatment all patients showed a significant reduction of pain (median pain score was 8(2) at baseline, 4(5) and 2(5) after 4 and 12 weeks, respectively; p=0.0014 and p<0.0001 vs baseline, respectively). R patients had lower pain VAS scores after 4 and 12 weeks of follow-up (p=0.0038 and p=0.0026) compared to NR. CGRP was significantly reduced in the whole cohort at 4 (p=0.0016) and 12 weeks (p=0.018) (Figure 1a). NR patients showed higher CGRP serum levels compared to R, after one month of baricitinib. Levels of CGRP significantly correlated with pain VAS (p=0.0152, r=0.22) (Figure 1b) and PGA (p=0.02, r=0.21), but not with ESR, CRP or disease activity (DAS28_CRP).Conclusion:With the same disease activity, we found higher levels of CGRP in active RA patients with higher levels of pain. Interestingly, CGRP was reduced to a greater extent in patients who responded to the JAK inhibitor. This preliminary result sheds light on a possible additional mechanism of baricitinib efficacy, linked to modulation of neurotransmission other than to control of inflammation. On the other hand, in NR patients, other mechanisms underlying pain may contribute to non-response to therapy.REFERENCES:[1] Walsh DA, et al. Nat Rev Rheumatol. Oct 2014;10(10):581-92.[2] Russell FA, et al. Physiol Rev. Oct 2014;94(4):1099-142.[3] Raap T, et al. J Rheumatol. Nov 2000;27(11):2558-65.Figure 1.a. Reduction of serum CGRP in patients treated with baricitinib, from baseline (T0) and after 1 and 3 months of treatment (T1, T3). b. correlation between levels of pain and serum CGRP.Acknowledgements:NIL.Disclosure of Interests:Cristina Garufi: None declared, Silvia Mancuso: None declared, Letizia Caruso: None declared, Fulvia Ceccarelli: None declared, Simona Truglia: None declared, Fabrizio Conti Eli Lilly, Abbvie, UCB, Pfizer, Galapagos, Francesca Romana Spinelli Eli Lilly, Abbvie, UCB, Galapagos

Background:The new classification criteria for antiphospholipid antibody syndrome (APS) have introduced many novelties, such as the presence of an entry criterion, the use of a scoring system, the introduction of previously “extra-criteria” manifestations, the weighting of thrombotic events according to thromboembolic and cardiovascular risk factors and the redefinition of obstetric APS (OAPS).Objectives:The aim of this study is to apply the latest classification criteria to a monocentric cohort of patients with diagnosis of APS and to compare it with the previous criteria.Methods:We retrospectively collected data from patients with clinical diagnosis of APS from the Lupus Clinic of Sapienza University of Rome and applied the new classification criteria and the previous 2006 “Sapporo” criteria.Results:In our cohort of 165 APS patients, 139 could be classified according to Sapporo criteria, while 106 patients fulfilled the new classification criteria. Among the 33 patients that could not be reclassified by the new criteria, 21 patients did not meet clinical domains (8 thrombotic APS patients did not reach enough points due to a high thromboembolic/cardiovascular risk and 13 OAPS patients due to the presence of poliabortivity or fetal death alone), while 18 lacked laboratory domains due to only IgM aPL positivity (Graph 1).Conclusion:In our cohort, the application of the new APS classification criteria identifies a smaller number of patients compared to the previous ones, confirming the reduced sensitivity of the former. The new criteria may lead to the exclusion of patients with specific clinical and laboratory characteristics, that require larger studies to be assessed.REFERENCES:NIL.Acknowledgements:NIL.Disclosure of Interests:None declared.