Understanding response to rituximab treatment in rheumatoid arthritis through immune fingerprinting of T and B cells

Despite the great research advances in dissecting the mechanisms underlying rheumatoid arthritis onset and development, the exact pathophysiology of this disease remains unsolved. In the past years, the introduction of biologicals, and in particular of therapeutic monoclonal antibodies (mAb), has constituted a major breakthrough in the clinical management of the disease. Although these new drugs have proven effective, in some patients, remission or low disease activity is only partially or temporally achieved. Understanding the mechanism behind this incomplete response might lead to improved therapies and in ultimately to improved quality of life for patients. This thesis describes our efforts to elucidate the mechanisms behind response to B-cell depletion therapy using rituximab in rheumatoid arthritis. We evaluated how both direct and indirect effects can influence clinical response to the treatment. To achieve this we applied AIRR sequencing or Adaptive Immune Receptor Repertoire sequencing, which allows to identify and quantify all T- and B-cell receptors within an individual. This methodology allowed us to fingerprint and monitor ongoing immune responses in cohorts of rheumatoid arthritis patients undergoing rituximab treatment

Understanding response to rituximab treatment in rheumatoid arthritis through immune fingerprinting of T and B cells

Despite the great research advances in dissecting the mechanisms underlying rheumatoid arthritis onset and development, the exact pathophysiology of this disease remains unsolved. In the past years, the introduction of biologicals, and in particular of therapeutic monoclonal antibodies (mAb), has constituted a major breakthrough in the clinical management of the disease. Although these new drugs have proven effective, in some patients, remission or low disease activity is only partially or temporally achieved. Understanding the mechanism behind this incomplete response might lead to improved therapies and in ultimately to improved quality of life for patients. This thesis describes our efforts to elucidate the mechanisms behind response to B-cell depletion therapy using rituximab in rheumatoid arthritis. We evaluated how both direct and indirect effects can influence clinical response to the treatment. To achieve this we applied AIRR sequencing or Adaptive Immune Receptor Repertoire sequencing, which allows to identify and quantify all T- and B-cell receptors within an individual. This methodology allowed us to fingerprint and monitor ongoing immune responses in cohorts of rheumatoid arthritis patients undergoing rituximab treatment

Effects of polysaccharides from Botryotinia fuckeliana (Botrytis cinerea) on in vitro culture of table and wine grapes (Vitis vinifera)

Shoots of several table and wine grape cultivars were cultured in vitro on a medium supplemented with polysaccharides obtained from a culture filtrate of Botryotinia fuckeliana through differential ethanolic precipitations. The general effects of polysaccharides resulted in leaf yellowness and in a reduction of fresh and dry weight. Differential response of assayed cultivars to polysaccharides seemed to be not related to their bunch susceptibility to grey mould under field conditions

A ready-to-use single- and Duplex-TaqMan-qPCR assay to detect and quantify the biocontrol agents Trichoderma asperellum and Trichoderma gamsii

Trichoderma asperellum strain icc012 and Trichoderma gamsii strain icc080, the microbial active ingredients of Remedier™ (ISAGRO, Novara, Italy), are biocontrol agents (BCAs) employable for crop protection against a wide range of fungal pathogens, including soil-borne pathogens and fungi involved in grapevine trunk disease. In this study, single and duplex real-time quantitative PCR (qPCR) methods to detect and quantify T. asperellum and T. gamsii were developed. Primers/probe sets were designed on the T. asperellum and T. gamsii rpb2 genes and tested for specificity on a panel of microorganisms commonly associated with grape wood and soil. No differences were observed comparing single- and duplex-qPCR assays on different BCAs, 1 pg of target DNA was detected approximately at Cq= 34. R2-values and the efficiency were always equal to 0.99 and > 80%, respectively. The detection limit of the duplex-qPCR assay on artificially inoculated samples was 2 × 103and 4 × 104conidia g-1of grape wood tissue and soil, respectively. The methods will be useful to better schedule BCA application in the field and in grapevine nurseries, as well as for investigating the dynamic of BCA populations

Identification of phenolic compounds from inflorescences of non-psychoactive Cannabis sativa L. by UHPLC-HRMS and in vitro assessment of the antiproliferative activity against colorectal cancer

Phenolic compounds from Cannabis sativa L. (Cannabaceae family), in particular cannflavins, are known to possess several biological properties. However, their antiproliferative activity, being of great interest from a medicinal chemistry point of view, has not been deeply investigated so far in the literature. In the light of this, the aim of this study was to obtain an enriched fraction of polyphenols (namely PEF) from inflorescences of a non-psychoactive C. sativa (hemp) variety and to evaluate its antiproliferative activity against cancer cells, capitalizing on a new and selective extraction method for hemp polyphenols, followed by preparative flash column chromatography. Untargeted metabolomics, using a new method based on ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC-HRMS), was applied here for the first time to fully characterize PEF. Then, the main phenolic compounds were quantified by HPLC-UV. The antiproliferative activity of PEF and of the isolated compounds was assessed in vitro for the first time against Caco-2 and SW480 human colon adenocarcinoma cell lines providing promising IC50 values, in comparison with the reference drug used in therapy for this cancer type. Based on these results, PEF can be considered as a new highly potential therapeutic product to be further investigated against colorectal cancer, thanks to the possible synergistic interaction of its compounds

PP272—Migraine and parthenolide inhibition of transient receptor potential ankyrin 1

2013 e103 emerged as a major complication of bortezomib therapy, which usually appears in the first courses of therapy with a number of sensory and painful symptoms, including reduced threshold to mechanical and cold stimuli. No satisfactory explanation or effective treatment exists for bortezomib-evoked CIPN. Patients (or Materials) and Methods: In this study, we evaluated whether TRPA1 acted as a critical mediator of CIPN by bortezomib or oxaliplatin in a mouse model system. Results: Our data demonstrated that CIPN hypersensitivity phenotype that was stably established by bortezomib could be transiently reverted by systemic or local treatment with the TRPA1 antagonist HC-030031. A similar effect was produced by the oxidative stress scavenger α -lipoic acid. Notably, the CIPN phenotype was abolished completely in mice that were genetically deficient in TRPA1, highlighting its essential role. Administration of bortezomib or oxaliplatin, which also elicits TRPA1-dependent hypersensitivity, produced a rapid, transient increase in plasma of carboxy-methyllysine, a byproduct of oxidative stress. Short-term systemic treatment with either HC-030031 or α -lipoic acid could completely prevent hypersensitivity if administered before the cytotoxic drug. Conclusion: Our findings highlight a key role for early activation/ sensitization of TRPA1 by oxidative stress by-products in producing CIPN. Furthermore, they suggest prevention strategies for CIPN in patients through the use of early, short-term treatments with TRPA1 antagonists. Disclosure of Interest: None declared

Helichrysum stoechas (L.) Moench reduces body weight gain and modulates mood disorders via inhibition of silent information regulator 1 (SIRT1) by arzanol

The prevalence of obesity is steadily rising, making safe and more efficient anti-obesity treatments an urgent medical need. Growing evidence correlates obesity and comorbidities, including anxiety and depression, with the development of a low-grade inflammation in peripheral and central tissues. We hypothesized that attenuating neuroinflammation might reduce weight gain and improve mood. We investigated the efficacy of a methanolic extract from Helichrysum stoechas (L.) Moench (HSE), well-known for its anti-inflammatory properties, and its main constituent arzanol (AZL). HPLC-ESI-MS2 and HPLC-UV were used to characterize the extract. HSE effects on mood and feeding behavior was assessed in mice. The mechanism of action of HSE and AZL was investigated in hippocampus samples and SH-SY5Y cells by western blotting and immunofluorescence. Oral administration of HSE for 3 weeks limited weight gain with no significant decrease in food intake. HSE produced an anxiolytic-like and antidepressant-like phenotype comparable to diazepam and amitriptyline, respectively, in the absence of locomotor and cognitive impairments and induced neuroprotective effects in glutamate-exposed SH-SY5Y cells. A dose-dependent reduction of SIRT1 expression was detected in SH-SY5Y cells and in hippocampal samples from HSE-treated mice. The inhibition of the SIRT1-FoxO1 pathway was induced in the hypothalamus. Molecular docking studies proposed a mechanism of SIRT1 inhibition by AZL, confirmed by the evaluation of inhibitory effects on SIRT1 enzymatic activity. HSE limited weight gain and comorbidities through an AZL-mediated SIRT1 inhibition. These activities indicate HSE an innovative therapeutic perspective for obesity and associated mood disorders

Absinthin, an agonist of the bitter taste receptor hTAS2R46, uncovers an ER-to-mitochondria Ca2–shuttling event

Type 2 taste receptors (TAS2R) are G protein-coupled receptors first described in the gustatory system, but have also been shown to have extra-oral localizations, including airway smooth muscle (ASM) cells, in which TAS2R have been reported to induce relaxation. TAS2R46 is an unexplored subtype that responds to its highly specific agonist absinthin. Here, we first demonstrate that, unlike other bitter-taste receptor agonists, absinthin alone (1 μM) in ASM cells does not induce Ca2+ signals, but reduces histamine-induced cytosolic Ca2+ increases. To investigate this mechanism, we introduced into ASM cells aequorin-based Ca2+ probes targeted to the cytosol, sub-plasma membrane domain, or the mitochondrial matrix. We show that absinthin reduces cytosolic histamine-induced Ca2+-rises and simultaneously increases Ca2+-influx into mitochondria. We found that this effect is inhibited by the potent human TAS2R46 (hTAS2R46) antagonist 3β-hydroxydihydrocostunolide and is no longer evident in hTAS2R46-silenced ASM cells, indicating that it is hTAS2R46-dependent. Furthermore, these changes were sensitive to the mitochondrial uncoupler carbonyl cyanide p-(trifluoromethoxy)phenyl-hydrazone (FCCP); the mitochondrial calcium uniporter inhibitor KB-R7943 (carbamimidothioic-acid); the cytoskeletal disrupter latrunculin; and an inhibitor of the exchange protein directly activated by cAMP (EPAC), ESI-09. Similarly, the β2 agonist salbutamol also could induce Ca2+ shuttling from cytoplasm to mitochondria, suggesting that this new mechanism might be generalizable. Moreover, forskolin and an EPAC activator mimicked this effect in HeLa cells. Our findings support the hypothesis that plasma membrane receptors can positively regulate mitochondrial Ca2+ uptake, adding a further facet to the ability of cells to encode complex Ca2+ signals