Tu1283 Proteolytic Stability of AVX-470, a Bovine Colostral Anti-TNF Antibody, Determined In Vitro and in Clinical Samples After Oral Administration to Patients With Ulcerative Colitis

SummaryImmune-mediated platelet activation is emerging as an important pathogenic mechanism of thrombosis. In vitro studies have suggested two distinct pathways for immune-mediated platelet activation; one involving clustering of platelet FcyRIIa, the other involving platelet-associated complement activation. HLA-related antibodies have been shown to cause platelet aggregation, but the mechanism has not been clarified. We evaluated the mechanism of platelet aggregation induced by HLA-related antibodies from nine patients. Antibody to platelet FcyRIIa failed to block platelet aggregation with 8/9 samples, indicating that engagement of platelet FcyRIIa is not necessary for the platelet aggregation induced by HLA-related antibodies. In contrast, platelet aggregation was blocked by antibodies to human C8 (5/7) or C9 (7/7). F(ab’)2 fragments of patient IgG failed to induce platelet activation although they bound to HLA antigen on platelets. Intact patient IgG failed to aggregate washed platelets unless aged serum was added. The activating IgG could be adsorbed by incubation with lymphocytes and eluted from the lymphocytes. These results indicate that complement activation is involved in the aggregation response to HLA-related antibodies. This is the first demonstration of complement-mediated platelet aggregation by clinical samples. Five of the patients developed thrombocytopenia in relationship to blood transfusion and two patients developed acute thromboembolic disease, suggesting that these antibodies and the complement-dependent pathway of platelet aggregation may be of clinical significance.

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Atorvastatin-Loaded Oleic Acid Nanoglobules for Oral Administration: In Vitro Characterization and Biopharmaceutical Evaluation

Current Nanoscience ◽

10.2174/1573413711309020007 ◽

2013 ◽

Vol 9 (2) ◽

pp. 202-210

Author(s):

Pradum Pundlikrao Ige ◽

Nilesh Ashok Bachhav ◽

Hitendra Shaligram Mahajan ◽

Pankaj Padmakar Nerkar ◽

Surendra Ganeshlal Gattani

Keyword(s):

Oleic Acid ◽

Oral Administration ◽

In Vitro Characterization

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Red Grape Polyphenol Oral Administration improves Immune Response in Women Affected by Nickel-Mediated Allergic Contact Dermatitis

Endocrine Metabolic & Immune Disorders - Drug Targets ◽

10.2174/1871530320666200313152648 ◽

2020 ◽

Vol 20 ◽

Author(s):

Thea Magrone ◽

Emilio Jirillo ◽

Manrico Magrone ◽

Matteo Antonio Russo ◽

Paolo Romita ◽

...

Keyword(s):

Contact Dermatitis ◽

Oral Administration ◽

Allergic Contact Dermatitis ◽

Laboratory Data ◽

Anti Oxidant ◽

Anti Inflammatory ◽

Red Grape ◽

Allergic Contact ◽

Drop Outs

Background: Our previous findings demonstrated that in vitro supplementation of polyphenols, extracted from seeds of red grape (Nero di Troia cultivar), to peripheral lymphomonocytes from patients affected by allergic contact dermatitis (ACD) to nickel (Ni) could reduce release of pro-inflammatory cytokines and nitric oxide (NO), while increasing levels of interleukin (IL)-10, an anti-inflammatory cytokine. Objective: To assess whether an intervention with oral administration of polyphenols leads to a reduction of peripheral biomarkers in ACD patients. Method: At T0, 25 patients affected by ACD to Ni were orally administered with 300 mg polyphenols prodie extracted from seeds of red grape (Nero di Troia cultivar) (NATUR-OX®) for 3 months (T1). Other 25 patients affected by ACD to Ni received placebo only for the same period of time. Serum biomarkers were analyzed at T0 and T1. In both groups seven drop outs were recorded. Result: At T1 in comparison to T0, in treated patients, values of IFN-γ, IL-4, IL-17, PTX3 and NO decreased, while IL-10 levels increased when compared with T0 values. Conversely, in placebo-treated patients no modifications of biomarkers were evaluated at T1. Conclusion: Present laboratory data rely on the anti-oxidant, anti-inflammatory and anti-allergic properties of polyphenols.

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Mutations in fbiD (Rv2983) as a Novel Determinant of Resistance to Pretomanid and Delamanid in Mycobacterium tuberculosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01948-20 ◽

2020 ◽

Vol 65 (1) ◽

pp. e01948-20

Author(s):

Dalin Rifat ◽

Si-Yang Li ◽

Thomas Ioerger ◽

Keshav Shah ◽

Jean-Philippe Lanoix ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Clinical Evidence ◽

Clinical Samples ◽

Loss Of Function ◽

Wild Type ◽

Content Type ◽

Solid Media ◽

High Level ◽

Level Resistance

ABSTRACTThe nitroimidazole prodrugs delamanid and pretomanid comprise one of only two new antimicrobial classes approved to treat tuberculosis (TB) in 50 years. Prior in vitro studies suggest a relatively low barrier to nitroimidazole resistance in Mycobacterium tuberculosis, but clinical evidence is limited to date. We selected pretomanid-resistant M. tuberculosis mutants in two mouse models of TB using a range of pretomanid doses. The frequency of spontaneous resistance was approximately 10−5 CFU. Whole-genome sequencing of 161 resistant isolates from 47 mice revealed 99 unique mutations, of which 91% occurred in 1 of 5 genes previously associated with nitroimidazole activation and resistance, namely, fbiC (56%), fbiA (15%), ddn (12%), fgd (4%), and fbiB (4%). Nearly all mutations were unique to a single mouse and not previously identified. The remaining 9% of resistant mutants harbored mutations in Rv2983 (fbiD), a gene not previously associated with nitroimidazole resistance but recently shown to be a guanylyltransferase necessary for cofactor F420 synthesis. Most mutants exhibited high-level resistance to pretomanid and delamanid, although Rv2983 and fbiB mutants exhibited high-level pretomanid resistance but relatively small changes in delamanid susceptibility. Complementing an Rv2983 mutant with wild-type Rv2983 restored susceptibility to pretomanid and delamanid. By quantifying intracellular F420 and its precursor Fo in overexpressing and loss-of-function mutants, we provide further evidence that Rv2983 is necessary for F420 biosynthesis. Finally, Rv2983 mutants and other F420H2-deficient mutants displayed hypersusceptibility to some antibiotics and to concentrations of malachite green found in solid media used to isolate and propagate mycobacteria from clinical samples.

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In-vitro and in-vivo metabolism of different aspirin formulations studied by a validated liquid chromatography tandem mass spectrometry method

Scientific Reports ◽

10.1038/s41598-021-89671-w ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Michele Dei Cas ◽

Jessica Rizzo ◽

Mariangela Scavone ◽

Eti Femia ◽

Gian Marco Podda ◽

...

Keyword(s):

Oral Administration ◽

Whole Blood ◽

Serum Levels ◽

Reversed Phase ◽

Weekly Treatment ◽

Low Dose Aspirin ◽

Liquid Chromatography Tandem Mass ◽

Enteric Coated

AbstractLow-dose aspirin (ASA) is used to prevent cardiovascular events. The most commonly used formulation is enteric-coated ASA (EC-ASA) that may be absorbed more slowly and less efficiently in some patients. To uncover these “non-responders” patients, the availability of proper analytical methods is pivotal in order to study the pharmacodynamics, the pharmacokinetics and the metabolic fate of ASA. We validated a high-throughput, isocratic reversed-phase, negative MRM, LC–MS/MS method useful for measuring circulating ASA and salicylic acid (SA) in blood and plasma. ASA-d4 and SA-d4 were used as internal standards. The method was applied to evaluate: (a) the "in vitro" ASA degradation by esterases in whole blood and plasma, as a function of time and concentration; (b) the "in vivo" kinetics of ASA and SA after 7 days of oral administration of EC-ASA or plain-ASA (100 mg) in healthy volunteers (three men and three women, 37–63 years). Parameters of esterases activity were Vmax 6.5 ± 1.9 and Km 147.5 ± 64.4 in plasma, and Vmax 108.1 ± 20.8 and Km 803.2 ± 170.7 in whole blood. After oral administration of the two formulations, tmax varied between 3 and 6 h for EC-ASA and between 0.5 and 1.0 h for plain-ASA. Higher between-subjects variability was seen after EC-ASA, and one subject had a delayed absorption over eight hours. Plasma AUC was 725.5 (89.8–1222) for EC-ASA, and 823.1(624–1196) ng h/mL (median, 25–75% CI) for plain ASA. After the weekly treatment, serum levels of TxB2 were very low (< 10 ng/mL at 24 h from the drug intake) in all the studied subjects, regardless of the formulation or the tmax. This method proved to be suitable for studies on aspirin responsiveness.

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1280. In-Vitro Activity of Cefiderocol, Imipenem/Relebactam, & Ceftazidime/Avibactam in Ceftolozane/Tazobactam Resistant Strains of Multidrug Resistant Pseudomonas aeruginosa

Open Forum Infectious Diseases ◽

10.1093/ofid/ofaa439.1463 ◽

2020 ◽

Vol 7 (Supplement_1) ◽

pp. S655-S655

Author(s):

Daniel Navas ◽

Angela Charles ◽

Amy Carr ◽

Jose Alexander

Keyword(s):

Multidrug Resistant ◽

Resistance Mechanisms ◽

Vitro Activity ◽

Clinical Isolates ◽

Resistance Testing ◽

Clinical Samples ◽

In Vitro Activity ◽

Carbapenemase Gene ◽

Resistant Strains

Abstract Background The activity of imipenem/relebactam (I/R), ceftazidime/avibactam (CZA) and cefiderocol (FDC) were evaluated against clinical isolates of multidrug resistant (MDR) strains of P. aeruginosa which was resistant to ceftolozane/tazobactam (C/T). The recent increase of MDR P. aeruginosa strains isolated from clinical samples has prompted research and development of new antimicrobials that can withstand its multiple resistance mechanisms. C/T is an effective option for treatment of MDR P. aeruginosa in our facility with only 10% of resistance in MDR strains, but the emergence of resistance may occur due to the presence of a carbapenemase gene or an ampC mutation. Methods Antimicrobial susceptibility testing for C/T Etest® (bioMérieux, Inc.) were performed on all MDR strains initially screened by the VITEK2® (bioMérieux, Inc.). 10% (n=20) of all MDR isolates were resistant to C/T by the CLSI 2019 breakpoints. These resistant isolates were tested for presence of a carbapenemase gene using the GeneXpert CARBA-R (Cepheid®) PCR and against CZA Etest® (bioMérieux, Inc.) I/R gradient strips (Liofilchem®) and FDC broth microdilution (Thermo Scientific™ Sensititre™). Results A total of 20 clinical isolates of MDR P. aeruginosa resistant to C/T were tested following standardized CLSI protocols and techniques. All 20 isolates were screened for the presence of a carbapenemase gene (blaVIM, blaNDM, blaKPC, blaOXA-48, blaIMP). A blaVIM gene was detected in 6 (30%) out of 20 isolates. FDC demonstrated the greatest activity with 85% (n=17) of susceptible isolates (CLSI MIC <4µg/dL). CZA (CLSI MIC <8µg/dL) and I/R (FDA MIC <2µg/dL) showed 15% (n=3) and 10% (n=2) of susceptible isolates respectively. FDC was active against all 6 blaVIM isolates, where all 6 strains were resistant to CZA and I/R as expected. 3 isolates tested non-susceptible against FDC; additional characterization was not performed at this time. Conclusion Based on these results, FDC demonstrated the greatest in-vitro activity against C/T resistant strains of MDR P. aeruginosa. FDC also demonstrated activity against all 6 MDR P. aeruginosa carrying blaVIM gene. FDC is a strong option to consider on MDR P. aeruginosa strains based on a resistance testing algorithm and a cost/effective protocol. Disclosures All Authors: No reported disclosures

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Splicing factor SRSF1 promotes breast cancer progression via oncogenic splice switching of PTPMT1

Journal of Experimental & Clinical Cancer Research ◽

10.1186/s13046-021-01978-8 ◽

2021 ◽

Vol 40 (1) ◽

Author(s):

Jun-Xian Du ◽

Yi-Hong Luo ◽

Si-Jia Zhang ◽

Biao Wang ◽

Cong Chen ◽

...

Keyword(s):

Breast Cancer ◽

Cancer Progression ◽

High Risk Group ◽

Clinical Samples ◽

Binding Motif ◽

Ki 67 ◽

Tissue Samples

Abstract Background Intensive evidence has highlighted the effect of aberrant alternative splicing (AS) events on cancer progression when triggered by dysregulation of the SR protein family. Nonetheless, the underlying mechanism in breast cancer (BRCA) remains elusive. Here we sought to explore the molecular function of SRSF1 and identify the key AS events regulated by SRSF1 in BRCA. Methods We conducted a comprehensive analysis of the expression and clinical correlation of SRSF1 in BRCA based on the TCGA dataset, Metabric database and clinical tissue samples. Functional analysis of SRSF1 in BRCA was conducted in vitro and in vivo. SRSF1-mediated AS events and their binding motifs were identified by RNA-seq, RNA immunoprecipitation-PCR (RIP-PCR) and in vivo crosslinking followed by immunoprecipitation (CLIP), which was further validated by the minigene reporter assay. PTPMT1 exon 3 (E3) AS was identified to partially mediate the oncogenic role of SRSF1 by the P-AKT/C-MYC axis. Finally, the expression and clinical significance of these AS events were validated in clinical samples and using the TCGA database. Results SRSF1 expression was consistently upregulated in BRCA samples, positively associated with tumor grade and the Ki-67 index, and correlated with poor prognosis in a hormone receptor-positive (HR+) cohort, which facilitated proliferation, cell migration and inhibited apoptosis in vitro and in vivo. We identified SRSF1-mediated AS events and discovered the SRSF1 binding motif in the regulation of splice switching of PTPMT1. Furthermore, PTPMT1 splice switching was regulated by SRSF1 by binding directly to its motif in E3 which partially mediated the oncogenic role of SRSF1 by the AKT/C-MYC axis. Additionally, PTPMT1 splice switching was validated in tissue samples of BRCA patients and using the TCGA database. The high-risk group, identified by AS of PTPMT1 and expression of SRSF1, possessed poorer prognosis in the stage I/II TCGA BRCA cohort. Conclusions SRSF1 exerts oncogenic roles in BRCA partially by regulating the AS of PTPMT1, which could be a therapeutic target candidate in BRCA and a prognostic factor in HR+ BRCA patient.

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Impact of 2′-Fucosyllactose on Gut Microbiota Composition in Adults with Chronic Gastrointestinal Conditions: Batch Culture Fermentation Model and Pilot Clinical Trial Findings

Nutrients ◽

10.3390/nu13030938 ◽

2021 ◽

Vol 13 (3) ◽

pp. 938

Author(s):

Jennifer Joan Ryan ◽

Andrea Monteagudo-Mera ◽

Nikhat Contractor ◽

Glenn R. Gibson

Keyword(s):

Ulcerative Colitis ◽

Batch Culture ◽

Pilot Trial ◽

Gastrointestinal Symptoms ◽

Short Chain Fatty Acids ◽

Open Label ◽

Gastrointestinal Conditions ◽

Fermentation Model ◽

Quality Of Life Index

Intestinal dysbiosis has been described in patients with certain gastrointestinal conditions including irritable bowel syndrome (IBS) and ulcerative colitis. 2′-fucosyllactose (2′-FL), a prebiotic human milk oligosaccharide, is considered bifidogenic and butyrogenic. To assess prebiotic effects of 2′-FL, alone or in combination with probiotic strains (potential synbiotics), in vitro experiments were conducted on stool from healthy, IBS, and ulcerative colitis adult donors. In anaerobic batch culture fermenters, Bifidobacterium and Eubacterium rectale-Clostridium coccoides counts, and short-chain fatty acids (SCFAs) including butyrate increased during fermentation with 2′-FL and some of the 2′-FL/probiotic combinations. In a subsequent open-label pilot trial, the effect of a 2′-FL-containing nutritional formula was evaluated in twelve adults with IBS or ulcerative colitis. Gastrointestinal Quality of Life Index (GIQLI) total and gastrointestinal symptoms domain scores, stool counts of Bifidobacterium and Faecalibacterium prausnitzii, and stool SCFAs including butyrate, increased after six weeks of intervention. Consistent with documented effects of 2′-FL, the batch culture fermentation experiments demonstrated bifidogenic and butyrogenic effects of 2′-FL during fermentation with human stool samples. Consumption of the 2′-FL-containing nutritional formula by adults with IBS or ulcerative colitis was associated with improvements in intra- and extra-intestinal symptoms, and bifidogenic and butyrogenic effects.

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Creatine Supplementation for Patients with Inflammatory Bowl Diseases: A Scientific Rationale for a Clinical Trial

Nutrients ◽

10.3390/nu13051429 ◽

2021 ◽

Vol 13 (5) ◽

pp. 1429

Author(s):

Theo Wallimann ◽

Caroline H. T. Hall ◽

Sean P. Colgan ◽

Louise E. Glover

Keyword(s):

Ulcerative Colitis ◽

Clinical Trial ◽

Crohn’S Disease ◽

Pilot Study ◽

Crohn's Disease ◽

Single Case ◽

Initial Period ◽

Creatine Supplementation

Based on theoretical considerations, experimental data with cells in vitro, animal studies in vivo, as well as a single case pilot study with one colitis patient, a consolidated hypothesis can be put forward, stating that “oral supplementation with creatine monohydrate (Cr), a pleiotropic cellular energy precursor, is likely to be effective in inducing a favorable response and/or remission in patients with inflammatory bowel diseases (IBD), like ulcerative colitis and/or Crohn’s disease”. A current pilot clinical trial that incorporates the use of oral Cr at a dose of 2 × 7 g per day, over an initial period of 2 months in conjunction with ongoing therapies (NCT02463305) will be informative for the proposed larger, more long-term Cr supplementation study of 2 × 3–5 g of Cr per day for a time of 3–6 months. This strategy should be insightful to the potential for Cr in reducing or alleviating the symptoms of IBD. Supplementation with chemically pure Cr, a natural nutritional supplement, is well tolerated not only by healthy subjects, but also by patients with diverse neuromuscular diseases. If the outcome of such a clinical pilot study with Cr as monotherapy or in conjunction with metformin were positive, oral Cr supplementation could then be used in the future as potentially useful adjuvant therapeutic intervention for patients with IBD, preferably together with standard medication used for treating patients with chronic ulcerative colitis and/or Crohn’s disease.

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Persimmon-derived tannin ameliorates the pathogenesis of ulcerative colitis in a murine model through inhibition of the inflammatory response and alteration of microbiota

Scientific Reports ◽

10.1038/s41598-021-86608-1 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Masahiro Kitabatake ◽

Yoko Matsumura ◽

Noriko Ouji-Sageshima ◽

Tatsuki Nishioka ◽

Atsushi Hara ◽

...

Keyword(s):

Ulcerative Colitis ◽

Immune Response ◽

Murine Model ◽

Control Diet ◽

Antimicrobial Activities ◽

Diet Group ◽

Chronic Inflammatory Bowel Disease ◽

Diospyros Kaki ◽

Colon Inflammation

AbstractUlcerative colitis (UC) is a chronic inflammatory bowel disease (IBD) induced by dysregulation of the immune response in the intestinal mucosa. Although the underlying mechanisms of UC development are not fully understood, disruption of gut microbiota, “dysbiosis”, is thought to lead to the development of IBD. Persimmon (Ebenaceae Diospyros kaki Thunb.)-derived tannin, which is a condensed polymeric tannin consisting of catechin groups, has antioxidant, anti-inflammatory, and antimicrobial activities. In this study, we assessed the effect of persimmon-derived tannin on a murine model of UC established by dextran sulfate sodium-induced colitis in female mice. Dietary supplementation of tannin significantly decreased disease activity and colon inflammation. A hydrolysate of tannin directly suppressed expression of inflammatory genes in macrophages in vitro. In faecal microbiota, the relative abundance of Bacteroides was increased significantly by tannin supplementation. Alpha-diversity indices in colitis-induced mice were significantly higher in the tannin diet group compared with the control diet group. Additionally, expansion of Enterobacteriaceae and Enterococcus, which is associated with disease progression of IBD, was remarkably suppressed in the tannin diet group. These results suggest that persimmon-derived tannin ameliorates colon inflammation in UC through alteration of the microbiota composition and immune response, which may be a promising candidate for IBD therapy.

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