Antibiotic use differentially affects the risk of anti-drug antibody formation during anti-TNFα therapy in inflammatory bowel disease patients: a report from the epi-IIRN

ObjectiveAnti-drug antibodies (ADA) to anti-tumour necrosis factor (anti-TNF) therapy drive treatment loss of response. An association between intestinal microbial composition and response to anti-TNF therapy was noted. We therefore aimed to assess the implications of antibiotic treatments on ADA formation in patients with inflammatory bowel disease (IBD).DesignWe analysed data from the epi-IIRN (epidemiology group of the Israeli IBD research nucleus), a nationwide registry of all patients with IBD in Israel. We included all patients treated with anti-TNF who had available ADA levels. Survival analysis with drug use as time varying covariates were used to assess the association between antibiotic use and ADA development. Next, specific pathogen and germ-free C57BL mice were treated with respective antibiotics and challenged with infliximab. ADA were assessed after 14 days.ResultsAmong 1946 eligible patients, with a median follow-up of 651 days from initiation of therapy, 363 had positive ADA. Cox proportional hazard model demonstrated an increased risk of ADA development in patients who used cephalosporins (HR=1.97, 95% CI 1.58 to 2.44), or penicillins with β-lactamase inhibitors (penicillin-BLI, HR=1.4, 95% CI 1.13 to 1.74), whereas a reduced risk was noted in patients treated with macrolides (HR=0.38, 95% CI 0.16 to 0.86) or fluoroquinolones (HR=0.20, 95% CI 0.12 to 0.35). In mice exposed to infliximab, significantly increased ADA production was observed in cephalosporin as compared with macrolide pretreated mice. Germ-free mice produced no ADA.ConclusionADA production is associated with the microbial composition. The risk of ADA development during anti-TNF therapy can possibly be reduced by avoidance of cephalosporins and penicillin-BLIs, or by treatment with fluoroquinolones or macrolides.

The Antitumor Activity of Sodium Selenite Alone and in Combination with Gemcitabine in Pancreatic Cancer: An In Vitro and In Vivo Study

Sodium selenite acts by depleting enzymes that protect against cellular oxidative stress. To determine its effect alone or in combination with gemcitabine (GMZ) in pancreatic cancer, we used PANC-1 and Pan02 cell lines and C57BL mice bearing a Pan02-generated tumor. Our results demonstrated a significant inhibition of pancreatic cancer cell viability with the use of sodium selenite alone and a synergistic effect when associated with GMZ. The molecular mechanisms of the antitumor effect of sodium selenite alone involved apoptosis-inducing factor (AIF) and the expression of phospho-p38 in the combined therapy. In addition, sodium selenite alone and in association with GMZ significantly decreased the migration capacity and colony-forming ability, reduced tumor activity in multicellular tumor spheroids (MTS) and decreased sphere formation of cancer stem cells. In vivo studies demonstrated that combined therapy not only inhibited tumor growth (65%) compared to the untreated group but also relative to sodium selenite or GMZ used as monotherapy (up to 40%), increasing mice survival. These results were supported by the analysis of C57BL/6 albino mice bearing a Pan02-generated tumor, using the IVIS system. In conclusion, our results showed that sodium selenite is a potential agent for the improvement in the treatment of pancreatic cancer and should be considered for future human clinical trials.

Ablation of Selenbp1 Alters Lipid Metabolism via the Pparα Pathway in Mouse Kidney

Selenium-binding protein 1 (Selenbp1) is a 2,3,7,8-tetrechlorodibenzo-p-dioxin inducible protein whose function is yet to be comprehensively elucidated. As the highly homologous isoform, Selenbp2, is expressed at low levels in the kidney, it is worthwhile comparing wild-type C57BL mice and Selenbp1-deficient mice under dioxin-free conditions. Accordingly, we conducted a mouse metabolomics analysis under non-dioxin-treated conditions. DNA microarray analysis was performed based on observed changes in lipid metabolism-related factors. The results showed fluctuations in the expression of numerous genes. Real-time RT-PCR confirmed the decreased expression levels of the cytochrome P450 4a (Cyp4a) subfamily, known to be involved in fatty acid ω- and ω-1 hydroxylation. Furthermore, peroxisome proliferator-activated receptor-α (Pparα) and retinoid-X-receptor-α (Rxrα), which form a heterodimer with Pparα to promote gene expression, were simultaneously reduced. This indicated that reduced Cyp4a expression was mediated via decreased Pparα and Rxrα. In line with this finding, increased levels of leukotrienes and prostaglandins were detected. Conversely, decreased hydrogen peroxide levels and reduced superoxide dismutase (SOD) activity supported the suppression of the renal expression of Sod1 and Sod2 in Selenbp1-deficient mice. Therefore, we infer that ablation of Selenbp1 elicits oxidative stress caused by increased levels of superoxide anions, which alters lipid metabolism via the Pparα pathway.

Encephalitozoon cuniculi takes advantage of efferocytosis to evade the immune response

Microsporidia are recognized as opportunistic pathogens in individuals with immunodeficiencies, especially related to T cells. Although the activity of CD8+ T lymphocytes is essential to eliminate these pathogens, earlier studies have shown significant participation of macrophages at the beginning of the infection. Macrophages and other innate immunity cells play a critical role in activating the acquired immunity. After programmed cell death, the cell fragments or apoptotic bodies are cleared by phagocytic cells, a phenomenon known as efferocytosis. This process has been recognized as a way of evading immunity by intracellular pathogens. The present study evaluated the impact of efferocytosis of apoptotic cells either infected or not on macrophages and subsequently challenged with Encephalitozoon cuniculi microsporidia. Macrophages were obtained from the bone marrow monocytes from C57BL mice, pre-incubated with apoptotic Jurkat cells (ACs), and were further challenged with E. cuniculi spores. The same procedures were performed using the previously infected Jurkat cells (IACs) and challenged with E. cuniculi spores before macrophage pre-incubation. The average number of spores internalized by macrophages in phagocytosis was counted. Macrophage expression of CD40, CD206, CD80, CD86, and MHCII, as well as the cytokines released in the culture supernatants, was measured by flow cytometry. The ultrastructural study was performed to analyze the multiplication types of pathogens. Macrophages pre-incubated with ACs and challenged with E. cuniculi showed a higher percentage of phagocytosis and an average number of internalized spores. Moreover, the presence of stages of multiplication of the pathogen inside the macrophages, particularly after efferocytosis of infected apoptotic bodies, was observed. In addition, pre-incubation with ACs or IACs and/or challenge with the pathogen decreased the viability of macrophages, reflected as high percentages of apoptosis. The marked expression of CD206 and the release of large amounts of IL-10 and IL-6 indicated the polarization of macrophages to an M2 profile, compatible with efferocytosis and favorable for pathogen development. We concluded that the pathogen favored efferocytosis and polarized the macrophages to an M2 profile, allowing the survival and multiplication of E. cuniculi inside the macrophages and explaining the possibility of macrophages acting as Trojan horses in microsporidiosis.

EXPERIMENTAL STUDY OF EKOM BIOTRONS, WHICH PROVIDE A REMOTE EFFECT OF SOME BIOLOGICAL OBJECTS ON OTHERS

Back in the middle of the last century, information began to appear about the existence of a certain electromagnetic field, called a biofield, which is emitted by biological objects - animals and plants. A scientist from China, Jiang Kanzhen Yu. V., suggested that if you concentrate this radiation from several plants or animals, this field will affect other biological objects. Further research proved this phenomenon. Jian Kanzhen designed the device that concentrated radiation from many biological objects using spherical copper mirrors. He called this device a BIOTRON. Another Russian scientist, E. V. Komrakov, developed and patented a modified Biotron device, more powerful, in 58 countries of the world, and called it BIOTRON-EKOM. Plant seedlings are placed in front of mirrors, and the radiation from them is focused on biological objects. It was found that when irradiated in a Biotron with seedlings of cereal plants, the average life span of C57Bl mice increased to 24.5%, and the nematodes Caenorhabditis elegans increased to 30.1% (the maximum life span was up to 23.2 and 25%, respectively) compared to the control groups. The acceleration of the growth of animal cells in the culture, an increase in the saturating density, an increase in the "total" and "stationary" life span of culture by 23 and 22%, respectively, and an increase in the germination energy of barley seeds by 7.5% were revealed.

Abstract 13695: Paracrine-mediated Rejuvenation of Aged Mesenchymal Stem Cells Involves Broad Transcriptional Modulation of Angiogenic Factors

Introduction: Angiogenesis induced by bone marrow mesenchymal stem cells (MSCs) obtained from aged mice is inferior to those obtained from young mice, but is improved following exposure to conditioned media (CM) from young MSCs. To define alterations in gene expression and signaling pathways underlying the observed angiogenic improvement, we characterized differences in cellular mRNA expression between “non-rejuvenated” and “rejuvenated” (exposed to CM from young MSCs) old MSCs. Methods: Replicates of 105 MSCs isolated from old (18-24 months) C57BL mice (n=6) were cultured separately, or in co-culture with MSCs from young (4-6 weeks, n=6) mice using 0.4μm Transwell plates that allow transfer of soluble factors, but not of cells. After 7d in culture, mRNA from old and rejuvenated MSCs was isolated and sequenced. Analysis was performed using open source Galaxy pipeline. Transcription factor (TF) and miRNA target enrichment analyses were performed using ChEA3 and MIENTURNET. Results: Of the 529 unique transcripts involved in angiogenesis (GO-ID 0001525), 98 differentially expressed transcripts (Bonferroni p < 0.0001) were identified. The rejuvenated MSCs showed significantly increased expression of 39 genes. The majority of these involved canonical angiogenic pathways and/or regulation of VEGF: JAK1, LOXL2, KLF4, BMP4, and ADM. Top enriched TFs and miRNAs included EPAS1 and miR-20a, respectively, both directly involved in VEGF signaling, along with SOX18, SNAI1, SOX7, miR-126a, and miR-499 (FDR < 0.05), all of which are known to promote either angiogenesis and/or stemness. Conclusions: Improved angiogenesis by old MSCs exposed to CM from young MSCs is accompanied by significant modulation of angiogenic mediators, crucial in both VEGF and non-VEGF signaling pathways. These changes suggest targets for transcriptional modification to improve angiogenesis and tissue repair in aged patients.

Experimental Screening Study of the Antiarhythmic Activity of Empagliflosin

The antiarrhythmic activity of the type 2 sodium glucose co-transporter inhibitor Empagliflozin was studied in a model induced by calcium chloride in C57BL mice. It was found that preliminary administration of Empagliflozin at a dose of 1 mg/kg prevented CaCl2-induced ventricular arrhythmia and death during four periods of the biological half-life of the drug.

ANTI-MELANOMA BIO-EFFICACY OF THE PLANT MADHUCA LONGIFOLIA AND ITS ENHANCEMENT USING BIOACTIVE PRINCIPLE LOADED GOLD NANOPARTICLE

Objective: Unexplored in-vivo anti-melanoma bio-efficacy of the plant Madhuca longifolia (bark) has been carried out against C57BL/6 mice. Methods: Optimized experimental conditions of phytofabrication of gold nanoparticles were as follows: flavonoid content (1 ml, 0.5 mg/ml), sodium tetrachloroaurate dihydrate solution (2 ml, 1 mM), and sonication (15 min, 20 KHz) at pH 4. The optical properties; ultraviolet-visible spectrophotometer (UV-Vis), particles size and zeta potential (Zetasizer), miller indices; X-ray diffraction (XRD), morphology; field emission-scanning electron microscope (FE-SEM), particle size; high resolution-transmission electron microscopy (HR-TEM), surface roughness; atomic force microscopy (AFM) and elemental composition; and energy dispersive X-rays (EDX) of flavonoid loaded gold nanoparticles. In-vivo anti-melanoma bio-efficacy has been carried out against C57BL mice. Radioisotopic, hematological, and histopathological studies were carried out using standard procedures. Results: Redox potential of the total flavonoid extracted from the bark of the plant (Madhuca longifolia) has been used for the fabrication of flavonoid loaded gold nanoparticles (F@AuNp) and confirmed for the first time their significant anti-melanoma bio-efficacy. The finding is supported by hematological and histopathological studies carried out in the organs (liver, kidney, and intestine) of C57BL mice. The significant enhancement in phytofabricated F@AuNp compared to native bark extract of the plant has been assigned to enhanced stay period and nanosizing, biocompatibility, nontoxic nature, and enhanced beneficial payload to the cancerous cells. Conclusion: Such phytofabricated gold nanoparticles possess an admirable prospect for the expansion of herbal nanomedicine for anti-melanoma bio-efficacy.

Ghrelin Receptor Signaling Is Not Required for Glucocorticoid-Induced Obesity in Male Mice

Chronically elevated levels of glucocorticoids increase food intake, weight gain, and adiposity. Similarly, ghrelin, a gut-secreted hormone, is also associated with weight gain, adiposity, and increased feeding. Here we sought to determine if corticosterone-induced metabolic and behavioral changes require functional ghrelin receptors (GHSR). To do this, we treated male C57BL mice with chronic corticosterone (CORT) mixed in their drinking water for 28 days. Half of these mice received the GHSR antagonist JMV2959 via osmotic minipumps while treated with CORT. In a second experiment, we gave the same CORT protocol to mice with a targeted mutation to the GHSR or their wild-type littermates. As expected, CORT treatment increased food intake, weight gain, and adiposity, but contrary to expectations, mice treated with a GHSR receptor antagonist or GHSR knockout (KO) mice did not show attenuated food intake, weight gain, or adiposity in response to CORT. Similarly, the effects of CORT on the liver were the same or more pronounced in GHSR antagonist-treated and GHSR KO mice. Treatment with JMV2959 did attenuate the effects of chronic CORT on glycemic regulation as determined by the glucose tolerance test. Finally, disruption of GHSR signaling resulted in behavioral responses associated with social withdrawal, potentially due to neuroprotective effects of GHSR activation. In all, we propose that blocking GHSR signaling helps to moderate glucose concentrations when CORT levels are high, but blocking GHSR signaling does not prevent increased food intake, weight gain, or increased adiposity produced by chronic CORT.