The Prognostic and Predictive Value of Human Gastrointestinal Microbiome and Exosomal mRNA Expression of PD-L1 and IFNγ for Immune Checkpoint Inhibitors Response in Metastatic Melanoma Patients: PROTOCOL TRIAL.

BACKGROUND: Immunotherapy has been successful in treating advanced melanoma, but a large proportion of patients do not respond to the treatment with immune checkpoint inhibitors (ICIs). Preclinical and small cohort studies suggest gastrointestinal microbiome composition and exosomal mRNA expression of PD-L1 and IFNγ from the primary tumor, stool and body fluids as potential biomarkers for response. METHODS: Patients treated with immune checkpoint inhibitors as a first line treatment for metastatic melanoma are recruted to this prospective study. Stool samples are submitted before the start of treatment, at the 12th (+/-2) week and 28th (+/-2) week, and at the occurrence of event (suspected disease progression/hyperprogression, immune-related adverse event (irAE), deterioration). Peripheral venous blood samples are taken additionally at the same time points for cytologic and molecular tests. Histological material from the tumor tissue is obtained before the start of immunotherapy treatment. Primary objectives are to determine whether the human gastrointestinal microbiome (bacterial and viral) and the exosomal mRNA expression of PD-L1 and IFNγ and its dynamics predicts the response to treatment with PD-1 and CTLA-4 inhibitors and its association with the occurrence of irAE. The response is evaluated radiologically with imaging methods in accordance with the irRECIST criteria. CONCLUSIONS: This is the first study to combine and investigate multiple potential predictive and prognostic biomarkers and their dynamics in first line ICI in metastatic melanoma patients.

Retrospective analysis of treatment-naive Slovenian patients with metastatic melanoma treated with pembrolizumab - real-world experience.

Background Based on recent data from clinical trials, the immune checkpoint inhibitor pembrolizumab prolongs survival and has a good toxicity profile in patients with advanced or metastatic melanoma. However, the question remains whether these results are transmitted into daily clinical practice. The aim of this study was to assess the efficacy and toxicity of pembrolizumab in treatment-naive patients with metastatic melanoma in everyday clinical practice in Slovenia and compare it to the results from clinical trials. Patients and methods This observational retrospective cohort study included 138 consecutive metastatic treatment-naive melanoma patients treated with pembrolizumab at the Institute of Oncology Ljubljana in Slovenia, from January 2016 to December 2018. Patient and treatment characteristics were retrospectively collected from hospital data base. Statistical data was obtained using the SPSS software version 22. Survival rate was calculated with the Kaplan-Meier method. Observation period took place between January 2016 and the end of June 2019. Results The estimated median overall survival (OS) was 25.1 months (95% CI, 14.6-35.6) and the median progression-free survival (PFS) was 10.7 months (95% CI, 5.9-15.4). Among all patients, 29 (21.0%) achieved complete response, 31 (22.5%) partial response and 23 (16.7%) reached stable disease. The number of organs with metastatic involvement and the level of baseline lactate dehydrogenase (LDH) concentration had significant influence on survival rates. Immune-related adverse events (irAE) were reported in 88 (63%) patients, while grade 3-4 irAE occurred in 12 (8.7%). Due to toxicity, 16 (11.6%) patients discontinued the treatment. Conclusions Our real-world data from single centre retrospective analysis of treatment-naive metastatic melanoma patients treated with pembrolizumab showed inferior median OS and similar median PFS, compared to the results from clinical trials. However, patients with normal serum levels of LDH and a small number of organs with metastatic involvement had comparable survival outcomes. Toxicity rates of pembrolizumab were quite similar. These results further support the use of pembrolizumab for metastatic treatment-naive melanoma patients.

Influence of different wild-garlic (Allium ursinum) extracts on the gastrointestinal system: spasmolytic, antimicrobial and antioxidant properties.

OBJECTIVES: As there are no previous studies of the European wild-garlic (Allium ursinum) effects on the gastrointestinal system, despite its traditional applications in gastrointestinal disorders' treatment and regular use in the human diet, we have quantified and compared spasmolytic, antimicrobial and antioxidant activities of its different leaf extracts. METHODS: Wild-garlic extracts were tested for spasmolytic activity on isolated rat ileum, antimicrobial activity on selected Gram-positive and Gram-negative bacteria and fungi by microdilution method and antioxidant capacity by DPPH radical-scavenging assay. KEY FINDINGS: Wild-garlic extracts were found to decrease ileal basal tone. As the relaxation of K+ -induced contractions was similar to one caused by papaverin, the observed spasmolytic effect was most likely mediated through Ca2+ -channel inhibition. Ethanolic extract (with the highest phenolic and high alk(en)yl cysteine sulphoxides' levels) produced the strongest spasmolytic activity. In case of acetylcholine-induced contractions, only hydromethanolic extract showed no statistical difference in comparison with positive control. All samples exhibited certain antioxidant potential and strong antimicrobial activity against tested enteropathogenic strains (Salmonella enteritidis was the most sensitive, followed by Escherichia coli, Proteus mirabilis and Enterococcus faecalis). CONCLUSION: Besides other already established health-promoting effects, wild garlic could be useful in treatment of mild gastrointestinal disturbances.

Hypotensive, cardiodepressant, and vasorelaxant activities of black currant (Ribes nigrum 'Ben Sarek') juice.

The aim of this study was to evaluate the effects of black currant (Ribes nigrum L. 'Ben Sarek') juice on the blood pressure and frequency of cardiac contractions, as well as vasomotor responses of rat aortic rings. Arterial blood pressure was measured directly from the carotid artery in the anaesthetized rabbits. The aortic rings were pre-contracted with KCl (80 mmol·L-1), after which black currant juice was added. An intravenous injection of black currant juice (0.33-166.5 mg·kg-1) induced a significant and dose-dependent decrease of rabbit arterial blood pressure and heart rate. The black currant juice decreased arterial blood pressure of rabbit by 22.33% ± 3.76% (p < 0.05) and heart rate by 17.18% ± 2.93% (p < 0.05). Cumulative addition of the black currant juice (0.01-3 mg·mL-1) inhibited concentration-dependent KCl induced contractions of the isolated rat aorta. The black currant juice, at the concentration of 3 mg·mL-1, caused a maximum relaxation of 21.75% ± 3.15% (p < 0.05). These results demonstrate that black currant juice can induce hypotension. The hypotensive effect of the black currant may occur as the consequence of its inhibitory activity on the rate of heart contraction and vasorelaxant effects.

Radiation protection from whole-body gamma irradiation (6.7 Gy): behavioural effects and brain protein-level changes by an aminothiol compound GL2011 in the Wistar rat.

GL2011 is a naturally occurring thiol compound and a series of thiol compounds have been proposed as radioprotectors. Radioprotective efficacy of a triple intraperitoneal dose of GL2011 of 100 mg/kg body weight of Wistar rats, 30 min prior to and 3 and 6 h following irradiation (6.7 Gy) was evaluated. Four groups of animals were used, vehicle-treated non-irradiated (VN), GL2011-treated and irradiated (GI), GL2011-treated and non-irradiated (GN) and vehicle-treated and irradiated (VI) (n = 30 per group). The radioprotective efficacy of GL2011 was determined by measuring 28-day survival and intestinal crypt cell survival. Neuroprotection in terms of behaviour was evaluated using the behavioural observational battery, open field test and elevated plus maze paradigm. An RNA microarray was carried out in order to show differences at the RNA level between VI and VN groups. Brain protein changes were identified using a gel-based proteomics method and major brain receptor complex levels were determined by blue-native gels followed by immunoblotting. 28-Day survival rate in VI was 30 %, in GI survival was 93 %, survival of VN and GN was 100 %. Jejunal crypt cell survival was significantly enhanced in GI. Protein-level changes of peroxiredoxin-5, Mn-superoxide dismutase 2, voltage-dependent anion-selective channel protein 1, septin 5 and dopamine D2 receptor complex levels were paralleling radiation damage and protection. Taken together, the findings demonstrate that GL2011 improves survival rates and jejunal crypt survival, provides partial neuroprotection at the behavioural level and modulates proteins known to be involved in protection against oxidative stress-mediated cell damage.

Increased survival after irradiation followed by regeneration of bone marrow stromal cells with a novel thiol-based radioprotector.

AIM: To investigate the survival of laboratory rats after irradiation and to study the cellularity of their bone marrow and the multipotential mesenchymal stem cells (BM-MSCs) in groups treated with or without a new thiol-based radioprotector (GM2011). METHODS: Animals were irradiated by a Cobalt gamma source at 6.7 Gy. Treated animals were given i.p. GM2011 30 minutes before and 3 and 7 hours after irradiation. Controls consisted of sham irradiated animals without treatment and animals treated without irradiation. After 30 days post-irradiation, animals were sacrificed and bone marrow cells were prepared from isolated femurs. A colony forming unit-fibroblast (CFU-F) assay was performed to obtain the number of BM-MSCs. RESULTS: In the treated group, 87% of animals survived, compared to only 30% in the non-treated irradiated group. Irradiation induced significant changes in the bone marrow of the treated rats (total bone marrow cellularity was reduced by~60%--from 63 to 28 cells × 10(6)/femur and the frequency of the CFU-F per femur by~70% - from 357 to 97), however GL2011 almost completely prevented the suppressive effect observed on day 30 post-irradiation (71 cells × 10(6)/femur and 230 CFU-F/femur). CONCLUSION: Although the irradiation dosage was relatively high, GL2011 acted as a very effective new radioprotector. The recovery of the BN-MSCs and their counts support the effectiveness of the studied radioprotector.

ATP and ADP hydrolysis in cell membranes from rat myometrium.

Extracellular nucleotides affect female reproductive functions, fertilization, and pregnancy. The aim of this study was to investigate biochemical characteristics of ATP and ADP hydrolysis and identify E-NTPDases in myometrial cell membranes from Wistar albino rats. The apparent K (m) values were 506.4 ± 62.1 and 638.8 ± 31.3 µM, with a calculated V (max) (app) of 3,973.0 ± 279.5 and 2,853.9 ± 79.8 nmol/min/mg for ATP and ADP, respectively. The enzyme activity described here has common properties characteristic for NTPDases: divalent cation dependence; alkaline pH optimum for both substrates, insensitivity to some of classical ATPase inhibitors (ouabain, oligomycine, theophylline, levamisole) and significant inhibition by suramine and high concentration of sodium azides (5 mM). According to similar apparent K(m) values for both substrates, the ATP/ADP hydrolysis ratio, and Chevillard competition plot, NTPDase1 is dominant ATP/ADP hydrolyzing enzyme in myometrial cell membranes. RT-PCR analysis revealed expression of three members of ectonucleoside triphosphate diphosphohydrolase family (NTPDase 1, 2, and 8) in rat uterus. These findings may further elucidate the role of NTPDases and ATP in reproductive physiology.

Beneficial effects of calcium oral coadministration in gentamicin-induced nephrotoxicity in rats.

Frequent therapeutical use of an aminoglycoside antibiotic gentamicin (GM) is limited by its nephrotoxic effects often characterized by both morphological and functional alterations of kidney leading to acute renal failure. The aim of this study was to examine the effect of dietary calcium supplementation on GM-induced nephrotoxicity in rats. Experiments were performed on 30 adult male Wistar rats divided into three groups of 10 animals each. G-group received GM intraperitoneally at a dose of 100 mg/kg; GCa-group received the same dose of GM concomitantly with 1 g/kg calcium carbonate given orally; and C-group, serving as control, received 1 mL/day of normal saline. All groups were treated during 8 consecutive days. Quantitative evaluation of GM-induced structural and functional changes of kidney was performed by histopathological, morphometrical, and biochemical analyses. Compared with control, G-group of rats were found to have diffusely and unequally thickened glomerular basement membrane with neutrophil cells infiltration. In addition, vacuolization of cytoplasm of proximal tubule cells with coagulation-type necrosis was observed. These GM-induced pathological lesions were significantly reduced in the rats of GCa-group. Morphometric analysis revealed statistically significant differences in the size of glomeruli (area, major and minor axes, perimeter), optical density, and roundness of glomeruli (p < 0.05) between G and GCa groups. Biochemical analysis showed significant elevation in blood urea and serum creatinine concentrations, whereas potassium concentration was lowered in G-group compared with the other groups (p < 0.01). It is concluded that oral supplementation of calcium during treatment with GM resulted in significant reduction of morphological and functional kidney alterations.