Morphological features of the large intestine in experimental acute generalized peritonitis against the background of streptozotocin-induced diabetes

The work is a fragment of the research project “Pathogenetic features of the allergic and inflammatory processes course and their pharmacocorrection”, conducted by the Department of Pathological Physiology, Danylo Halytskyi Lviv National Medical University (state registration No. 0116U004503). The aim of the work: to study the morphological features of the colon in the dynamics of the development of experimental acute peritonitis on the background of streptozotocin-induced diabetes mellitus. Materials and Methods. The experiment used 48 white male rats. Diabetes mellitus in experimental animals was simulated by a single intraperitoneal injection of streptozotocin (Sigma) at a rate of 60 mg/kg, acute disseminated peritonitis – the introduction of 0.5 ml of 10 % filtered fecal suspension into the abdominal cavity. A morphological study of the colon in animals removed from the experiment on the first, third and seventh days of acute peritonitis on the background of concomitant diabetes mellitus was performed. Results and Discussion. Morphological examination of the colon of animals on the first day of experimental acute peritonitis in conditions of concomitant streptozotocin-induced diabetes mellitus revealed an increase in the size of the crypts due to stroma edema and lymphohistiocytic infiltration, slight perivascular edema in the subclavian edema. On the third day, thickening of the mucous membrane of the colon, a sharp increase in the depth of the crypts, uneven blood supply to the vessels in the submucosal layer with a predominance of perivascular edema were verified. On the seventh day, a decrease in the thickness of the mucous membrane due to the expansion of the crypts was visualized. Part of the epitheliocytes was in a phase of increased secretory activity, the other part was dystrophically altered, which stimulated increased lymphocytic and histiocytic infiltration. These changes were accompanied by activation of reactive processes and hyperplasia of lymphoid follicles on the first day of peritonitis on the background of streptozotocin-induced diabetes mellitus, and as the severity of purulent inflammation – hypoplasia of the lymphoid tissue and a decrease in local reactivity(the third and seventh days of the development of acute peritoneal burning in conditions of combined pathology).

Application of chitosan microparticles against human norovirus

Human norovirus (HuNoV) is the leading causative agent of foodborne outbreaks and is associated with the second most prevalent cause of waterborne infections in the United States. The goal of this research was to investigate the antiviral activity of chitosan microparticles (CM) against HuNoV GII.4 Sydney and its cultivable surrogate, Tulane virus (TuV), in suspensions mimicking fecally-contaminated water. CM was prepared by crosslinking chitosan molecules with sodium sulfate, and then its anti-noroviral activity was assessed using infectivity assay on TuV and RT-qPCR on TuV and HuNoV. A 3% CM suspension in PBS (pH 7.2) showed binding to TuV particles but with a negligible impact on virus infectivity (p>0.05). TuV and HuNoV suspended in fecal suspensions showed a 1.5-log10 reduction in genomic copies per ml following a 10-min contact time (p<0.05). Despite the negligible impact on viral infectivity, CM moderately binds to virus particles and helps purify environmental water by removing infectious virus particles. In this study, TuV served as a suitable surrogate for HuNoV by showing a similar log10 reduction in fecal suspension. Overall, the outcomes of thisresearch highlight the potential application of CM as a novel, natural treatment to minimize the spread of water-transmitted viral pathogens.

Gut Microbiota Dysbiosis Accelerates Prostate Cancer Progression Through Increased LPCAT1 Expression and Enhanced DNA Repair Pathways

Gut microbiota dysbiosis is related to cancer development and progression. Our previous study showed that Ruminococcus was more abundant in CRPC (Castration-resistant prostate cancer) than HSPC (Hormone-sensitive prostate cancer) individuals. Here, we determined the potential mechanism of microbiota dysbiosis in prostate cancer (PCa) progression. Metagenomics was used to verify the gut microbial discrepancies between CRPC and HSPC individuals. Fecal microbiota transplantation (FMT) was performed by transferring the fecal suspension of CRPC or HSPC individuals to TRAMP mice. Afterwards, the mice’s prostate histopathology and gut microbiota composition were determined. Since Ruminococcus was demonstrated to correlate with phospholipid metabolism, we used lipidomics to examine the mice’s fecal lipid profiles. The expression of LPCAT1 the key enzyme for phospholipid remodeling in mice prostate was also examined. Meanwhile, both microbial functions prediction and LPCAT1 GSEA analysis (Gene Set Enrichment Analysis) indicated DNA repair pathways, we further determined the expressions of RAD51 and DNA-PKcs in mice prostate. The results showed that gut Ruminococcus was significantly more abundant in CRPC individuals. FMT using CRPC feces accelerated mice’s PCa progression and increased their gut Ruminococcus abundance. Majority of fecal lipids including lysophosphatidylcholine and phosphatidylcholine were upregulated in CRPC FMT treated mice, accompanied with enhanced expressions of LPCAT1, RAD51, and DNA-PKcs in mice prostate. We reported an abundant colonization of Ruminococcus in the gut of CRPC individuals and mice receiving their fecal suspensions, and revealed the promotive capability of Ruminococcus in PCa progression via upregulating LPCAT1 and DNA repair protein expressions. The bacterium and its downstream pathways may become the targets of therapies for PCa in the future.

Development of a reference standard for the detection and quantification of Mycobacterium avium subsp. paratuberculosis by quantitative PCR

Quantitative PCR (qPCR) has become a frequently employed direct method for the detection and quantification of Mycobacterium avium subsp. paratuberculosis (MAP). The quantity of MAP determined by qPCR, however, may be affected by the type of qPCR quantification standard used (PCR product, plasmid, genomic DNA) and the way in which standard DNA quantity is determined (absorbance, fluorescence). In practice, this can be reflected in the inability to properly compare quantitative data from the same qPCR assays in different laboratories. Thus, the aim of this study was to prepare a prototype of an international MAP reference standard, which could be used to calibrate routinely used qPCR quantification standards in various laboratories to promote clinical data comparability. Considering stability, storage and shipment issues, a lyophilised fecal suspension artificially contaminated with a MAP reference strain was chosen as the most suitable form of the standard. The effect of five types of lyophilisation matrices on standard stability was monitored on 2-weeks interval basis for 4 months by F57 qPCR. The lyophilisation matrix with 10% skimmed milk provided the best recovery and stability in time and was thus selected for subsequent comparative testing of the standard involving six diagnostic and research laboratories, where DNA isolation and qPCR assay procedures were performed with the parallel use of the identical supplied genomic DNA solution. Furthermore, the effect of storage conditions on the standard stability was tested for at least 6 months. The storage at room temperature in the dark and under light, at + 4 °C, − 20 °C and − 80 °C showed no significant changes in the stability, and also no substantial changes in MAP viability were found using phage amplification assay. The prepared MAP quantification standard provided homogeneous and reproducible results demonstrating its suitability for utilisation as an international reference qPCR standard.

Experimental Validation of the Use of Immobilized Form of Miramistin in the Treatment of Advanced Peritonitis

Introduction. Mortality in generalized peritonitis ranges from 16% to 30%.The aim of research was to experimentally study the effect of the immobilized form of 0.01% miramistin on the basis of sodium salt of carboxymethylcellulose on the course of the inflammatory process in generalized purulent peritonitis.Materials and methods. The experimental study included 288 male Wistar rats, divided into 3 groups, 96 animals each. Animals of the 1st group (control) were exposed to laparotomy and lavage of the abdominal cavity with saline under aseptic conditions 24 hours after the introduction of fecal suspension into the abdominal cavity. Simultaneously, animals of the 2nd group (comparison) underwent a thorough sanitation of the abdominal cavity with saline with removal of purulent effusion and fibrin films at the first stage, and at the second stage, they were injected 5 ml of an aqueous solution of 0.01% miramistin. In animals of the 3rd (experimental) group, 5 ml of 0.01% miramistin gel was evenly distributed over the entire surface of the peritoneum after laparotomy and sanitation of the abdominal cavity with saline solution. The anti-inflammatory activity of the dosage forms was assessed by the dynamics of leukocytosis and the leukocyte index of intoxication, and the anti-inflammatory activity was assessed by the dynamics of the number of microorganisms in the abdominal exudate. The lethality of animals in each group was estimated. The following areas were taken for histological examination: small and large intestine, parietal peritoneum, pancreas, liver.Results. The anti-inflammatory activity of the immobilized form of 0.01% miramistin on the basis of sodium carboxymethylcellulose was superior to the aqueous solution of miramistin 0.01% on the 1st day - 1.3 times, on the 3rd day - 1.6 times, on the 7th day - 1.5 times. Antimicrobial activity in animals of the experimental group was 1.3 times higher on the 1st day, 1.9 times higher on the 3rd day, and 1.7 times higher on the 7th day than in the comparison group. The mortality rate in animals of the experimental group was 1.5 times lower on the 1st day, and 1.4 times lower on the 3rd and 7th days than in animals of the comparison group. On the first day, the morphological picture of peritonitis in animals of the experimental and comparison groups had no significant differences. On the 3rd day in animals of the comparison group, the inflammatory process in the abdominal cavity was pronounced, and in the experimental group, the intensity of peritonitis began to decrease, and by the 7th day it was completely eliminated.Conclusion. The results of the study allow recommending the use of the immobilized form of 0.01% miramistin based on sodium carboxymethylcellulose in the treatment of generalized peritonitis.

Maternal Gut Microbiota Transplantation Undermined the Lipid and Glucose Metabolism of Newborns in a Piglet Model

BackgroundThe present study was conducted to explore the maternal gut microbiota transplantation on the lipid and glucose metabolism of newborns in a piglet model. Six hysterectomy-derived germ-free (GF) Bama piglets were reared in three sterile isolators and were orally inoculated with healthy sow fecal suspension on day 7 after birth, which considers as fecal microbiota transplanted (FMT) group. Another six piglets from a natural birth and reared in conventional (CV) environments was regarded as CV group. The FMT piglets were hand-fed Co60-γ-irradiated sterile milk powder, CV piglets were reared by lactating Bama sows and both were weaned at day 21. Then, all piglets were housed separately and fed sterile feed for another 21 days. ResultsWe observed that transplanted with sow fecal microbiota increased the content of lipid in liver (P < 0.05), and upregulated the mRNA abundances of genes related to lipid anabolism and catabolism in liver and longissimus dorsi of newborn piglets (P < 0.05). Meanwhile, the concentrations of adiponectin, GLP-1, and insulin in serum and the activity of CPT-1 in liver were lower in FMT piglets (P < 0.05). Besides, transplanted with sow fecal microbiota enhanced the concentration of glycogen in liver (P < 0.05), while upregulated the mRNA expressions of genes related to glycogenesis and glycogenolysis in liver and longissimus dorsi of newborn piglets (P < 0.05). Moreover, the pathway of AMPK was stimulated by sow fecal microbiota transplantation (P < 0.10). In addition, the microbial structure between FMT and CV piglets was marked differently (P < 0.05). Furthermore, transplanted with sow fecal microbiota markedly activated the metabolic pathway of bile secretion in newborn piglets (P < 0.05). ConclusionsCollectively, healthy sow gut microbiota transplanted to newborn germ-free piglets might undermine the homeostasis of glucose and lipid metabolism, and increased the content of lipid, and decreased the concentration of glycogen in liver. It is concluded that transplanted with maternal gut microbiota might induce diseases related to glucose and lipid metabolism in newborns.

Fecal Microbiota Transplant: Latest Addition to Arsenal Against Recurrent Clostridium Difficile Infection

: An infectious disease of colon, recurrent Clostridium difficile infection (RCDI) is hitherto considered insurmountable leading to significant morbidity and mortality. Gut dysbiosis, generally resulting from frequent use of antibiotics is considered to be responsible for the etiopathogenesis of rCDI. Ironically, the conventional treatment strategies for the disease also include the use of anti-infective drugs such as metronidazole, vancomycin and fidaxomycin. As a result of the efforts to overcome the limitations of these treatment options to control recurrence of disease, Fecal Microbiota Transplant (FMT) has emerged as an effective and safe alternative. It is pertinent to add here that FMT is defined as the process of engraftment of fecal suspension from the healthy person into the gastrointestinal tract of the diseased individual aiming at the restoration of gut microbiota. FMT has proved to be quite successful in the treatment of recurrent and resistant Clostridium difficile infections (RCDI). In last three decades a lot of information has been generated on the use of FMT for RCDI. A number of clinical trials have been reported with generally very high success rates. However, very small number of patents could be found in the area indicating that there still exists lacuna in the knowledge about FMT with respect to its preparation, regulation, mode of delivery and safety. The current review attempts to dive deeper to discuss the patents available in the area while supporting the information contained therein with the non-patent literature.

Alteration of fecal microbiota by fucoxanthin results in prevention of colorectal cancer in AOM/DSS-treated mice

Fucoxanthin (Fx), a marine carotenoid found in edible brown algae, is well known for having anti-cancer properties. The gut microbiota has been demonstrated as a hallmark for colorectal cancer (CRC) progression in both humans and rodents. However, it remains unclear whether the gut microbiota is associated with the anti-cancer effect of Fx. We investigated the chemopreventive potency of Fx and its effect on gut microbiota in a mouse model of inflammation-associated CRC (by azoxymethane [AOM]/ dextran sulfate sodium [DSS] treatment). Fx administration (30 mg/kg body weight) during a 14-week period significantly inhibited the multiplicity of colorectal adenocarcinoma in mice. The number of apoptosis-like cleaved caspase-3 high cells increased significantly in both colonic adenocarcinoma and mucosal crypts. Fx administration significantly suppressed Bacteroidlales (f_ui; g_ui) (0.3-fold) and Rikenellaceae (g_ui) (0.6-fold) and increased Lachnospiraceae (g_ui) (2.2-fold), compared with those of control mice. Oral administration of a fecal suspension obtained from Fx-treated mice, aimed to enhance Lachnospiraceae, suppress the number of colorectal adenocarcinomas in AOM/DSS-treated mice with a successful increase in Lachnospiraceae in the gut. Our findings suggested that an alteration in gut microbiota by dietary Fx might be an essential factor in the cancer chemopreventive effect of Fx in AOM/DSS-treated mice.

A key role of gut microbiota-vagus nerve axis and gut microbiota-spleen axis in sleep deprivation-mediated aggravation of systemic inflammation after LPS administration

Background: Sleep deprivation (SD) is shown to be correlated with exacerbated systemic inflammation after sepsis. However, the underlying mechanisms remain unclear. Methods: In this study, mice were intraperitoneally injected with lipopolysaccharide (LPS) followed by 3 consecutive days of SD. The subdiaphragmatic vagotomy (SDV) was performed 2 weeks before LPS injection. The pseudo germ-free mouse model was created by administering antibiotics for 14 consecutive days, and then fecal microbiota transplant (FMT) was performed by gavaging supernatant from fecal suspension of septic mice with or without SD into pseudo germ-free mice with or without SDV or splenectomy. Splenectomy was performed 14 days prior to antibiotics administration.Results: We found that SD after LPS administration increased the plasma levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), reduced IL-10 plasma leve, increased spleen weight, and promoted pathological injury and IL-6 expression in lung, liver and kidney. Post-septic sleep deprivation had no effects on the diversity of gut microbiota. However, the relative abundance of Proteobacteria and its subgroups were increased in septic mice received SD. Pseudo germ-free mice transplanted with fecal bacteria from septic mice subjected to SD developed splenomegaly, systemic inflammation, organ inflammation and damage as their donors do. Intriguingly, SDV abolished the aggravated effects of SD on splenomegaly and inflammatory organ injury in septic mice received SD or in pseudo germ-free mice transplanted with fecal bacteria from septic mice subjected to SD. Furthermore, Splenectomy also abrogated the increase in IL-6 and TNF-α plasma levels and the decrease in IL-10 plasma level in pseudo germ-free mice transplanted with fecal bacteria from septic mice subjected to SD. Conclusions: Taken together, our results suggest that gut microbiota-vagus nerve axis and gut microbiota-spleen axis could play key roles in modulating systemic inflammation induced by SD after LPS administration.

Intestine wall histostructure peculiarities with peritonitis and mechanical intestine obstruction (experimental study)

Today, the histological criteria for differential diagnosis of dynamic ileus due to peritonitis and mechanical obstruction of the intestine remain undeveloped. In this regard, the aim of the work was to establish the difference in morphological changes occurring in the intestinal wall during dynamic and mechanical ileus in the experiment. The experiment was conducted on 33 sexually mature Wistar rats. In 15 animals of the first group, mechanical ileus was modeled by ligation of the lumen of the small intestine at the middle of the distance between the duodenojejunal junction and the ileocecal angle. In 15 rats of the second group, a dynamic ileus model was formed in the form of peritonitis by introducing fecal suspension into the lumen of the abdominal cavity. The control group included 3 animals who underwent laparotomy without the formation of mechanical ileus and peritonitis. For histological examination, fragments of the intestinal wall were sampled 1 cm above the site of the obstruction with mechanical ileus and the portion of the small intestine with peritonitis. Statistical processing was performed in an Excel package using parametric statistics methods. It was stated that with mechanical ileus purulent inflammation develops in the intestine wall beginning from the mucous membrane spreading over wall thickness which can cause its destruction within 48 hours; with dynamical ileus purulent inflammation develops in the intestine wall, it captures particularly serous and muscle layers without causing violations of mucosa cover structure and without intestine wall destruction within 48 hours. Under experimental dynamic ileus, changes in the mucous membrane were reactive in nature and consisted of manifestations of compensatory-adaptive and regenerative processes in response to a violation of the trophism of various structures of the intestinal wall.