Evaluation of the Treatment with Akkermansia muciniphila BAA-835 of Chemotherapy-induced Mucositis in Mice.

Mucositis is a high-incidence side effect in cancer patients undergoing chemotherapy. Next-generation probiotics are emerging as new therapeutic tools for managing various disorders. Studies have demonstrated the potential of Akkermansia muciniphila to increase the efficiency of anticancer treatment and to mitigate mucositis. Due to the beneficial effect of A. muciniphila on the host, we evaluated the dose-response, the microorganism viability, and the treatment protocol of A. muciniphila BAA-835 in a murine model of chemotherapy-induced mucositis. Female Balb/c mice were divided into groups that received either sterile 0.9% saline or A. muciniphila by gavage. Mucositis was induced using a single intraperitoneal injection of 5-fluorouracil. The animals were euthanized three days after the induction of mucositis, and tissue and blood were collected for analysis. Prevention of weight loss and small intestine shortening and reduction of neutrophil and eosinophil influx were observed when animals were pretreated with viable A. muciniphila at 1010 colony-forming units per mL (CFU/mL). The A. muciniphila improved mucosal damage by preserving tissue architecture and increasing villus height and goblet cell number. It also improved the integrity of the epithelial barrier, decreasing intestinal permeability and bacterial translocation. In addition, the treatment prevented the expansion of Enterobacteriaceae. The immunological parameters were also improved by decreasing the expression of pro-inflammatory cytokines (IL6, IL1ß, and TNF) and increasing IL10. In conclusion, pretreatment with 1010 CFU/mL of viable A. muciniphila effectively controlled inflammation, protected the intestinal mucosa and the epithelial barrier, and prevented Enterobacteriaceae expansion in treated mice.

Evaluation of a Functional Craft Wheat Beer Fermented with Saccharomyces cerevisiae UFMG A-905 to treat Salmonella Typhimurium infection in mice.

Functional foods containing probiotics are generally administered as dairy products. Non-dairy beverages are another possibility, but probiotic functionality must be confirmed in such vehicles. In the present study, a craft wheat beer brewed with the probiotic yeast Saccharomyces cerevisiae UFMG A-905 (905) was evaluated in a murine model of Salmonella Typhimurium infection. Unfiltered or filtered beer brewed with 905, a commercial wheat beer used as a negative control, or saline were administered orally to mice before and during oral S. Typhimurium challenge. High fecal levels of yeast were only counted in mice treated with the unfiltered 905 beer, which also had reduced mortality and body weight loss due to S. Typhimurium infection. Increased levels of intestinal IgA, translocation to liver and spleen, liver and intestinal lesions, pro-inflammatory cytokines in liver and ileum, and hepatic and intestinal myeloperoxidase and eosinophilic peroxidase activities were observed in animals infected with S. Typhimurium. All these parameters were reduced by the treatment with unfiltered 905 beer. In conclusion, the results show that a craft wheat beer brewed with S. cerevisiae UFMG A-905 maintained the probiotic properties of this yeast when administered orally to mice challenged with S. Typhimurium.

Anti-Mayaro virus activity of a hydroethanolic extract from Fridericia chica (Bonpl.) L. G. Lohmann leaves.

ETHNOPHARMACOLOGICAL RELEVANCE: Mayaro fever is a neglected tropical disease. The region of the most significant circulation of the Mayaro virus (MAYV) is the Amazon rainforest, situated in remote areas that are difficult to access and where medicine is scarce. Thus, the regional population uses plants as an alternative for the treatment of various diseases. Fridericia chica is an endemic plant of tropical regions used in traditional medicine to treat fever, malaise, inflammation, and infectious diseases such as hepatitis B. However, its antiviral activity is poorly understood. AIM OF THE STUDY: This study aimed to investigate the anti-MAYV activity of the hydroethanolic extract of the leaves of Fridericia chica (HEFc) in mammalian cells and its possible mechanism of action. MATERIALS AND METHODS: The antiviral activity of HEFc was studied using Vero cell lines against MAYV. The cytotoxicity and antiviral activity of the extract were evaluated by the 3-(4, 5- dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay. The overall antiviral activity was confirmed by the plaque forming units (PFU) method. Then, the effects of HEFc on MAYV multiplication kinetics, virus adsorption, penetration, and post-penetration, and its virucidal activity were determined in Vero cells using standard experimental procedures. RESULTS: HEFc exerted a effect against viral infection in Vero cells at a non-cytotoxic concentration, and no virion was detected in the supernatant in a dose-dependent and selective manner. HEFc inhibited MAYV in the early and late stages of the viral multiplication cycle. The extract showed significant virucidal activity at low concentrations and did not affect adsorption or viral internalization stages. In addition, HEFc reduced virions at all post-infection times investigated. CONCLUSIONS: HEFc has good antiviral activity against MAYV, acting directly on the viral particles. This plant extract possesses an excellent and promising potential for developing effective herbal antiviral drugs.

Antarctic Strain of Rhodotorula mucilaginosa UFMGCB 18,377 Attenuates Mucositis Induced by 5-Fluorouracil in Mice.

Mucositis is one of the most strenuous side effects caused by chemotherapy drugs, such as 5-fluorouracil (5-FU), during the treatment of several types of cancers. The disease is so prevalent and aggressive that many patients cannot resist such symptoms. However, despite its frequency and clinical significance, there is no effective treatment to prevent or treat mucositis. Thus, the use of probiotics as an adjuvant for the treatment has gained prominence. In the present study, we evaluated the effectiveness of oral administration of the Antarctic strain of Rhodotorula mucilaginosa UFMGCB 18,377 as an alternative to minimize side effects of 5-FU-induced mucositis in mice. Body weight, food consumption, stool consistency, and presence of blood in the feces were assessed daily in mice orally treated or not with the yeast and submitted or not to experimental mucositis. Blood, bones, and intestinal tissues and fluid were used to determine intestinal permeability and immunological, microbiological, and histopathological parameters. Treatment with R. mucilaginosa UFMGCB 18,377 was able to decrease clinical signs of the disease, such as reduction of food intake and body weight loss, and also decreased the number of intestinal enterobacteria and intestinal length shortening. Additionally, treatment was able to decrease the levels of MPO and EPO activities and inflammatory infiltrates, as well as the histopathological lesions characteristic of mucositis in the jejunum and ileum. Results of the present study showed that the oral administration of R. mucilaginosa UFMGCB 18,377 protected mice against mucositis induced by 5-FU.

Splenic, hepatic, renal and pulmonary clearance dysfunction associated with high-energy X-rays.

PURPOSE: To verify the high-energy X-rays effects on the blood clearance of colloidal particles by the spleen, liver, kidneys, and lungs. MATERIALS AND METHODS: Seventeen male Wistar rats were distributed into three groups. Group 1 (n = 5) - control - non-irradiated animals, group 2 (n = 6) - irradiated animals studied 24 h after irradiation, and group 3 (n = 6) - irradiated animals studied 48 h after irradiation. The animals were anesthetized and irradiated with a non-fractionated 8 Gy dose in the abdominal region divided into two parallel and opposite fields, 4 Gy was given to the anteroposterior and 4 Gy to the posteroanterior. This high dose of high-energy X-rays causes extensive cell killing, tissue disorganization and break down cell to cell communication. According to the groups, 50 µCi of technetium-phytate were injected into the right internal jugular vein. After 30 minutes, the liver, spleen, kidneys, and lungs were removed. The clot was harvested from the abdominal cavity two minutes after the sectioning of the abdominal aorta and cava vein. The organs and clot were placed into plastic flasks to be weighed and studied for the emission of radioactivity in a gamma radiation detector. The uptake function of each organ was calculated based on the count of gamma rays emitted per minute and normalized with the organ mass, having as a reference the radioactivity count of a standard sample. The arithmetic mean of each organ uptake was calculated and compared among the groups. RESULTS: After irradiation, the spleen uptake of colloidal radiopharmaceutical was greater, while the hepatic, renal, and pulmonary uptake were lower. The renal uptake decreased slower than the hepatic and pulmonary uptake. CONCLUSIONS: A single high dose of high-energy X-rays enhances the splenic clearance function, while it reduces the hepatic, renal, and pulmonary clearance until 48 h after irradiation, with a rapid deterioration of the hepatic and pulmonary uptake function.

Lyophilized Symbiotic Mitigates Mucositis Induced by 5-Fluorouracil.

Mucositis is an adverse effect of cancer chemotherapies using 5-Fluorouracil (5-FU). It is characterized by mucosal inflammation, pain, diarrhea, and weight loss. Some studies reported promising healing effects of probiotic strains, when associated with prebiotics, as adjuvant treatment of mucositis. We developed a lyophilized symbiotic product, containing skimmed milk, supplemented with whey protein isolate (WPI) and with fructooligosaccharides (FOS), and fermented by Lactobacillus casei BL23, Lactiplantibacillus plantarum B7, and Lacticaseibacillus rhamnosus B1. In a mice 5-FU mucositis model, this symbiotic lyophilized formulation was able to reduce weight loss and intestinal permeability. This last was determined in vivo by quantifying blood radioactivity after oral administration of 99mTc-DTPA. Finally, histological damages caused by 5-FU-induced mucositis were monitored. Consumption of the symbiotic formulation caused a reduced score of inflammation in the duodenum, ileum, and colon. In addition, it decreased levels of pro-inflammatory cytokines IL-1ß, IL-6, IL-17, and TNF-α in the mice ileum. The symbiotic product developed in this work thus represents a promising adjuvant treatment of mucositis.

Use of chitosan as pharmaceutical excipient in ocular drug delivery systems: Sterilization and pharmacokinetics.

The use of chitosan as a pharmaceutical excipient in the ocular field is already established. Nevertheless, some aspects related to its ocular administration, such as sterilization and excipient's pharmacokinetics, remain unclear. So, in this study, we evaluated those two relevant aspects, related to chitosan administration in eye. We used chitosan-based ocular inserts (CI) as formulation model. CI were produced by solvent/casting method and sterilized by saturated steam. Sterilization was confirmed by direct inoculation of inserts in suitable microbiological growth media. Physicochemical characterization of inserts before and after sterilization was performed. Results suggested that, although steam sterilization changed the arrangement of the matrix, the heat and the humidity did not modify the structure of the main polymeric chain. Pharmacokinetics of CI radiolabeled with technetium-99m (99m Tc) was assessed by scintigraphic images and ex vivo biodistribution study, after ocular administration in male Wistar rats. Scintigraphic and images analysis and ex vivo biodistribution study showed that the insert remained mainly in the eye until 6 hr after administration and its degradation products began to migrate to the abdominal cavity after 18 hr. Together, these data represent an important step forward the manufacturing and the clinical application of CI in the ophthalmic field.

Chitosan/hydroxyethyl cellulose inserts for sustained-release of dorzolamide for glaucoma treatment: In vitro and in vivo evaluation.

Eye drops containing hydrophilic drugs are commonly used to reduce intraocular pressure (IOP) in glaucoma patients, but compliance to the treatement is commonly reduced by frequent dosing and eventual systemic side effects. Sustained-release drug delivery systems, such as ocular inserts, can reduce dosing, limit systemic exposure, reduce side effects, and, then, improve patient adherence to therapy. Here, we developed and evaluated chitosan/hydroxyethyl cellulose-based ocular inserts for sustained release of dorzolamide, a hydrophilic drug. Dorzolamide inserts (DI) were produced by solvent/casting method and characterized by various physicochemical techniques. Pharmacokinetics studies were performed using scintigraphic images and ex vivo biodistribution. The effectiveness of inserts was tested in glaucomatous rats. Characterization studies showed that the drug strongly interacted with the polymeric matrix, but in vitro results showed that DI took only 3 h to release 75% of dorzolamide entraped. However, scintigraphic images and ex vivo biodistribution studies revealed that more than 50% of 99mTc-dorzolamide remained in the eye after 18 h of DI administration, while only about 30% of the drug remained in the eye after drops instilation. DI exerted significant hypotensive effect for two weeks, after single administration, while IOP values remained high in placebo and untreated groups. Eye drops were effective only during the treatment period. Only DI treatment prevented retinal ganglion cells death. Altogether, these findings evidenced the potential application of polymeric-based inserts for sustained release of dorzolamide in glaucoma management.

Mandible protraction alters Type I collagen, osteocalcin and osteonectin gene expression in adult mice condyle.

Mandible condyle remodeling is a great challenge on craniofacial growth studies. The great majority of the reports deals with growing period. However, there is a great necessity to clarify the importance of functional stimulation on adult mandible condyle remodeling. By using an adult mouse model, we investigated the influence of mandible forwarding on condyle remodeling and gene expression by bone forming cells. Tomographic and scintigraphic evaluations showed sagittal growth and cell activity enhancement. RT-PCR showed that Type I collagen, osteocalcin and osteonectin expression level can be altered. We showed that functional stimulation is necessary to maintain the regular gene expression by condyle bone forming cells in adult mice. It opens new frame for further investigations aiming new clinical approaches to temporomandibular joint problems treatment, as well as mandible retrusion treatment.

Preliminary data of the antipancreatic tumor efficacy and toxicity of long-circulating and pH-sensitive liposomes containing cisplatin.

PURPOSE: Pancreatic cancer is the fourth most common cause of cancer-related death in the USA. This is mainly because of the chemoresistance of this type of tumor; thus, the development of novel therapeutic modalities is needed. METHODS: Long-circulating and pH-sensitive liposomes containing cisplatin (SpHL-CDDP) were administered systemically into pancreatic tumor-bearing mice for a period of 14 days. The antitumor efficacy and toxicity of this new treatment method on the basis of cisplatin-loaded liposomes was compared with the classical free-CDDP method. Tc-HYNIC-ßAla-bombesin(7-14) tumor uptake and histopathologic findings were used to monitor and compare the two treatment modalities. RESULTS: The antitumor activity of SpHL-CDDP treatment was shown by (a) decrease in tumor volume, (b) development of tumor necrotic areas, and (c) decrease in Tc-HYNIC-ßAla-bombesin(7-14) tumor uptake. Toxicity was evaluated by the development of inflammation and necrotic areas in the kidneys, liver, spleen, and intestine: toxic effects were greater with free-CDDP than SpHL-CDDP. CONCLUSION: SpHL-CDDP showed significant antitumor activity in pancreatic cancer-bearing mice, with lower toxicity in comparison with free-CDDP.

Antileishmanial activity and evaluation of the mechanism of action of strychnobiflavone flavonoid isolated from Strychnos pseudoquina against Leishmania infantum.

The present study aimed to investigate the in vitro antileishmanial activity of strychnobiflavone flavonoid against Leishmania infantum, as well as its mechanism of action, and evaluate the ex vivo biodistribution profile of the flavonoid in naive BALB/c mice. The antileishmanial activity (IC50 value) of strychnobiflavone against stationary promastigote and amastigote-like stages of the parasites was of 5.4 and 18.9 µM, respectively; with a 50% cytotoxic concentration (CC50) value of 125.0 µM on murine macrophages, resulting in selectivity index (SI) of 23.2 and 6.6, respectively. Amphotericin B, used as a positive control, presented SI values of 7.6 and 3.3 for promastigote and amastigote-like stages of L. infantum, respectively. The strychnobiflavone was also effective in reducing in significant levels the percentage of infected macrophages, as well as the number of amastigotes per macrophage, after the treatment of infected macrophages using the flavonoid. By using different fluorescent probes, we investigated the bioenergetics metabolism of L. infantum promastigotes and demonstrated that the flavonoid caused the depolarization of the mitochondrial membrane potential, without affecting the production of reactive oxygen species. In addition, using SYTOX(®) green as a fluorescent probe, the strychnobiflavone demonstrated no interference in plasma membrane permeability. For the ex vivo biodistribution assays, the flavonoid was labeled with technetium-(99m) and studied in a mouse model by intraperitoneal route. After a single dose administration, the scintigraphic images demonstrated a highest uptake by the liver and spleen of the animals within 60 min, resulting in low concentrations after 24 h. The present study therefore demonstrated, for the first time, the antileishmanial activity of the strychnobiflavone against L. infantum, and suggests that the mitochondria of the parasites may be the possible target organelle. The preferential distribution of this compound into the liver and spleen of the animals could warrant its employ in the treatment of visceral leishmaniasis.

Evolving role of radiolabeled particles in detecting infection and inflammation, preliminary data with 99mTc-phytate in rats.

PURPOSE: The aim of this study was to evaluate the ability of phytate radiolabeled with technetium-99m (Tc-phytate) to identify inflammatory processes. MATERIALS AND METHODS: Radiolabeling efficiency analyses were carried out by thin-layer chromatography on silica gel strips, yielding a radiochemical purity of 92%. In addition, the partition coefficient of Tc-phytate, obtained in a mixture of n-octanol/water (1 : 1), showed hydrophilic features of the radiopharmaceutical. After Tc-phytate was administered into the tail vein of healthy and inflammation focus-bearing rats, induced, in the right tight, by zymosan suspension at 5% (w/v), blood clearance evaluation was performed and showed a short plasma half-life (2.7 min). In the inflammation focus-bearing rats, Tc-phytate scintigraphic images were obtained at 2, 4, and 8 h after radiotracer injection. RESULTS: A significant radiopharmaceutical uptake was found in mononuclear phagocyte system organs (liver and spleen) and in the inflammation focus (compared with contralateral muscle). Histopathological analysis showed an intense mononuclear infiltration in the inflamed muscle, suggesting that macrophages may be responsible for the greater radiotracer uptake in the inflamed site. Furthermore, the target-to-nontarget ratio (%ID/g of inflamed muscle-to-%ID/g of control muscle ratio) obtained by scintigraphic images performed at 2 h after the radiotracer injection was 10.24±3.49, remaining without any significant difference at 4 and 8 h. CONCLUSION: Inflammation focus was evident in the scintigraphic images from 2 to 8 h after Tc-phytate administration, suggesting that this radiopharmaceutical could be a potential alternative to identify inflamed regions.

An optimized nanoparticle delivery system based on chitosan and chondroitin sulfate molecules reduces the toxicity of amphotericin B and is effective in treating tegumentary leishmaniasis.

Amphotericin B (AmpB) is active against leishmaniasis, but its use is hampered due to its high toxicity observed in patients. In this study, a nanoparticles-delivery system for AmpB (NQC-AmpB), containing chitosan and chondroitin sulfate molecules, was evaluated in BALB/c mice against Leishmania amazonensis. An in vivo biodistribution study, including biochemical and toxicological evaluations, was performed to evaluate the toxicity of AmpB. Nanoparticles were radiolabeled with technetium-99m and injected in mice. The products presented a similar biodistribution in the liver, spleen, and kidneys of the animals. Free AmpB induced alterations in the body weight of the mice, which, in the biochemical analysis, indicated hepatic and renal injury, as well as morphological damage to the kidneys of the animals. In general, no significant organic alteration was observed in the animals treated with NQC-AmpB. Mice were infected with L. amazonensis and treated with the nanoparticles or free AmpB; then, parasitological and immunological analyses were performed. The NQC-AmpB group, as compared to the control groups, presented significant reductions in the lesion size and in the parasite burden in all evaluated organs. These animals presented significantly higher levels of IFN-γ and IL-12, and low levels of IL-4 and IL-10, when compared to the control groups. The NQC-AmpB system was effective in reducing the infection in the animals, and proved to be effective in diminishing the toxicity evoked by AmpB, which was observed when it was administered alone. In conclusion, NQC-AmpB could be considered a viable possibility for future studies in the treatment of leishmaniasis.

Dietary glutamine prevents the loss of intestinal barrier function and attenuates the increase in core body temperature induced by acute heat exposure.

Dietary glutamine (Gln) supplementation improves intestinal function in several stressful conditions. Therefore, in the present study, the effects of dietary Gln supplementation on the core body temperature (T core), bacterial translocation (BT) and intestinal permeability of mice subjected to acute heat stress were evaluated. Male Swiss mice (4 weeks old) were implanted with an abdominal temperature sensor and randomly assigned to one of the following groups fed isoenergetic and isoproteic diets for 7 d before the experimental trials: group fed the standard AIN-93G diet and exposed to a high ambient temperature (39°C) for 2 h (H-NS); group fed the AIN-93G diet supplemented with l-Gln and exposed to a high temperature (H-Gln); group fed the standard AIN-93G diet and not exposed to a high temperature (control, C-NS). Mice were orally administered diethylenetriaminepentaacetic acid radiolabelled with technetium (99mTc) for the assessment of intestinal permeability or 99mTc-Escherichia coli for the assessment of BT. Heat exposure increased T core (approximately 41°C during the experimental trial), intestinal permeability and BT to the blood and liver (3 h after the experimental trial) in mice from the H-NS group relative to those from the C-NS group. Dietary Gln supplementation attenuated hyperthermia and prevented the increases in intestinal permeability and BT induced by heat exposure. No correlations were observed between the improvements in gastrointestinal function and the attenuation of hyperthermia by Gln. Our findings indicate that dietary Gln supplementation preserved the integrity of the intestinal barrier and reduced the severity of hyperthermia during heat exposure. The findings also indicate that these Gln-mediated effects occurred through independent mechanisms.

Antiglaucomatous effects of the activation of intrinsic Angiotensin-converting enzyme 2.

PURPOSE: To evaluate the effects of the activation of endogenous angiotensin-converting enzyme 2 (ACE2) using the compound diminazene aceturate (DIZE) in an experimental model of glaucoma in Wistar rats. METHODS: DIZE (1 mg/kg) was administered daily, either systemically or topically, and the IOP was measured weekly. To examine the role of the Mas receptor in the effects of DIZE, the Ang-(1-7) antagonist A-779 was co-administered. Drainage of the aqueous humor was evaluated by using scintigraphy. The analysis of ACE2 expression by immunohistochemistry and the counting of retinal ganglion cells (RGCs) were performed in histologic sections. Additionally, the nerve fiber structure was evaluated by transmission electron microscopy. RESULTS: The systemic administration and topical administration (in the form of eye drops) of DIZE increased the ACE2 expression in the eyes and significantly decreased the IOP of glaucomatous rats without changing the blood pressure. Importantly, this IOP-lowering action of DIZE was similar to the effects of dorzolamide. The antiglaucomatous effects of DIZE were blocked by A-779. Histologic analysis revealed that the reduction in the number of RGCs and the increase in the expression of caspase-3 in the RGC layer in glaucomatous animals were prevented by DIZE. This compound also prevented alterations in the cytoplasm of axons in glaucomatous rats. In addition to these neuroprotective effects, DIZE facilitated the drainage of the aqueous humor. CONCLUSIONS: Our results evidence the pathophysiologic relevance of the ocular ACE2/Ang-(1-7)/Mas axis of the renin-angiotensin system and, importantly, indicate that the activation of intrinsic ACE2 is a potential therapeutic strategy to treat glaucoma.

Mitochondrial calcium regulates rat liver regeneration through the modulation of apoptosis.

UNLABELLED: Subcellular Ca(2+) signals control a variety of responses in the liver. For example, mitochondrial Ca(2+) (Ca(mit)(2+)) regulates apoptosis, whereas Ca(2+) in the nucleus regulates cell proliferation. Because apoptosis and cell growth can be related, we investigated whether Ca(mit)(2+) also affects liver regeneration. The Ca(2+)-buffering protein parvalbumin, which was targeted to the mitochondrial matrix and fused to green fluorescent protein, was expressed in the SKHep1 liver cell line; the vector was called parvalbumin-mitochondrial targeting sequence-green fluorescent protein (PV-MITO-GFP). This construct properly localized to and effectively buffered Ca(2+) signals in the mitochondrial matrix. Additionally, the expression of PV-MITO-GFP reduced apoptosis induced by both intrinsic and extrinsic pathways. The reduction in cell death correlated with the increased expression of antiapoptotic genes [B cell lymphoma 2 (bcl-2), myeloid cell leukemia 1, and B cell lymphoma extra large] and with the decreased expression of proapoptotic genes [p53, B cell lymphoma 2-associated X protein (bax), apoptotic peptidase activating factor 1, and caspase-6]. PV-MITO-GFP was also expressed in hepatocytes in vivo with an adenoviral delivery system. Ca(mit)(2+) buffering in hepatocytes accelerated liver regeneration after partial hepatectomy, and this effect was associated with the increased expression of bcl-2 and the decreased expression of bax. CONCLUSION: Together, these results reveal an essential role for Ca(mit)(2+) in hepatocyte proliferation and liver regeneration, which may be mediated by the regulation of apoptosis.