Blockage of the fractalkine pathway reduces hyperalgesia and prevents morphological glial alterations-Comparison between inflammatory and neuropathic orofacial pain in male rats.

This study aimed to evaluate the effects of inhibitors of the fractalkine pathway in hyperalgesia in inflammatory and neuropathic orofacial pain in male rats and the morphological changes in microglia and satellite glial cells (SGCs). Rats were submitted to zymosan-induced arthritis of the temporomandibular joint or infraorbital nerve constriction, and treated intrathecally with a P2 X7 antagonist, a cathepsin S inhibitor or a p-38 mitogen-activated protein kinase (MAPK) inhibitor. Mechanical hyperalgesia was evaluated 4 and 6 h following arthritis induction or 7 and 14 days following nerve ligation. The expression of the receptor CX3 CR1 , phospho-p-38 MAPK, ionized calcium-binding adapter molecule-1 (Iba-1), and glutamine synthetase and the morphological changes in microglia and SGCs were evaluated by confocal microscopy. In both inflammatory and neuropathic models, untreated animals presented a higher expression of CX3 CR1 and developed hyperalgesia and up-regulation of phospho-p-38 MAPK, which was prevented by all drugs (p < .05). The number of microglial processes endpoints and the total branch length were lower in the untreated animals, but the overall immunolabeling of Iba-1 was altered only in neuropathic rats (p < .05). The mean area of SGCs per neuron was significantly altered only in the inflammatory model (p < .05). All morphological alterations were reverted by modulating the fractalkine pathway (p < .05). In conclusion, the blockage of the fractalkine pathway seemed to be a possible therapeutic strategy for inflammatory and neuropathic orofacial pain, reducing mechanical hyperalgesia by impairing the phosphorylation of p-38 MAPK and reverting morphological alterations in microglia and SGCs.

Inflammatory breast cancer microenvironment repertoire based on DNA methylation data deconvolution reveals actionable targets to enhance the treatment efficacy.

BACKGROUND: Although the clinical signs of inflammatory breast cancer (IBC) resemble acute inflammation, the role played by infiltrating immune and stromal cells in this aggressive disease is uncharted. The tumor microenvironment (TME) presents molecular alterations, such as epimutations, prior to morphological abnormalities. These changes affect the distribution and the intricate communication between the TME components related to cancer prognosis and therapy response. Herein, we explored the global DNA methylation profile of IBC and surrounding tissues to estimate the microenvironment cellular composition and identify epigenetically dysregulated markers. METHODS: We used the HiTIMED algorithm to deconvolve the bulk DNA methylation data of 24 IBC and six surrounding non-tumoral tissues (SNT) (GSE238092) and determine their cellular composition. The prognostic relevance of cell types infiltrating IBC and their relationship with clinicopathological variables were investigated. CD34 (endothelial cell marker) and CD68 (macrophage marker) immunofluorescence staining was evaluated in an independent set of 17 IBC and 16 non-IBC samples. RESULTS: We found lower infiltration of endothelial, stromal, memory B, dendritic, and natural killer cells in IBC than in SNT samples. Higher endothelial cell (EC) and stromal cell content were related to better overall survival. EC proportions positively correlated with memory B and memory CD8+ T infiltration in IBC. Immune and EC markers exhibited distinct DNA methylation profiles between IBC and SNT samples, revealing hypermethylated regions mapped to six genes (CD40, CD34, EMCN, HLA-G, PDPN, and TEK). We identified significantly higher CD34 and CD68 protein expression in IBC compared to non-IBC. CONCLUSIONS: Our findings underscored cell subsets that distinguished patients with better survival and dysregulated markers potentially actionable through combinations of immunotherapy and epigenetic drugs.

The role of adiposity, adipokines and polymorphisms of leptin and adiponectin in myelodysplastic syndromes.

The aim of the present study was to investigate the relationship between leptin and adiponectin gene polymorphisms, circulating levels of leptin and adiponectin, adiposity and clinical markers in patients with myelodysplastic syndrome (MDS). This cross-sectional study was conducted with 102 adults and elderly MDS patients and 102 age- and sex-matched controls. Clinical characteristics, co-morbidities, anthropometric data, laboratory evaluation and genetic analysis (polymorphisms -2548G > A/rs7799039 of the LEP gene and +276G > T/rs1501299 of the ADIPOQ gene) were investigated. Serum leptin was higher and adiponectin lower in MDS when compared with controls. There was a significant positive correlation between serum leptin levels and BMI (r = 0·264, P = 0·025), waist circumference (r = 0·235, P = 0·047), body fat percentage (BF %) (r = 0·373, P = 0·001) and the fat mass index (FMI) (r = 0·371, P < 0·001). A lower mean adiponectin was found among patients with high BF %, higher visceral adiposity index and metabolic syndrome. A significant association was found between the AA genotype (mutant) of the LEP polymorphism rs7799039 and male sex and blast excess (≥ 5 %). In addition, a significant association was observed between the TT genotype (mutant) of the ADIPOQ rs1501299 polymorphism and Fe overload. These results demonstrate the importance of a comprehensive and systematic evaluation in patients with MDS in order to identify and control negative factors not related to the disease at an early stage.

Umbelliferone reduces inflammation and ligature-induced osteoclastic alveolar bone resorption in mice.

AIMS: This study aimed to investigate the effects of Umbelliferone (UMB) on the inflammation underlying alveolar bone resorption in mouse periodontitis. METHODS: Male Swiss mice subjected to a ligature of molars were grouped as non-treated (NT), received UMB (15, 45, or 135 mg/kg) or saline daily for 7 days, respectively, and were compared with naïve mice as control. Gingival tissues were evaluated by myeloperoxidase (MPO) activity and interleukin-1ß level by ELISA. The bone resorption was directly assessed on the region between the cement-enamel junction and the alveolar bone crest. Microscopically, histomorphometry of the furcation region, immunofluorescence for nuclear factor-kappa B (NF-ĸB), and immunohistochemistry for tartrate-resistant acid phosphatase (TRAP), and cathepsin K (CTSK) were performed. Systemically, body mass variation and leukogram were analyzed. RESULTS: Periodontitis significantly increased MPO activity, interleukin-1ß level, and NF-ĸB+ immunofluorescence, and induced severe alveolar bone and furcation resorptions, besides increased TRAP+ and CTSK+ cells compared with naïve. UMB significantly prevented the inflammation by reducing MPO activity, interleukin-1ß level, and NF-ĸB+ intensity, besides reduction of resorption of alveolar bone and furcation area, and TRAP+ and CTSK+ cells compared with the NT group. Periodontitis or UMB treatment did not affect the animals systemically. CONCLUSION: UMB improved periodontitis by reducing inflammation and bone markers.

Oral carcinogenesis triggers a nociceptive behavior and c-Fos expression in rats' trigeminal pathway.

OBJECTIVE: To recognize changes that occur along the trigeminal pathway in oral cancer in order to establish an effective approach to pain control. METHODS: Wistar rats were divided into control and 4-NQO groups for 8, 12, 16, or 20 weeks. 4-NQO suspension was administered on the animals' tongues. Mechanical hyperalgesia, assessment of facial expressions, and an open-field test were performed. After euthanasia, the animals' tongues were removed for macro- and microscopic analysis. c-Fos expression was analyzed in the trigeminal pathway structures. RESULTS: 4-NQO induced time-dependent macroscopic lesions that were compatible with neoplastic tumors. Histopathological analysis confirmed oral squamous cell carcinoma in 50% of the animals on the 20th week. There was a significant nociceptive threshold reduction during the first two weeks, followed by a threshold return to the baseline levels, decreasing again from the 12th week. Facial nociceptive expression scores were observed on the 20th week, while increased grooming and exploratory activity were observed on the 8th week. Trigeminal ganglion showed an increased c-Fos immunoexpression on the 20th week, and in the trigeminal subnucleus caudalis, it occurred on the 16th and 20th. The long-term carcinogenic exposure caused changes in the nociceptive behavior and c-Fos expression in the rats' trigeminal pathway.

Effect of CYB2B6 (c.516G>T), CYP2C9 (c.1075A>C) and UGT1A9 (c.98T>C) polymorphisms on propofol pharmacokinetics in patients submitted to colonoscopy: a cohort study.

BACKGROUND: The aim of this study was to investigate the effect of CYB2B6 (c.516G>T, rs3745274), CYP2C9 (c.1075A>C, rs1057910) and UGT1A9 (c.98T>C, rs72551330) polymorphisms on the pharmacokinetics of single-drug propofol in adult patients undergoing intravenous sedation. METHODS: In this prospective clinical study, a total of 124 patients undergoing anaesthesia with propofol, as a single drug, were evaluated when undergoing colonoscopy procedure. Clinical variables were obtained from the patient's anamnesis prior to performing the anaesthetic procedure, in the moment of the patient's loss of consciousness, during the colonoscopy exam (recorded every 5 min) and in the awakening time. RESULTS: Polymorphic genotypes for the rs3745274 and rs1057910 polymorphisms were associated with bispectral index, target-controlled infusion (TCI)/effector concentration of propofol and TCI/plasma concentration of propofol values. Based on multivariate analysis, it was observed that weight, age, surgery time, systolic blood pressure and the rs1057910 polymorphism corresponded to predictive values for the dose of propofol used. Weight (B = 4.807±0.897), age (B = 1.834±0.834) and duration of surgery (B = 8.164±1.624) corresponded to factors associated with increased propofol dose, while systolic blood pressure (B = -1.892±0.679) and the genotypes (AA vs CA) of the single nucleotide polymorphism (SNP) rs1057910 CYPP2C9 gene (B = -74.161±26.820) decreased the total dose of propofol used. CONCLUSION: We concluded that the rs1057910 and rs3745274 polymorphisms affect the metabolism of propofol in patients exclusively submitted to this drug. Thus, the knowledge of the polymorphic genotypes of the CYPP2C9 and CYB2B6 genes may be predictive of different metabolising phenotypes, suggesting expected behaviours of BIS parameter in the anaesthetic procedure, which contributes to safer monitoring by anaesthesiologists during the clinical intervention.

Effect of CYB2B6 (c.516G>T), CYP2C9 (c.1075A>C) and UGT1A9 (c.98T>C) polymorphisms on propofol pharmacokinetics in patients submitted to colonoscopy: a cohort study.

BACKGROUND: The aim of this study was to investigate the effect of CYB2B6 (c.516G>T, rs3745274), CYP2C9 (c.1075A>C, rs1057910) and UGT1A9 (c.98T>C, rs72551330) polymorphisms on the pharmacokinetics of single-drug propofol in adult patients undergoing intravenous sedation. METHODS: In this prospective clinical study, a total of 124 patients undergoing anaesthesia with propofol, as a single drug, were evaluated when undergoing colonoscopy procedure. Clinical variables were obtained from the patient's anamnesis prior to performing the anaesthetic procedure, in the moment of the patient's loss of consciousness, during the colonoscopy exam (recorded every 5 min) and in the awakening time. RESULTS: Polymorphic genotypes for the rs3745274 and rs1057910 polymorphisms were associated with bispectral index, target-controlled infusion (TCI)/effector concentration of propofol and TCI/plasma concentration of propofol values. Based on multivariate analysis, it was observed that weight, age, surgery time, systolic blood pressure and the rs1057910 polymorphism corresponded to predictive values for the dose of propofol used. Weight (B = 4.807±0.897), age (B = 1.834±0.834) and duration of surgery (B = 8.164±1.624) corresponded to factors associated with increased propofol dose, while systolic blood pressure (B = -1.892±0.679) and the genotypes (AA vs CA) of the single nucleotide polymorphism (SNP) rs1057910 CYPP2C9 gene (B = -74.161±26.820) decreased the total dose of propofol used. CONCLUSION: We concluded that the rs1057910 and rs3745274 polymorphisms affect the metabolism of propofol in patients exclusively submitted to this drug. Thus, the knowledge of the polymorphic genotypes of the CYPP2C9 and CYB2B6 genes may be predictive of different metabolising phenotypes, suggesting expected behaviours of BIS parameter in the anaesthetic procedure, which contributes to safer monitoring by anaesthesiologists during the clinical intervention.

Plasma IL-33 levels are decreased in patients with high-risk myelodysplastic syndrome and show no correlation with pro-inflammatory IL-6 levels.

Aberrant inflammatory signaling has been shown to be a key pathogenic driver in myelodysplastic syndromes (MDS). Abnormal IL-33 expression has been implicated in inflammatory, immune-related disorders and, some tumors. However, its role in MDS remains widely unknown. This study aimed to evaluate the relationship between plasma levels of IL-33, clinical and prognostic data and, IL-6 levels in 101 patients with MDS. A comparative group of 59 healthy individuals was also evaluated. Plasma levels of cytokines were determined by enzyme-linked immunosorbent assay. Lower levels of IL-33 were found in patients with MDS when compared to the control group (p = 0.001), mainly in patients with more advanced stages of the disease and worse prognosis. No significant correlation between the levels of IL-33 and IL-6 was observed (r = 0.175; p = 0.081). These results reinforce the close association between immunological disorders and the pathogenesis of MDS. A greater understanding of the role of inflammatory cytokines in the disease can potentially provide new diagnosis and prognosis markers and new therapeutic targets.

Phosphoinositide 3-Kinase Gamma Inhibition Protects From Anthracycline Cardiotoxicity and Reduces Tumor Growth.

BACKGROUND: Anthracyclines, such as doxorubicin (DOX), are potent anticancer agents for the treatment of solid tumors and hematologic malignancies. However, their clinical use is hampered by cardiotoxicity. This study sought to investigate the role of phosphoinositide 3-kinase γ (PI3Kγ) in DOX-induced cardiotoxicity and the potential cardioprotective and anticancer effects of PI3Kγ inhibition. METHODS: Mice expressing a kinase-inactive PI3Kγ or receiving PI3Kγ-selective inhibitors were subjected to chronic DOX treatment. Cardiac function was analyzed by echocardiography, and DOX-mediated signaling was assessed in whole hearts or isolated cardiomyocytes. The dual cardioprotective and antitumor action of PI3Kγ inhibition was assessed in mouse mammary tumor models. RESULTS: PI3Kγ kinase-dead mice showed preserved cardiac function after chronic low-dose DOX treatment and were protected against DOX-induced cardiotoxicity. The beneficial effects of PI3Kγ inhibition were causally linked to enhanced autophagic disposal of DOX-damaged mitochondria. Consistently, either pharmacological or genetic blockade of autophagy in vivo abrogated the resistance of PI3Kγ kinase-dead mice to DOX cardiotoxicity. Mechanistically, PI3Kγ was triggered in DOX-treated hearts, downstream of Toll-like receptor 9, by the mitochondrial DNA released by injured organelles and contained in autolysosomes. This autolysosomal PI3Kγ/Akt/mTOR/Ulk1 signaling provided maladaptive feedback inhibition of autophagy. PI3Kγ blockade in models of mammary gland tumors prevented DOX-induced cardiac dysfunction and concomitantly synergized with the antitumor action of DOX by unleashing anticancer immunity. CONCLUSIONS: Blockade of PI3Kγ may provide a dual therapeutic advantage in cancer therapy by simultaneously preventing anthracyclines cardiotoxicity and reducing tumor growth.

Dual effect of silymarin on experimental non-alcoholic steatohepatitis induced by irinotecan.

Irinotecan-based regimens are commonly used for treatment of colorectal cancer, which is limited by mucositis and non-alcoholic steatohepatitis (NASH). Silymarin (SIL) prevents fatty liver disease in the clinical setting and in models of liver damage induced chemically. This study investigated the possible effect of SIL on irinotecan (IRI)-induced NASH. Swiss female mice were injected with saline (SAL 5ml/kg i.p.), IRI (50mg/kg i.p.), SIL (150mg/kg p.o.) or IRI (50mg/kg i.p.)+(SIL 1.5, 15 or 150mg/kg p.o.) thrice/week/7weeks. On the seventh week, blood samples were collected for transaminases assay and livers were collected for histopathology, measurement of the total lipids, malondyadehyde (MDA), non-protein sulfhydryl groups (NPSH), cytokines (IL-1ß, IL 6 and IL-10), 3-nitrotyrosine (N-Tyr) and toll-like receptor 4 (TLR4) immunoexpression, quantification of NF-kB, α-smooth muscle actin (α-SMA), and Escherichia coli 16S rRNA gene (RRS) expression. IRI increased liver transaminases, neutrophil infiltration, lipid accumulation, MDA, IL-1ß and IL-6 levels, N-Tyr and TLR4 immunostaining, NF-kB, α-SMA expression and RRS versus the SAL group (p<0.05). Additionally, SIL (1.5mg/kg) improved these parameters (p<0.05), except neutrophil infiltration and RSS versus the IRI group. Furthermore, the SIL (15mg/kg) only improved the inflammatory parameters, the expression of α-SMA and RRS versus the IRI group (p<0.05). The higher dose of SIL (150mg/kg) was even more deleterious than the intermediate dose. Therefore, silymarin showed a dual effect on liver damage induced by IRI. Hepatoprotection seems to involve the inhibition of oxidative stress and protein nitrosylation, preventing activation of hepatic fibrosis mechanisms.

Chronic treatment with zoledronic acid increases inflammatory markers in periodontium of rats.

BACKGROUND: Bisphosphonates (BF) rise proinflammatory markers and irreversibly bind to bone. Chronically, BF can lead to an inflammatory status and can increase the local oxidative stress in periodontium. Therefore, the objective of this study was to evaluate whether the chronic infusion of Zoledronic Acid (ZA) increases inflammatory markers in periodontium of rats. METHODS AND RESULTS: Chronically, infusion therapy was performed with ZA (0.04, 0.2 or 1 mg/kg or saline) by four doses in over a 70-day period to analyze periodontium of the first right inferior molar using histologic, histochemical (toluidine blue), and immunohistochemical (CD68, tumor necrosis factor-α (TNF-α), interleukin-1beta (IL-1ß), inducible nitric oxide synthase (iNOS) and nuclear factor kappa B (NF-kB)) tests. The experiment was replicated (ZA 0.2 mg/kg versus saline) for myeloperoxidase (MPO) assay and dose TNF-α, IL-1ß, malondialdehyde (MDA) and glutathione (GSH) in gingiva of the same tooth. Despite there is no alteration in mast cells (P = .608) and CD68 mononuclear-positive cells (P = .351), in the periodontium of the ZA-treated group, was observed an increase in the presence of inflammatory cells (P = .001) and cytoplasmic immunostaining for TNF-α (P = .003), IL-1b (P = .004), iNOS (P = .008), and NF-kB (P =  .025). Levels of MPO (P < .001), TNF-α (P = .002), IL-1ß (P < .001), and GSH (P = .005) were augmented in gingiva of ZA-treated group but MDA (P = .993) levels and NF-kB nuclear staining (P = .923) were not altered. CONCLUSIONS: Chronic treatment with ZA increase proinflammatory cytokines and the number of inflammatory cells in periodontium of rats and GSH are expressed probably in a compensatory manner.

Side-Effects of Irinotecan (CPT-11), the Clinically Used Drug for Colon Cancer Therapy, Are Eliminated in Experimental Animals Treated with Latex Proteins from Calotropis procera (Apocynaceae).

Intestinal mucositis (IM) is the critical side effect of irinotecan (CPT-11), which is the front-line drug used for the treatment of colorectal cancer. This study aimed to evaluate the effectiveness of latex proteins (LP) from Calotropis procera to prevent IM and diarrhea in animals. Swiss mice were treated daily with saline or LP (1, 5, or 50 mg/kg, i.v.) 24 h prior to CTP-11 (75 mg/kg/4 days, i.p) and for additional 6 days. Animal survival, body weight variation, and diarrhea were registered. After animal sacrifice (day 7 post first injection of CPT-11), intestinal samples were collected to study morphology and inflammatory parameters. Animals given LP exhibited improved parameters (survival, body weight, and absence of diarrhea) as compared with the CPT-11 control. The severity of IM observed in animals given CPT-11 was reduced in animals treated with LP. Treatment with LP also prevented the reduction in the villus/crypt ratio promoted by CPT-11. The rise in MPO activity and pro-inflammatory cytokines, over-contractility of the smooth muscle, and diarrhea were all abrogated in LP-treated mice. Markedly reduced immunostaining intensity for COX-2, TNF-α, IL-1ß, iNOS, and NF-κB was observed in the intestinal tissue of animals treated with LP. The side-effects of CPT-11 were eliminated by LP treatment in experimental animals and improved clinical parameters characteristic of IM All known biochemical pathogenesis. Copyright © 2016 John Wiley & Sons, Ltd.

Acute and neuropathic orofacial antinociceptive effect of eucalyptol.

Terpenes have a wide range of pharmacological properties, including antinociceptive action. The anti-inflammatory and antinociceptive effects of eucalyptol are well established. The purpose of this study was to evaluate the antinociceptive effect of eucalyptol on acute and neuropathic orofacial pain in rodent models. Acute orofacial and corneal nociception was induced with formalin, capsaicin, glutamate and hypertonic saline in mice. In another series, animals were pretreated with capsazepine or ruthenium red to evaluate the involvement of TRPV1 receptors in the effect of eucalyptol. In a separate experiment, perinasal tissue levels of IL-1ß, TNF-α and IFN-γ were measured. Rats were pretreated with eucalyptol before induction of temporomandibular joint pain with formalin or mustard oil. In another experiment, rats were submitted to infraorbital nerve transection (IONX) to induce chronic pain, followed by induction of mechanical hypersensitivity using Von Frey hairs. Locomotor performance was evaluated with the open-field test, and molecular docking was conducted on the TRPV1 channel. Pretreatment with eucalyptol significantly reduced formalin-induced nociceptive behaviors in all mouse strains, but response was more homogenous in the Swiss strain. Eucalyptol produced antinociceptive effects in all tests. The effect was sensitive to capsazepine but not to ruthenium red. Moreover, eucalyptol significantly reduced IFN-γ levels. Matching the results of the experiment in vivo, the docking study indicated an interaction between eucalyptol and TRPV1. No locomotor activity changes were observed. Our study shows that eucalyptol may be a clinically relevant aid in the treatment of orofacial pain, possibly by acting as a TRPV1 channel antagonist.

Is There an Interplay between Environmental Factors, Microbiota Imbalance, and Cancer Chemotherapy-Associated Intestinal Mucositis?

Interindividual variation in drug efficacy and toxicity is a significant problem, potentially leading to adverse clinical and economic public health outcomes. While pharmacogenetics and pharmacogenomics have long been considered the primary causes of such heterogeneous responses, pharmacomicrobiomics has recently gained attention. The microbiome, a community of microorganisms living in or on the human body, is a critical determinant of drug response and toxicity. Factors such as diet, lifestyle, exposure to xenobiotics, antibiotics use, illness, and genetics can influence the composition of the microbiota. Changes in the intestinal microbiota are particularly influential in drug responsiveness, especially in cancer chemotherapy. The microbiota can modulate an individual's response to a drug, affecting its bioavailability, clinical effect, and toxicity, affecting treatment outcomes and patient quality of life. For instance, the microbiota can convert drugs into active or toxic metabolites, influencing their efficacy and side effects. Alternatively, chemotherapy can also alter the microbiota, creating a bidirectional interplay. Probiotics have shown promise in modulating the microbiome and ameliorating chemotherapy side effects, highlighting the potential for microbiota-targeted interventions in improving cancer treatment outcomes. This opinion paper addresses how environmental factors and chemotherapy-induced dysbiosis impact cancer chemotherapy gastrointestinal toxicity.

Combining cashew gum with cyclophosphamide in murine melanoma model: A strategy for the reduction of side effects.

The understanding of cancer immunity and antitumor factors generated by natural polysaccharides is not yet fully comprehended. Polysaccharides, like cashew gum (CG), can exhibit immunomodulatory action and may assist in the antitumor process and side effects relieve. This study aimed to determine the antitumor effect of CG alone or in combination with cyclophosphamide (CTX), and its interactions with immune cells, in a murine melanoma model, using the B16-F10 cell line. Tumor growth inhibition, hematological, histopathological, ELISA, flow cytometry, immunofluorescence, and qRT-PCR analyses were performed to elucidate the antitumor potential, involvement of immune cells, and potential toxic effects. CG showed significant tumor growth inhibition, reaching up to 42.9 % alone and 51.4 % in combination with CTX, with mild toxicity to organs. CG enhanced leukocyte count, even in the presence of CTX. Furthermore, CG influenced the activation of tumor-associated macrophages (TAM), characterized by an increase in Il4, as well as a reduction in Ifng, Il1b, Tgfb, and Il6 gene expression. Nevertheless, these effects did not compromise the antitumor activity of CG. In summary, the combination of CG with CTX is a promising approach for leukopenia, one of the most important side effects of cancer treatment and deserves further investigation.

Paraprobiotic Enterococcus faecalis EC-12 prevents the development of irinotecan-induced intestinal mucositis in mice.

AIMS: This study tested the protective effect of purified paraprobiotic Enterococcus faecalis (EC-12) and an E. faecalis-based formulation (Med LanS) on irinotecan-induced intestinal mucositis murine model. MAIN METHODS: C57BL/6 male mice received saline, irinotecan (75 mg/Kg, i.p.), EC-12 (0.3, 1, or 3 × 107 CFU/Kg, p.o.) + irinotecan or Med Lan-S (3 × 107 CFU/Kg, p.o.) + irinotecan. Body mass variation was assessed daily, and blood samples were collected for evaluating bacteremia and leukocyte count. The ileum was harvested for myeloperoxidase assay, histopathology, quantitative PCR, and immunofluorescence for macrophages (F4/80), TLR4, and IL-18 binding protein (IL-18BP). KEY FINDINGS: The best therapeutic strategy was EC-12 administration at 3 × 107 CFU/Kg, starting 1 week before irinotecan. EC-12 and Med Lan-S did not prevent the irinotecan-induced body mass loss or leukopenia but attenuated the neutrophil infiltration in the intestine and increased the villus/crypt ratio (P < 0.05). Additionally, EC-12 and Med Lan-S reduced the mRNA expression of Cldn-2, Ocln, and Tlr4 versus the irinotecan group (P < 0.05). Irinotecan also augmented the expression of Il-18, IL-18BP, the immunofluorescence of F4/80, and TLR4, while only EC-12 prevented the expression of all these markers. Remarkably, EC-12 and Med Lan inhibited the irinotecan-induced bacterial translocation to the blood. SIGNIFICANCE: Paraprobiotic E. faecalis EC-12 prevents the development of intestinal mucositis by downregulating the inflammatory response. Med Lan-S also protects from mucositis. Possibly, the complexity of the formulation accounts for an innate immune-driven protective mechanism.

A Novel Murine Model of a High Dose Brachytherapy-Induced Actinic Proctitis.

Background: Radiation proctitis affects 1-20% of cancer patients undergoing radiation exposure due to pelvic malignancies, including prostate, gynecological and rectum cancers. The patients manifest rectal discomfort, pain, discharge, and bleeding. Notably, the efficacy of prophylactic measures remains controversial due to the lack of adequate animal models that mimic this condition. Objective: The present study then aimed to develop a murine model of high-dose-rate (HDR) brachytherapy-induced proctitis. Material/Methods: C57BL/6 male mice were subjected to HDR (radiation source: iridium-192 [Ir-192]) through a cylindrical propylene tube inserted 2 cm far from the anal verge into the rectum. The animals received radiation doses once a day for three consecutive days (fractions of 9.5 Grays [Gy]), 3.0 mm far from the applicator surface. The sham group received only the applicator with no radiation source. The survival rate was recorded, and a colonoscopy was performed to confirm the tissue lesion development. Following euthanasia, samples of the rectum were collected for histopathology, cytokines dosage (IL-6 and KC), and immunohistochemical analysis (TNF-α and COX-2). Results: HDR significantly reduced animals' survival ten days post first radiation exposure (14% survival vs. 100% in the non-irradiated group). Day seven was then used for further investigation. Mice exposed to radiation presented with rectum injury confirmed by colonoscopy and histopathology (P < 0.05 vs. the control group). The tissue damage was accompanied by an inflammatory response, marked by increased KC and IL-6 tissue levels, and immunostaining for TNF-α and COX-2 (P < 0.05 vs. control group). Conclusions: We established a novel animal model of actinic proctitis induced by HDR brachytherapy, marked by inflammatory damage and low animal mortality.

Role of capsaicin-sensitive primary afferent neurons and non-protein sulphydryl groups on gastroprotective effect of amifostine against ethanol-induced gastric damage in rats.

BACKGROUND: Amifostine has been widely tested as a cytoprotective agent against a number of aggressors in different organs. Recently, a gastroprotective effect was observed for this drug in a model of indomethacin-induced gastric injury. Our objective was to investigate the effect of amifostine on ethanol-induced gastric injury and the role played in this mechanism by afferent sensory neurons, non-protein sulfhydryl groups, nitric oxide, ATP-sensitive potassium channels, and cyclooxygenase-2. METHODS: Rats were treated with amifostine (22.5, 45, 90, or 180 mg/kg, PO or SC). After 30 min, the rats received absolute ethanol (5 ml kg(-1), PO). One hour later, gastric damage was quantified with a planimeter. Samples from the stomach were also taken for histopathological assessment and for assays of non-protein sulfhydryl groups. The other groups were pretreated with L-NAME (10 mg kg(-1), IP), glibenclamide (10 mg kg(-1), PO), or celecoxib (10 mg kg(-1), PO). After 30 min, the animals were given amifostine (90 mg kg(-1), PO or SC), followed 30 min later by gavage with absolute ethanol (5 ml kg(-1)). Other rats were desensitized with capsaicin (125 mg kg(-1), SC) 8 days prior to amifostine treatment. RESULTS: Amifostine administration PO and SC significantly and dose-dependently reduced ethanol-induced macroscopic and microscopic gastric damage by restoring glutathione levels in the stomach mucosa. Amifostine-promoted gastroprotection against ethanol-induced stomach injury was reversed by pretreatment with neurotoxic doses of capsaicin, but not by L-NAME, glibenclamide, or celecoxib. CONCLUSIONS: Amifostine protects against ethanol-induced gastric injury by increasing glutathione levels and stimulating the afferent sensory neurons in the stomach.

Cancerous and non-neoplastic stem cells in the stomach similarly express CD44 and CD133.

CD44 and CD133 have been considered as cancer stem cell (CSC) markers. Stem cell markers are rarely described in healthy stomach tissues. However, the clinicopathological and prognostic value of CD44 and CD133 in gastric cancer remains controversial. This study investigated the expression of CD44 and CD133 in gastric cancer and non-neoplastic gastric mucosa. We used samples of primary gastric adenocarcinomas (n = 69), metastatic lymph nodes (n = 30), intestinal metaplasia (n = 17), and histologically normal gastric tissues of surgical margins (n = 54). The expression of CD44 and CD133 were studied in samples by immunohistochemistry. Fisher's exact test and a logistic regression model were used in this study. CD44 expression was observed in 12% of samples with intestinal metaplasia, 20% with lymph node metastases, 22% with normal mucosa, to 30% of samples with primary tumors. Most of these positive tumors showed immunostaining in less than 4% of cancerous cells, mainly in the diffuse type. CD133 expression was observed in 7% (intestinal metaplasia) to 46% (normal mucosa). In the positive cases of cancer (24%), in most of them, less than 3% of cells were marked. CD44 and CD133 expression in the histologically normal gastric mucosa was restricted to the deeper regions of the gastric crypts at the level where stem cells and progenitor cells are usually found. CD44 and CD133 expression occurs in few gastric cancer cells, mainly in diffuse carcinomas, and are expressed in histologically normal gastric mucosae. None of the markers are specific for cancer and are also present in intestinal metaplasia and the normal mucosa.

Moderate Physical Exercise Activates ATR2 Receptors, Improving Inflammation and Oxidative Stress in the Duodenum of 2K1C Hypertensive Rats.

Background: In addition to the cardiovascular and renal systems, the gastrointestinal tract also contains angiotensin ATR1a, ATR1b, and ATR2. We previously observed that the 2Kidney-1Clip hypertension model elicits physical exercise and gastrointestinal dysmotility, which is prevented by renin-angiotensin system blockers. Here, we investigate the effect of physical exercise on inflammation, stress biomarkers, and angiotensin II receptors in the duodenum of 2K1C rats. Methods: Arterial hypertension was induced by the 2K1C surgical model. The rats were allocated in Sham, 2K1C, or 2K1C+Exercise groups. One week after surgery, they were submitted to a physical exercise protocol (running 5x/week, 60min/day). Next, we assessed their intestinal contractility, cytokine levels (TNF-α, IL-1ß, and IL-6), oxidative stress levels (MPO, GSH, MDA, and SOD), and the gene expression of angiotensin receptors (ATR1A, ATR1B, and ATR2). Results: In comparison with the Sham group, the 2K1C arterial hypertension decreased (p<0.05) the intestinal contractility. In comparison with 2K1C, the 2K1C+Exercise group exhibited lower (p<0.05) MPO activity (22.04±5.90 vs. 78.95±18.09 UMPO/mg tissue) and higher (p<0.05) GSH concentrations in intestinal tissues (67.63±7.85 vs. 31.85±5.90mg NPSH/mg tissue). The 2K1C+Exercise group showed lower (p<0.05) cytokine levels in the intestine than 2K1C rats. In comparison with the Sham group, the 2K1C+Exercise rats showed higher (p<0.05) gene expression of ATR2 in the duodenum. Conclusion: 2K-1C hypertension elicits an oxidative stress and inflammation process in the duodenum. Physical exercise modulates the expression twice as much of ATR2 receptors, suggesting possible anti-inflammatory and antioxidant effects induced by exercise.