Recombinant probiotic Lactococcus lactis delivering P62 mitigates moderate colitis in mice.

Introduction and objective: p62 is a human multifunctional adaptor protein involved in key cellular processes such as tissue homeostasis, inflammation, and cancer. It acts as a negative regulator of inflammasome complexes. It may thus be considered a good candidate for therapeutic use in inflammatory bowel diseases (IBD), such as colitis. Probiotics, including recombinant probiotic strains producing or delivering therapeutic biomolecules to the host mucosal surfaces, could help prevent and mitigate chronic intestinal inflammation. The objective of the present study was to combine the intrinsic immunomodulatory properties of the probiotic Lactococcus lactis NCDO2118 with its ability to deliver health-promoting molecules to enhance its protective and preventive effects in the context of ulcerative colitis (UC). Material and methods: This study was realized in vivo in which mice were supplemented with the recombinant strain. The intestinal barrier function was analyzed by monitoring permeability, secretory IgA total levels, mucin expression, and tight junction genes. Its integrity was evaluated by histological analyses. Regarding inflammation, colonic cytokine levels, myeloperoxidase (MPO), and expression of key genes were monitored. The intestinal microbiota composition was investigated using 16S rRNA Gene Sequencing. Results and discussion: No protective effect of L. lactis NCDO2118 pExu:p62 was observed regarding mice clinical parameters compared to the L. lactis NCDO2118 pExu: empty. However, the recombinant strain, expressing p62, increased the goblet cell counts, upregulated Muc2 gene expression in the colon, and downregulated pro-inflammatory cytokines Tnf and Ifng when compared to L. lactis NCDO2118 pExu: empty and inflamed groups. This recombinant strain also decreased colonic MPO activity. No difference in the intestinal microbiota was observed between all treatments. Altogether, our results show that recombinant L. lactis NCDO2118 delivering p62 protein protected the intestinal mucosa and mitigated inflammatory damages caused by dextran sodium sulfate (DSS). We thus suggest that p62 may constitute part of a therapeutic approach targeting inflammation.

Anti-Inflammatory, Antimicrobial, Antioxidant and Photoprotective Investigation of Red Propolis Extract as Sunscreen Formulation in Polawax Cream.

Many activities have been described for propolis, including, antiviral, antibacterial, antifungal, anti-inflammatory, immunoregulatory, antioxidant and wound healing properties. Recently, propolis has been highlighted due to its potential application in the pharmaceutical and cosmetic industries, motivating a better understanding of its antioxidant and anti-inflammatory activities. Propolis and its main polyphenolic compounds presented high antioxidant activity, and effectiveness as broad spectrum UVB and UVA photoprotection sunscreens. Through a qualitative phytochemical screening, the ethanolic red propolis extracts (EEPV) (70% at room temperature and 70% at a hot temperature) presented a positive result for flavonoids and terpenoids. It presented an antioxidant activity for reducing 50% of DPPH of 17 and 12 µg/mL for extraction at room temperature and at a hot temperature, respectively. The UPLC-QTOF-MS/MS analysis allowed the annotation of 40 substances for EEPV-Heated and 42 substances for EEPV-Room Temperature. The IC50 results of the ABTS scavenging activity was 4.7 µg/mL for both extractions, at room temperature and at a hot temperature. Additionally, we also evaluated the cytotoxic profile of propolis extracts against macrophage (RAW 264.7 cells) and keratinocytes (HaCaT cells), which showed non-cytotoxic doses in cell viability assays even after a long period of exposure. In addition, propolis extracts showed antibacterial activity for Gram-positive bacteria (Staphylococcus aureus and Staphylococcus epidermidis), demonstrating potential biological activity for the creation of formulations aimed at disease control and prevention.

Paraprobiotic Lacticaseibacillus rhamnosus Protects Intestinal Damage in an Experimental Murine Model of Mucositis.

Intestinal mucositis (IM) is a common side effect resulting from cancer treatment. However, the management so far has not been very effective. In the last years, the role of the gut microbiota in the development and severity of mucositis has been studied. Therefore, the use of probiotics and paraprobiotics could have a potential therapeutic effect on IM. The aim of our study was to investigate the impact of the administration of Lacticaseibacillus rhamnosus (L. rhamnosus) CGMCC1.3724 and the paraprobiotic on IM in mice. For 13 days, male Balb/c mice were divided into six groups: control (CTL) and mucositis (MUC)/0.1 mL of saline; CTL LrV and MUC LrV/0.1 mL of 108 CFU of viable Lr; CTL LrI and MUC LrI/0.1 mL of 108 CFU of inactivated Lr. On the 10th day, mice from the MUC, MUC LrV, and MUC LrI groups received an intraperitoneal injection (300 mg/kg) of 5-fluorouracil to induce mucositis. The results showed that the administration of the chemotherapeutic agent increased the weight loss and intestinal permeability of the animals in the MUC and MUC LrV groups. However, administration of paraprobiotic reduced weight loss and maintained PI at physiological levels. The paraprobiotic also preserved the villi and intestinal crypts, reduced the inflammatory infiltrate, and increased the mucus secretion, Muc2 gene expression, and Treg cells frequency.

Açaí (Euterpe oleracea Martius) Promotes Jejunal Tissue Regeneration by Enhancing Antioxidant Response in 5-Fluorouracil-Induced Mucositis.

Intestinal mucositis (IM) caused by antineoplastic chemotherapy is characterized by an important inflammatory process, which may compromise ongoing treatment. Our study aimed to investigate the effect of Açaí (Euterpe oleracea Martius) on the antioxidant response in BALB/c mice pretreated with Açaí pulp (200 g/kg) for 14 day. A group of animals receiving a single intraperitoneal injection of 5-FU (200 mg/kg) were euthanized on day three (D3) or seven (D7) after administration, the distal jejunum was isolated for the analyses of histology, superoxide dismutase (SOD) and catalase (CAT) enzyme activities, and concentration of total sulfhydryl groups (GSH). Seven days after induction, the intake of Açaí by the IM group almost completely regenerated tissue histology. Notably, SOD activity decreased in the MUC + Açaí group (D3). CAT activity reduced on D3 and D7 in the IM groups and Açaí treatment groups, respectively. No change was observed in the total GSH concentration at the tissue level. Our results demonstrated the protective effect of Açaí pulp components on intestinal damage induced by 5-FU, as well as the ability to control the response to oxidative stress, in order to mobilize defense pathways and promote tissue repair.

Host dysbiosis negatively impacts IL-9-producing T-cell differentiation and antitumour immunity.

BACKGROUND: Host-microbiota interactions shape T-cell differentiation and promote tumour immunity. Although IL-9-producing T cells have been described as potent antitumour effectors, their role in microbiota-mediated tumour control remains unclear. METHODS: We analysed the impact of the intestinal microbiota on the differentiation of colonic lamina propria IL-9-producing T cells in germ-free and dysbiotic mice. Systemic effects of the intestinal microbiota on IL-9-producing T cells and the antitumour role of IL-9 were analysed in a model of melanoma-challenged dysbiotic mice. RESULTS: We show that germ-free mice have lower frequency of colonic lamina propria IL-9-producing T cells when compared with conventional mice, and that intestinal microbiota reconstitution restores cell frequencies. Long-term antibiotic treatment promotes host dysbiosis, diminishes intestinal IL-4 and TGF-ß gene expression, decreases the frequency of colonic lamina propria IL-9-producing T cells, increases the susceptibility to tumour development and reduces the frequency of IL-9-producing T cells in the tumour microenvironment. Faecal transplant restores intestinal microbiota diversity, and the frequency of IL-9-producing T cells in the lungs of dysbiotic animals, restraining tumour burden. Finally, recombinant IL-9 injection enhances tumour control in dysbiotic mice. CONCLUSIONS: Host-microbiota interactions are required for adequate differentiation and antitumour function of IL-9-producing T cells.

Effect of Conjugated Linoleic Acid-enriched Butter After 24 hours of Intestinal Mucositis Induction.

Mucositis is the most common side effect due to chemotherapy or radiotherapy. It refers to the inflammation of intestinal mucous membranes, and it is associated with complications such as diarrhea, weight loss, and increased intestinal permeability (IP). This study was designed to evaluate the effect of diet containing conjugated linoleic acid (CLA)-enriched butter on intestinal damage and inflammatory response after 24 h of 5-fluorouracil (5-FU)-induced mucositis. Mice were divided into four groups: CTL; CLA; 5-FU, and CLA 5-FU, and they were fed for 31 days. On the 30th experimental day, mucositis was induced by unique injection of 300 mg/kg of 5-FU. After 24 h (31st experimental day), IP was evaluated; ileum and fecal material were collected to determine cytokine level and myeloperoxidase (MPO) activity and secretory immunoglobulin A (sIgA). The 5-FU group showed an increase in IP and MPO activity (CTL vs. 5-FU: P < 0.05). Additionally, increased levels of IP and MPO were observed in CLA 5-FU group compared to those in the test groups (P < 0.05). Animals in the CLA 5-FU group showed reduced concentrations of sIgA (CTL vs. CLA 5-FU: P < 0.05). CLA-enriched butter exacerbating the 5-FU-induced intestinal damage. Safety concerns regarding the use of CLA require further investigation.

The absence of microbiota delays the inflammatory response to Cryptococcus gattii.

The inflammatory response plays a crucial role in infectious diseases, and the intestinal microbiota is linked to maturation of the immune system. However, the association between microbiota and the response against fungal infections has not been elucidated. Our aim was to evaluate the influence of microbiota on Cryptococcus gattii infection. Germ-free (GF), conventional (CV), conventionalized (CVN-mice that received feces from conventional animals), and LPS-stimulated mice were infected with C. gattii. GF mice were more susceptible to infection, showing lower survival, higher fungal burden in the lungs and brain, increased behavioral changes, reduced levels of IFN-γ, IL-1ß and IL-17, and lower NFκBp65 phosphorylation compared to CV mice. Low expression of inflammatory cytokines was associated with smaller yeast cells and polysaccharide capsules (the main virulence factor of C. gattii) in the lungs, and less tissue damage. Furthermore, macrophages from GF mice showed reduced ability to engulf, produce ROS, and kill C. gattii. Restoration of microbiota (CVN mice) or LPS administration made GF mice more responsive to infection, which was associated with increased survival and higher levels of inflammatory mediators. This study is the first to demonstrate the influence of microbiota in the host response against C. gattii.

L-arginine supplementation prevents increases in intestinal permeability and bacterial translocation in male Swiss mice subjected to physical exercise under environmental heat stress.

Dietary supplementation with l-arginine has been shown to improve the intestinal barrier in many experimental models. This study investigated the effects of arginine supplementation on the intestinal permeability and bacterial translocation (BT) induced by prolonged physical exercise under heat stress. Under anesthesia, male Swiss mice (5-wk-old) were implanted with an abdominal sensor to record their core body temperature (T(core)). After recovering from surgery, the mice were divided into 3 groups: a non-supplemented group that was fed the standard diet formulated by the American Institute of Nutrition (AIN-93G; control), a non-supplemented group that was fed the AIN-93G diet and subjected to exertional hyperthermia (H-NS), and a group supplemented with l-arginine at 2% and subjected to exertional hyperthermia (H-Arg). After 7 d of treatment, the H-NS and H-Arg mice were forced to run on a treadmill (60 min, 8 m/min) in a warm environment (34°C). The control mice remained at 24°C. Thirty min before the exercise or control trials, the mice received a diethylenetriamine pentaacetic acid (DTPA) solution labeled with technetium-99m ((99m)Tc-DTPA) or (99m)Tc-Escherichia coli by gavage to assess intestinal permeability and BT, respectively. The H-NS mice terminated the exercise with T(core) values of ∼40°C, and, 4 h later, presented a 12-fold increase in the blood uptake of (99m)Tc-DTPA and higher bacterial contents in the blood and liver than the control mice. Although supplementation with arginine did not change the exercise-induced increase in T(core), it prevented the increases in intestinal permeability and BT caused by exertional hyperthermia. Our results indicate that dietary l-arginine supplementation preserves the integrity of the intestinal epithelium during exercise under heat stress, acting through mechanisms that are independent of T(core) regulation.

Efeito de dois probióticos, Saccharomyces boulardii e Saccharomyces cerevisiae linhagem UFMG 905, na resposta inflamatória induzida por Salmonella enterica subsp. enterica sorovar. Typhimurium / Effect of two probiotics, Saccharomyces boulardii and Saccharomyces cerevisiae strain UFMG 905, on the inflammatory response induced by Salmonella enterica subsp. enterica serovar. typhimurium

Probióticos são definidos como microrganismos viáveis que exibem um efeito benéfico na saúde do hospedeiro, quando ingeridos em quantidades suficientes. Muitas espécies de bactérias são usadas para esse fim e a única levedura utilizada como probióticos em seres humanos é Saccharomyces boulardii. Resultados anteriores obtidos em nosso laboratório mostraram que a levedura Saccharomyces cerevisiae linhagem UFMG 905, isolada da produção de cachaça, foi capaz de colonizar e sobreviver no trato gastrintestinal de camundongos, isentos de germes e convencionais, respectivamente, além de proteger esses animais contra um desafio oral com Salmonella enterica subsp. enterica sorovar Typhimurium e Clostridium difficile. Na primeira parte desse trabalho, os efeitos de S. cerevisiae UFMG 905 na translocação de Salm. Typhimurium para os linfonodos mesentéricos, placas de Peyer, baço e fígado, assim como o efeito no sistema imune, pela contagem de células de Küpffer, produção de imunoglobulinas e clareamento de Escherichia coli B41 da corrente sanguínea, foram avaliados em camundongos gnotobióticos e/ou convencionais. O tratamento com a levedura reduziu significantemente a translocação de Salm. Typhimurium para o fígado de animais gnotobióticos e, para todos os órgãos testados, em animais convencionais. O número de células de Küpffer (por 100 hepatócitos) foi significantemente maior (P < 0,05) em camundongos monoassociados com a levedura (52,9 ± 15,7) que em camundongos isentos de germes (38,1 ± 9,0). Provavelmente, como uma consequência do aumento do número de células de Küpffer, o clareamento de E. coli B41 da corrente sanguínea foi mais eficiente nos animais monoassociados com a levedura, quando comparado com os animais controle isentos de germes. Foram observados maiores níveis de sIgA no conteúdo intestinal e de IgA e IgM no soro (P < 0,05) nos camundongos monoassociados com a levedura que no grupo isento de germes. Concluindo, a proteção observada contra a bactéria enteropatogênica em nosso estudo anterior foi, provavelmente, devida à modulação local e sistêmica do sistema imune de camundongos tratados com S. cerevisiae UFMG 905. Na segunda parte desse trabalho, estudou-se os efeitos de S. boulardii e S. cerevisiae UFMG 905 na inflamação e na sinalização celular induzidos pela Salm. Typhimurium 14028, em células T84. Observou-se que os dois probióticos mantiveram a resistência elétrica transepitelial e diminuiram, significativamente, a secreção de IL-8 em células T84 infectadas por Salm. Typhimurium 14028 (P < 0,05). Saccharomyces cerevisiae UFMG 905 e S. boulardii diminuiram, significativamente, os níveis de invasão pela Salm. Typhimurium 14028 (P < 0,05), mas nenhum efeito das leveduras no xxxii crescimento ou adesão bacteriana em células T84 foi observado. Diferentemente de S. boulardii, S. cerevisiae UFMG 905 não foi capaz de diminuir a ativação de Rac1 e Cdc42 em células infectadas por Salm. Typhimurium 14028. Essas duas proteínas são Rho-GTPases ativadas pela bactéria e estão envolvidas na internalização de bactérias invasivas. A ligação de Salm. Typhimurium 14028 à superfície de S. cerevisiae UFMG 905 e S. boulardii, e não às células epiteliais do intestino, pode ser responsável pela diminuição da invasão e ativação de MAPKs (mitogen-activated protein kinases) e, conseqüentemente, pela diminuição dos níveis de IL-8, uma vez que esse processo diminui o número de bactérias ligadas às células T84. Entretanto, a diminuição dos níveis de IL-8 também pode ser explicada por uma imunomodulação, via secreção de citocinas anti-inflamatórias, como a IL-10, mas essa hipótese não foi testada nesse trabalho. A presença das leveduras em células infectadas com Salm. Typhimurium 14028 também reduziu e/ou inibiu a fosforilação das MAPKs ERK1/2, p38 e JNK, mas não diminuiu a ligação do fator de transcrição para IL-8 (NF-κB) ao DNA, sugerindo que a diminuição dos níveis de IL-8 não foi devido à inibição do fator de transcrição para IL-8, mas, provavelmente, ao efeito das leveduras na inibição da MAPK p38, que é a proteína responsável pela estabilização do mRNA para IL-8. Uma outra explicação é o fato de as duas leveduras inibirem a MAPK JNK, uma vez que AP-1 (outro fator de transcrição para IL-8) é dependente da fosforilação dessa proteína. Finalmente, S. cerevisiae UFMG 905 e S. boulardii inibem, também, a ativação de mecanismos antiinflamatórios (a via PI3K/Akt) induzidos por Salm. Typhimurium 14028 em células T84. Concluindo, S. cerevisiae UFMG 905 e S. boulardii apresentaram um efeito protetor e modulador na função de barreira e na sinalização celular induzida em células T84 pela Salm. Typhimurium 14028.

Probiotics are defined as viable microorganisms that exhibit a beneficial effect on the host health when ingested in adequate amounts. Many species of bacteria are used for this purpose and the only one yeast used as probiotic in humans is Saccharomyces boulardii. Previous results in our laboratory showed that Saccharomyces cerevisiae strain UFMG 905, isolated from "cachaça" production, was able to colonize and survive in the gastrointestinal tract of germ-free and conventional mice, respectively, and to protect these animals against oral challenge with Salmonella enterica subsp. enterica serovar Typhimurium and Clostridium difficile. In the first part of this work, the effects of S. cerevisiae UFMG 905 on the translocation of Salm. Typhimurium to mesenteric lymph nodes, Peyer's patches, spleen and liver, as well as on the immune system by number of Küpffer cells, immunoglobulin production and clearance of Escherichia coli B41, were evaluated in gnotobiotic and/or conventional mice. The treatment with the yeast reduced significantly the translocation of Salm. Typhimurium to liver in gnotobiotic animals and to all the organs tested in conventional mice. The number of Küpffer cells per 100 hepatocytes in liver was significantly higher (P < 0.05) in yeast mono-associated mice (52.9 ± 15.7) than in germ-free controls (38.1 ± 9.0). Probably, as a consequence, clearance of E. coli B41 from the bloodstream was more efficient in yeast mono-associated animals when compared to germ-free mice. Higher levels (P < 0.05) of sIgA in intestinal content and of IgA and IgM in serum were observed in yeast mono-associated mice when compared to germ-free group. Concluding, the protection against pathogenic bacteria observed in a previous study was probably due to a modulation of both local and systemic immunity of mice treated with S. cerevisiae UFMG 905. In a second part of this work, we have studied the effects of S. boulardii and S. cerevisiae UFMG 905 on the inflammation and signal transduction induced by Salm. Typhimurium ATCC 14028 in T84 cells. We have observed that both probiotics maintained the transmonolayer electrical resistance and significantly diminished IL-8 secretion in Salm. Typhimurium 14028-infected T84 cells (P < 0.05). Saccharomyces cerevisiae UFMG 905 and S. boulardii also decreased significantly the levels of Salm. Typhimurium 14028 invasion (P < 0.05), but had no effect on Salm. Typhimurium 14028 growth or adhesion to T84 cells. Differently from S. boulardii, S. cerevisiae UFMG 905 was not implicated in the diminution of the activation of Rac1 and Cdc42 in Salm. Typhimurium 14028-infected cells, which are Rho-GTPases activated by the bacteria involved in the internalization of invasive bacteria. The binding of Salm. Xxxv Typhimurium 14028 to S. cerevisiae UFMG 905 and S. boulardii surface instead of T84 cells may be responsible for the diminution of invasion and activation of MAPKs (mitogen-activated protein kinases) and consequently by the diminution of IL-8 levels, since this process diminishes the number of bacteria bound to T84 cells. However, the diminution of IL-8 may also be explained by an immunomodulation through secretion of anti-inflammatory cytokines, such as IL-10, but this hypothesis was not tested in this work. The presence of the yeasts in Salm. Typhimurium 14028-infected cells also reduced and/or inhibited the phosphorylation of ERK1/2, p38 and JNK MAPKs, but did not inhibit NF-κB DNA binding activity, suggesting that the diminution of IL-8 levels was not due to inhibition of the IL-8 transcription factor but more probably to the effects of the yeasts on the p38 MAPK inhibition, which is responsible for the stabilization of IL-8 mRNA. The inhibition of JNK by the yeasts may be another explanation, once AP-1, another IL-8 transcription factor, is dependent on the phosphorylation of this protein. Finally, S. cerevisiae UFMG 905 and S. boulardii inhibited the activation of anti-inflammatory mechanism (PI3K/Akt pathway) induced by Salm. Typhimurium 14028 in T84 cells. Concluding, S. cerevisiae UFMG 905 and S. boulardii showed a protective and modulating effect on barrier function and signal transduction pathway in T84 cells when infected by Salm. Typhimurium 14028.

Efeito do método de conservação na viabilidade, reativação, colonização intestinal e efeito imunomodulador de dois produtos* probióticos a base de leveduras / Effect of the conservation method on the viability, reactivation kinetic, intestinal colonization, and immunomodulation of two yeast-based probiotic products

Objetivo: O estudo teve como objetivos comparar a viabilidade, cineticas de reativação e de colonização intestinal, e efeito imunomodulador de dois produtos probioticos comercializados no Brasil nas formas liofilizada e em suspensão aquosa, contendo Saccharomyces boulardii e Saccharomyces cerevisiae, respectivamente.Metodos : As leveduras Saccharomyces boulardii e Saccharomyces cerevisiae foram fornecidas nas suas formas comerciais liofilizada e em suspensão aquosa, respectivamente. Os dois produtos são comercializados no Brasil e depois de obtidos em farmácia, eles foram codificados como as letras A (lote No 1040276, fabricado em março 2004) e B (lote N° 040 30 33, também fabricado em março 2004), respectivamente. Os experimentos foram iniciados em junho de 2004, sendo que os prazos de validade indicados nos produtos eram de março de 2006 e setembro de 2005 para os produtos A e B, respectivamente. Resultados: somente o produto A apresentopu contagens iguais ou superiores ao indicado na bula, sendo que esses níveis se mantiveram estáveis ao longo dos seis meses. Em conclusão, o produto A apresentou melhores características de viabilidade, reativação, colonização e imunomodulação e esses fatos devem ser ligados, em parte, aos métodos diferentes de conservação dos produtos probióticos.