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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.
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PURPOSE: Anticancer-drug efficacy seems to involve the direct interaction with host immune cells. Although topoisomerase I (Top I) inhibitors have been suggested to block LPS-evoked inflammation, the interaction between these drugs and toll-like receptor 4 (TLR4) is unaddressed. METHODS: SN-38, the active metabolite of the Top I inhibitor irinotecan, and TLR4 interaction was assessed using the in vitro luciferase nuclear factor-κB reporter assay, neutrophil migration to murine air-pouch, in silico simulation, and the thermal shift assay (TSA). Topotecan was used as a positive anti-inflammatory control. RESULTS: Non-cytotoxic concentrations of SN-38 attenuated LPS (a TLR4 agonist)-driven cell activation without affecting peptidoglycan (a TLR2 agonist)-activating response. Similarly, topotecan also prevented LPS-induced inflammation. Conversely, increasing concentrations of LPS reversed the SN-38 inhibitory effect. In addition, SN-38 abrogated LPS-dependent neutrophil migration and reduced TNF-α, IL-6, and keratinocyte chemoattractant levels in the air-pouch model, but failed to inhibit zymosan (a TLR2 agonist)-induced cell migration. A two-step molecular docking analysis indicated two potential binding sites for the SN-38 in the MD-2/TLR4 complex, the hydrophobic MD-2 pocket (binding energy of - 8.1 kcal/mol) and the rim of the same molecule (- 6.9 kcal/mol). The topotecan also bound to the MD-2 pocket. In addition, not only the lactone forms, but also the carboxylate conformations of both Top I inhibitors interacted with the MD-2 molecule. Furthermore, the TSA suggested the interaction of SN-38 with MD-2. CONCLUSIONS: Therefore, SN-38 inhibits acute inflammation by blocking LPS-driven TLR4 signaling. This mechanism seems to be shared by other Top I inhibitors.
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Inflamación/tratamiento farmacológico , Irinotecán/uso terapéutico , Receptor Toll-Like 4/genética , Inhibidores de Topoisomerasa I/uso terapéutico , Animales , Humanos , Irinotecán/farmacología , Masculino , Ratones , Inhibidores de Topoisomerasa I/farmacologíaRESUMEN
Sepsis, an overwhelming inflammatory response syndrome secondary to infection, is one of the costliest and deadliest medical conditions worldwide. Neutrophils are classically considered to be essential players in the host defense against invading pathogens. However, several investigations have shown that impairment of neutrophil migration to the site of infection, also referred to as neutrophil paralysis, occurs during severe sepsis, resulting in an inability of the host to contain and eliminate the infection. On the other hand, the neutrophil antibacterial arsenal contributes to tissue damage and the development of organ dysfunction during sepsis. In this review, we provide an overview of the main events in which neutrophils play a beneficial or deleterious role in the outcome of sepsis.
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Cistite hemorrágica (CH) induzida por ifosfamida (IFO) é uma importante complicação clínica em pacientes com câncer. Atualmente, mesna e hiper-hidratação são utilizadas como profilaxia, a despeito de ainda ser observada CH através de cistoscopia e histopatologia mesmo com essas medidas. A participação de interleucina-1 (IL-1) e fator de necrose tumoral (TNF) na patogênese da CH provê alvos para o tratamento dessa doença. Assim, esse trabalho objetivou avaliar o efeito protetor do antagonista do receptor da IL-1 (anakinra) e do anticorpo anti-TNF-alfa (infliximabe) nas respostas inflamatórias, nociceptivas e funcionais da CH experimental induzida por IFO em camundongos. Foram utilizados camundongos Swiss, C57BL6, IL-1R-/-, CASP1-/-, TNFR1-/-, TNFR1/R2-/-. Os animais WT foram submetidos ao tratamento com anakinra 100 mg/kg i.p. ou infliximabe 5 mg/kgi.p. ou salina i.p., foram tratados 1h após com IFO 400 mg/kg i.p., e 12 h após a IFO foi realizado o sacrifício, com excisão das bexigas para avaliaçãomacroscópica, histopatológica, permeabilidade vascular, mieloperoxidase, contratilidade, cistometrografia e citometria de fluxo para neutrófilos e macrófagos. Alguns animais, antes do sacrifício, foram submetidos a avaliação de nocicepção visceral. Anakinra foi capaz de atenuar hemorragia, edema, infiltrado neutrofílico, hipernocicepção visceral e disfunção vesical...
Hemorrhagic cystitis (HC) induced by ifosfamide (IFO) is an importantclinical complication in patients with cancer. Despite prophylaxis, HC isobserved. The role of interleukin-1 (IL-1) and tumor necrosis factor (TNF) in the pathogenesis of HC provides targets for treatment. Thus, this study aimed to evaluate the protective effect of the IL-1 receptor antagonist (anakinra) and anti-TNF-alpha antibody (infliximab) in experimental HC-induced by IFO in mice. Swiss , C57BL6 , IL -1R-/-, CASP1-/-, TNFR1-/-, TNFR1/R2-/-mice were used. Animals were submitted to pre-treatment with anakinra 100 mg/ kg, ip or infliximab 5 mg/ Kg, ip, or saline ip, 1h after, they were treated with IFO 400 mg/ kg ip, and 12 h after IFO injection they were killed. Then, it was performed resection of the bladder for macroscopic and histopathological evaluation, vascular permeability assay, myeloperoxidase assay, muscle contractility, cistometrogram and flow cytometry to neutrophils andmacrophages. Some animals prior to death, were subjected to evaluation of visceral nociception. Anakinra was able to attenuate hemorrhage, edema, neutrophil infiltration, visceral hypernociception and bladder dysfunction...
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Humanos , Cistitis , Quimioterapia , Ifosfamida , Proteína Antagonista del Receptor de Interleucina 1 , Anticuerpos MonoclonalesRESUMEN
Introdução: O câncer colorretal (CCR) é a terceira neoplasia mais diagnosticada no mundo. A doença inflamatória intestinal é uma importante causa de CCR com patogênese atrelada à inflamação crônica. A sepse é uma doença grave que modifica a resposta imune do hospedeiro. Objetivou-se estudar se animais sobreviventes à sepse apresentam alteração na iniciação tumoral e crescimento tumoral do CCR. Material e métodos: Camundongos C57Bl6 machos foram submetidos ao modelo de ligadura e punção do ceco (CLP), e após 15 dias, os sobreviventes e um grupo controle receberam azoximetano (AOM, 10 mg/kg, i.p.) e 3 ciclos de 5 dias de dextran sulfato de sódio (DSS 2%, ad libitum) nos dias 20-25, 40-45, 60-65 pós-CLP. Foi avaliada a colite e o desenvolvimento de tumores por colonoscopia. Foram quantificadas citocinas nos dias 0 12, 52 e 65 e de células T reguladoras (Treg) nos dias 12 e 65. Dois grupos de animais C57 receberam ciclofosfamida (CYP) (100 mg/kg, i.p.) e dois grupos de animais com expressão do receptor de toxina diftérica (DTX) sob regulação do gene Foxp3 receberam DTX (0,5 mg/animal, i.p.) antes do protocolo AOM/DSS. Outro grupo de animais, foi submetido posteriormente à CLP após AOM/DSS, bem como um grupo de animais foi injetado células de CCR murino (MC38luc) 105 células no subcutâneo e outro grupo, 106 células no baço no 15 dias pós-CLP. Resultados: Animais sobreviventes à sepse apresentaram redução da colite, além de menor incidência, número de tumores e carga tumoral que animais controle. Os animais pós-sepse tiveram menor produção de IFN-γ nos cólon no dia 12, de IL-1ß, TNF, IL-6, IL-17, KC e MIG dias 12 e 65, e IL-33, TGF e GM-CSF no dia 65 quando comparados a animais controle. Além disso, animais pós-sepse apresentaram maior quantidade de Treg no D15, contudo nos dias 12 e 65 observou-se um aumento significativo dessas células nos animais controle comparado ao pós-sepse. Curiosamente, animais pós-sepse que receberam CYP e DTX apresentaram colite e tumores de maneira semelhante aos animais controle. Por outro lado, camundongos pós-sepse tratados previamente com AOM/DSS apresentaram aumento na carga tumoral 30 dias após a CLP quando comparados aos controles. Além disso, no modelo de tumor subcutâneo e esplênico, foi observado maior crescimento tumoral em animais pós-sepse em comparação aos controles. Conclusões: A sepse promove inibição da inflamação intestinal murina e do CCR associado à colite, de maneira dependente de células Treg.
Introduction: Colorectal cancer (CRC) is the third most diagnosed neoplasm in the world. Inflammatory bowel disease is an important cause of CRC with pathogenesis linked to chronic inflammation. Sepsis is a life-threatening disease that modify the host immune response. We aimed to study whether sepsis-surviving mice showed alterations of colorectal cancer initiation and tumor progression. Materials and methods: C57Bl6 male mice were submitted to cecal ligation and puncture (CLP) model, and after 15 days, sepsis-surviving mice and one control group received azoxymethane (AOM, 10 mg/kg, ip) and dextran sodium sulfate (DSS 2%, ad libitum, 3 cycles of 5 days) on days 20-25, 40-45, 60-65 post-CLP. Colitis and tumor development were evaluated by colonoscopy. Cytokines were quantified on days 0, 12, 52 and 65 by Milliplex and Treg cells on days 12 and 65 by flow cytometry. Two groups of C57 animals received cyclophosphamide (CYP) (100 mg/kg, ip), and two groups of animals expressing diphtheria toxin (DTX) receptor under regulation of Foxp3 received DTX (0.5 mg/animal, ip) before the AOM/DSS protocol. Another animal cohort was submitted to CLP after receiving AOM/DSS, as well as, two other groups were injected with murine CRC cells (MC38luc) 105 cells subcutaneously or 106 cells intra-splenically 15 days post-CLP. Results: Sepsis-surviving animals showed reduced colitis, besides lower incidence, number of tumors and tumor load than control animals. Sepsis-surviving animals have lower production of IFN-γ in the colon on day 12, and of IL-1ß, TNF, IL-6, IL-17, KC and MIG on days 12 and 65, in addition to IL-33, TGF and GM-CSF on day 65 than control animals. In addition, post-sepsis mice had a higher amount of Treg on D15, however on days 12 and 65 a significant increase of these cells occurs in control animals compared to post-sepsis. Interestingly, post-sepsis animals receiving CYP and DTX developed colitis and tumors in a similar pattern as control animals. Conversely, post-sepsis mice previously submitted to OM/DSS showed an increase in tumor load 30 days after CLP when compared to controls. Furthermore, in the subcutaneous and intra-splenic model, it was observed increased tumor growth in post-sepsis animals when compared to the controls. Conclusions: Sepsis promotes inhibition of murine intestinal inflammation and colitis-associated CRC in a Treg dependent manner.