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AIMS: Immune checkpoint inhibitors (ICIs) are antineoplastic drugs designed to activate the immune system's response against cancer cells. Evidence suggests that they may lead to immune-related adverse events, particularly when combined (e.g., anti-CTLA-4 plus anti-PD-1), sometimes resulting in severe conditions such as myocarditis. We aimed to investigate whether a previously sustained cardiac injury, such as pathological remodelling due to hypertension, is a prerequisite for ICI therapy-induced myocarditis. METHODS: We evaluated the cardiotoxicity of ICIs in a hypertension (HTN) mouse model (C57BL/6). Weekly doses were administered up to day 21 after the first administration. Our analysis encompassed the following parameters: (i) survival and cardiac pathological remodelling, (ii) cardiac function assessed using pressure-volume (PV)-loops, with brain natriuretic peptide (BNP) serving as a marker of haemodynamic dysfunction and (iii) cardiac inflammation (cytokine levels, infiltration, and cardiac antigen autoantibodies). RESULTS: After the first administration of ICI combined therapy, the treated HTN group showed a 30% increased mortality (P = 0.0002) and earlier signs of hypertrophy and pathological remodelling compared with the untreated HTN group. BNP (P = 0.01) and TNF-α (<0.0001) increased 2.5- and 1.7-fold, respectively, in the treated group, while IL-6 (P = 0.8336) remained unchanged. Myocarditis only developed in the HTN group treated with ICIs on day 21 (score >3), characterised by T cell infiltration and increased cardiac antigen antibodies (86% showed a titre of 1:160). The control group treated with ICI was unaffected in any evaluated feature. CONCLUSIONS: Our findings indicate that pre-existing sustained cardiac damage is a necessary condition for ICI-induced myocarditis.

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A key problem in colorectal cancer (CRC) is the development of resistance to current therapies due to the presence of cancer stem cells (CSC), which leads to poor prognosis. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a protein that activates apoptosis in cancer cells through union with TRAIL death receptors. Cell therapies as delivery systems can produce soluble TRAIL (sTRAIL) and full-length TRAIL (flTRAIL), showing a high capacity to produce apoptosis in vitro and in vivo assays. However, the apoptotic activity of TRAIL as monotherapy had limitations, so it is important to explore other ways to enhance susceptibility to TRAIL. This study evaluated the cytotoxic and proapoptotic activity of soluble TRAIL overexpressed by mesenchymal stem cells (MSC) in an oxaliplatin-resistant CRC cell line. Bone marrow-MSC were lentiviral transduced for soluble TRAIL expression. DR5 death receptor expression was determined in Caco-2 and CMT-93 CRC cell lines. Sensitivity to first-line chemotherapies and recombinant TRAIL was evaluated by half-maximal inhibitory concentrations. Cytotoxic and proapoptotic activity of soluble TRAIL-MSC alone and combined with chemotherapy pre-treatment was evaluated using co-cultures. Caco-2 and CMT-93 cell lines expressed 59.08 ± 5.071 and 51.65 ± 11.99 of DR5 receptor and had IC50 of 534.15 ng/mL and 581.34 ng/mL for recombinant murine TRAIL (rmTRAIL), respectively. This finding was classified as moderate resistance to TRAIL. The Caco-2 cell line showed resistance to oxaliplatin and irinotecan. MSC successfully overexpressed soluble TRAIL and induced cancer cell death at a 1:6 ratio in co-culture. Oxaliplatin pre-treatment in the Caco-2 cell line increased the cell death percentage (50%) and apoptosis by sTRAIL. This finding was statistically different from the negative control (p < 0.05), and activity was even higher with the oxaliplatin-flTRAIL combination. Thus, oxaliplatin increases apoptotic activity induced by soluble TRAIL in a chemoresistant CRC cell line.

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Parkinson's disease (PD) is characterized by the loss of dopaminergic neurons in the substantia nigra pars compact (SNpc), and no effective treatment has yet been established to prevent PD. Neurotrophic factors, such as cerebral dopamine neurotrophic factor (CDNF), have shown a neuroprotective effect on dopaminergic neurons. Previously, we developed a cell-penetrating-peptide-based delivery system that includes Asn194Lys mutation in the rabies virus glycoprotein-9R peptide (mRVG9R), which demonstrated a higher delivery rate than the wild-type. In this study, using a mouse PD-like model, we evaluated the intrastriatal mRVG9R-KP-CDNF gene therapy through motor and cognitive tests and brain cell analysis. The mRVG9R-KP-CDNF complex was injected into the striatum on days 0 and 20. To induce the PD-like model, mice were intraperitoneally administered Paraquat (PQ) twice a week for 6 weeks. Our findings demonstrate that mRVG9R-KP-CDNF gene therapy effectively protects brain cells from PQ toxicity and prevents motor and cognitive dysfunction in mice. We propose that the mRVG9R-KP-CDNF complex inhibits astrogliosis and microglia activation, safeguarding dopaminergic neurons and oligodendrocytes from PQ-induced damage. This study presents an efficient CDNF delivery system, protecting neurons and glia in the nigrostriatal pathway from PQ-induced damage, which is known to lead to motor and cognitive dysfunction in neurodegenerative diseases such as PD.

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BACKGROUND: Cancer treatment has many side effects; therefore, more efficient treatments are needed. Mesenchymal stem cells (MSC) have immunoregulatory properties, tumor site migration and can be genetically modified. Some proteins, such as soluble TRAIL (sTRAIL) and interleukin-12 (IL-12), have shown antitumoral potential, thus its combination in solid tumors could increase their activity. MATERIALS AND METHODS: Lentiviral transduction of bone marrow MSC with green fluorescent protein (GFP) and transgenes (sTRAIL and IL-12) was confirmed by fluorescence microscopy and Western blot. Soluble TRAIL levels were quantified by ELISA. Lymphoma L5178Y cells express a reporter gene (GFP/mCherry), and TRAIL receptor (DR5). RESULTS: An in vivo model showed that combined treatment with MSC expressing sTRAIL+IL-12 or IL-12 alone significantly reduced tumor volume and increased survival in BALB/c mice (p < 0.05) with only one application. However, at the histological level, only MSC expressing IL-12 reduced tumor cell infiltration significantly in the right gastrocnemius compared with the control group (p < 0.05). It presented less tissue dysplasia confirmed by fluorescence and hematoxylin-eosin dye; nevertheless, treatment not inhibited hepatic metastasis. CONCLUSIONS: MSC expressing IL-12, is or combination with BM-MSC expressing sTRAIL represents an antitumor strategy for lymphoma tumors since they increase survival and reduce tumor development. However, the combination did not show significative additive effect. The localized application did not inhibit metastasis but reduced morphological alterations of tissue associated with liver metastasis.

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The environment, containing pollutants, toxins, and transition metals (copper, iron, manganese, and zinc), plays a critical role in neurodegenerative disease development. Copper occupational exposure increases Parkinson's disease (PD) risk. Previously, we determined the mechanisms by which copper induces dopaminergic cell death in vitro. The copper transporter protein 1 (Ctr1) overexpression led to intracellular glutathione depletion potentiating caspase-3 mediated cell death; oxidative stress was primarily cytosolic, and Nrf2 was upregulated mediating an antioxidant response; and protein ubiquitination, AMPK-Ulk1 signaling, p62, and Atg5-dependent autophagy were increased as a protective mechanism. However, the effect of chronic copper exposure on the neurodegenerative process has not been explored in vivo. We aimed to elucidate whether prolonged copper treatment reproduces PD features and mechanisms during aging. Throughout 40 weeks, C57BL/6J male mice were treated with copper at 0, 100, 250, and 500 ppm in the drinking water. Chronic copper exposure altered motor function and induced dopaminergic neuronal loss, astrocytosis, and microgliosis in a dose-dependent manner. α-Synuclein accumulation and aggregation were increased in response to copper, and the proteasome and autophagy alterations, previously observed in vitro, were confirmed in vivo, where protein ubiquitination, AMPK phosphorylation, and the autophagy marker LC3-II were also increased by copper exposure. Finally, nitrosative stress was induced by copper in a concentration-dependent fashion, as evidenced by increased protein nitration. To our knowledge, this is the first study combining chronic copper exposure and aging, which may represent an in vivo model of non-genetic PD and help to assess potential prophylactic and therapeutic approaches. DATA AVAILABILITY: The data underlying this article are available in the article.

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BACKGROUND: Hyaluronic acid (HA) filler application is one of the most frequent minimally invasive aesthetic procedures used worldwide. Its properties and characterization, performance, effects in other tissues, and response to complication treatments have been studied in several animal models. This review aims to categorize animal models considering the advantages and disadvantages regarding the purpose of the study. METHODS: Literature research was made using MEDLINE via PubMed by two reviewers using keywords "hyaluronic acid" "filler" and "animal model". Full-text articles published in English and with an in vivo animal model were included for data extraction. RESULTS: The rat model was the most common animal used to evaluate properties or characteristics and degradation of HA fillers. Rabbits were preferred for evaluating HA embolism treatments; however, anatomical names of the arteries differ in some studies. Mice and rats used as vascular occlusion model are challenging due to the size of the vessels and viscosity of the filler. CONCLUSION: There is a wide variability of options of in vivo animal models to evaluate HA fillers. The animal characteristics, laboratory resources, and HA properties should be considered in accordance with the objective of the study, when choosing the ideal model. LEVEL OF EVIDENCE III: This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

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Biometals are all metal ions that are essential for all living organisms. About 40% of all enzymes with known structures require biometals to function correctly. The main target of damage by biometals is the central nervous system (CNS). Biometal dysregulation (metal deficiency or overload) is related to pathological processes. Chronic occupational and environmental exposure to biometals, including iron and copper, is related to an increased risk of developing Parkinson's disease (PD). Indeed, biometals have been shown to induce a dopaminergic neuronal loss in the substantia nigra. Although the etiology of PD is still unknown, oxidative stress dysregulation, mitochondrial dysfunction, and inhibition of both the ubiquitin-proteasome system (UPS) and autophagy are related to dopaminergic neuronal death. Herein, we addressed the involvement of redox-active biometals, iron, and copper, as oxidative stress and neuronal death inducers, as well as the current metal chelation-based therapy in PD.

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Peroxisomicine A1 (PA1) is a toxin isolated from the Karwinskia genus plants whose target organs are the liver, kidney, and lung. In vitro studies demonstrated the induction of apoptosis by PA1 in cancer cell lines, and in vivo in the liver. Apoptosis has a wide range of morphological features such as cell shrinkage, plasma membrane blistering, loss of microvilli, cytoplasm, and chromatin condensation, internucleosomal DNA fragmentation, and formation of apoptotic bodies that are phagocytized by resident macrophages or nearby cells. Early stages of apoptosis can be detected by mitochondrial alterations. We investigated the presence of apoptosis in vivo at the morphological, ultrastructural, and biochemical levels in two target organs of PA1: kidney and lung. Sixty CD-1 mice were divided into three groups (n = 20): untreated control (ST), vehicle control (VH), and PA1 intoxicated group (2LD50). Five animals of each group were sacrificed at 4, 8, 12, and 24 h post-intoxication. Kidney and lung were examined by morphometry, histopathology, ultrastructural, and DNA fragmentation analysis. Pre-apoptotic mitochondrial alterations were present at 4 h. Apoptotic bodies were observed at 8 h and increased over time. TUNEL positive cells were detected as early as 4 h, and the DNA ladder pattern was observed at 12 h and 24 h. The liver showed the highest value of fragmented DNA, followed by the kidney and the lung. We demonstrated the induction of apoptosis by a toxic dose of PA1 in the kidney and lung in vivo. These results could be useful in understanding the mechanism of action of this compound at toxic doses in vivo.

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Hair graying, a prototypical sign of human aging, is a progressive loss of pigmentation from growing hair shafts caused by disease and as a side effect of medications. Cerebrolysin is a neuropeptide preparation that mimics the effect of endogenous neurotrophic factors. Cerebrolysin has been widely used in neurologic conditions, such as cerebral stroke, Alzheimer's disease, and dementia, among others. Cerebrolysin treatment has achieved to regain or maintain the cognitive ability of affected patients; however, up to date, there are no reports about the reactivation of hair pigmentation. We describe a previously not described effect occurring on patients receiving Cerebrolysin treatment for neurologic diseases and whether this effect is associated in reactivation of melanocytes and melanin expression. Here, we report five patients (mean age, 70.6 years), who also had age-related hair graying and scalp hair repigmentation during Cerebrolysin treatment. Macroscopic analysis revealed hair repigmentation consisted in diffuse darkening of the scalp hair. Impregnation and immunostaining analysis were performed on scalp biopsies taken before and after Cerebrolysin treatment; the results showed greater melanin and melanocyte marker MART-1/Melan-A staining following Cerebrolysin treatment. We present, to our knowledge, the first report on hair repigmentation is a previously not described effect occurring following Cerebrolysin treatment.

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Encephalitozoon cuniculi spores cause severe granulomatous inflammation in the brain where mononuclear cells and macrophages infiltrate. Here, we orally infected New Zealand white rabbits with 1 × 106E. cuniculi viable spores to study the recruitment and localization of macrophages in brain granulomas. At day 30 post-infection, the positive phenotype markers iNOS (M1) and Arg-1 (M2) were located in the periphery and center of granulomas, respectively. Live intracytoplasmic spores were found only in positive Arg-1 cells. This is the first work to describe the recruitment and distribution of M1 and M2 macrophages in the brain granulomas of rabbits infected with E. cuniculi.

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