ABSTRACT
BACKGROUND: Low muscle mass quantity and quality (myosteatosis) can be evaluated by computed tomography (CT) by measuring skeletal muscle area and muscular attenuation, respectively, at the third lumbar vertebra. We aimed to define cut-off points of skeletal muscle area and muscular attenuation to predict mortality in non-dialysis chronic kidney disease (CKD) patients. METHODS: We conducted a retrospective study including non-dialysis CKD patients over two years, who underwent an opportunistic computed tomography within a two year period, and for whom creatinine was measured within 90 days of CT. Skeletal muscle area was normalized for stature to calculate the skeletal muscle index. Area under the receiver operating characteristic (AuROC) curve and Youden's index were used, to identify the cut-point, separately according to sex. RESULTS: One hundred sixty-seven patients (50.9% male, mean age of 68.3 ± 16.4 years) were included, most with CKD stages 3 and 4. During a median follow-up of 4.9 (4.2) years, 39 (23.4%) patients died. Muscular attenuation showed a better ability to predict mortality (AuROC curve 0.739 [95% CI 0.623-0.855] in women and 0.744 in men [95% CI 0.618-0.869]) than skeletal muscle index (AuROC curve 0.491 [95% CI 0.332-0.651] in women and 0.711 [95% CI 0.571-0.850] in men). For muscular attenuation, the best cut-off values to predict mortality were 27.56 Hounsfield units in women and 24.58 Hounsfield units in men. For skeletal muscle index, the best cut-off values were 38.47 cm2/m2 in women and 47.81 cm2/m2 in men. In univariable Cox-regression both low muscle mass and myosteatosis were associated with increased mortality. In multivariable Cox-regression models only myosteatosis maintained a significant association with mortality (Hazard Ratio 2.651 (95% CI 1.232-5.703, p = 0.013)). CONCLUSIONS: We found sex-specific cut-off values for muscle parameters using CT analysis in non-dialysis CKD patients that were associated with mortality. In this population, myosteatosis may be more closely associated with mortality than muscle quantity.
ABSTRACT
Skeletal muscle regeneration after injury is a complex process involving inflammatory signaling and myoblast activation. Pro-inflammatory cytokines like tumor necrosis factor-alpha (TNF-α) are key mediators, but their effects on gene expression in proliferating myoblasts are unclear. We performed the RNA sequencing of TNF-α treated C2C12 myoblasts to elucidate the signaling pathways and gene networks regulated by TNF-α during myoblast proliferation. The TNF-α (10 ng/mL) treatment of C2C12 cells led to 958 differentially expressed genes compared to the controls. Pathway analysis revealed significant regulation of TNF-α signaling, along with the chemokine and IL-17 pathways. Key upregulated genes included cytokines (e.g., IL-6), chemokines (e.g., CCL7), and matrix metalloproteinases (MMPs). TNF-α increased myogenic factor 5 (Myf5) but decreased MyoD protein levels and stimulated the release of MMP-9, MMP-10, and MMP-13. TNF-α also upregulates versican and myostatin mRNA. Overall, our study demonstrates the TNF-α modulation of distinct gene expression patterns and signaling pathways that likely contribute to enhanced myoblast proliferation while suppressing premature differentiation after muscle injury. Elucidating the mechanisms involved in skeletal muscle regeneration can aid in the development of regeneration-enhancing therapeutics.
Subject(s)
Cell Proliferation , Myoblasts , Signal Transduction , Tumor Necrosis Factor-alpha , Myoblasts/metabolism , Tumor Necrosis Factor-alpha/metabolism , Tumor Necrosis Factor-alpha/pharmacology , Cell Proliferation/drug effects , Animals , Mice , Cell Line , Chemokines/metabolism , Chemokines/genetics , Cytokines/metabolism , Cytokines/genetics , Gene Expression Regulation/drug effectsABSTRACT
Phagocytosis, an essential process for host defense, requires the coordination of a variety of signaling reactions. MT-II, an enzymatically inactive Lys49 phospholipase A2 (PLA2) homolog, and MT-III, a catalytically-active Asp49 PLA2, are known to activate phagocytosis in macrophages. In this study, the signaling pathways mediating phagocytosis, focusing on protein kinases, were investigated. Macrophages from male Swiss mice peritoneum were obtained 96 h after intraperitoneal thioglycolate injection. Phagocytosis was evaluated using non-opsonized zymosan particles in the presence or absence of specific inhibitors, as well as PKC and PKC-α localization by confocal microscopy. Moreover, protein kinase C (PKC) activity was assessed by γP32 ATP in macrophages stimulated by both PLA2s. Data showed that both sPLA2s increased phagocytosis. Cytochalasin D, staurosporine/H7, wortmannin, and herbimycin, inhibitors of actin polymerization, PKC, phosphoinositide 3-kinase (PI3K), and protein tyrosine kinase (PTK), respectively, significantly reduced phagocytosis induced by both PLA2s. PKC activity was increased in macrophages stimulated by both PLA2s. Actin polymerization and talin were evidenced by immunofluorescence and talin was recruited 5 min after both PLA2s stimulation. PKC and PKC-α localization within the cell were increased after 60 min of MT-II and MT-III stimulation. These data suggest that the effect of both PLA2s depends on actin cytoskeleton rearrangements and the activation of PKC, PI3K, and PTK signaling events required for phagocytosis.
Subject(s)
Phagocytosis , Protein Kinase C-alpha , Signal Transduction , Animals , Phagocytosis/drug effects , Mice , Signal Transduction/drug effects , Male , Protein Kinase C-alpha/metabolism , Macrophages/drug effects , Phospholipases A2, Secretory/metabolism , Snake Venoms/toxicity , Rifabutin/analogs & derivatives , Rifabutin/pharmacologyABSTRACT
Introduction: Acute liver failure (ALF), although rare in children, is a complex progressive pathology, with multisystem involvement and high mortality. Isolated variables or those included in prognostic scores have been studied, to optimize organ allocation. However, its validation is challenging. This study aimed to assess the accuracy of several biomarkers and scores as predictors of prognosis in pediatric ALF (PALF). Methods: An observational study with retrospective data collection, including all cases of ALF, was defined according to the criteria of the Pediatric Acute Liver Failure Study Group, admitted to a pediatric intensive care unit (PICU) for 28 years. Two groups were defined: spontaneous recovery (SR) and non-SR (NSR) - submitted to liver transplantation (LT) or death at PICU discharge. Results: Fifty-nine patients were included, with a median age of 24 months, and 54% were female. The most frequent etiologies were metabolic (25.4%) and infectious (18.6%); 32.2% were undetermined. SR occurred in 21 patients (35.6%). In NSR group (N = 38, 64.4%), 25 required LT (42.4%) and 19 died (32.2%), 6 (15.7%) of whom after LT. The accuracy to predict NSR was acceptable for lactate at admission (AUC 0.72; 95% CI: 0.57-0.86; p = 0.006), ammonia peak (AUC 0.72; 95% CI: 0.58-0.86; p = 0.006), and INR peak (AUC 0.70; 95% CI: 0.56-0.85; p = 0.01). The cut-off value for lactate at admission was 1.95 mmol/L (sensitivity 78.4% and specificity 61.9%), ammonia peak was 64 µmol/L (sensitivity 100% and specificity 38.1%), and INR peak was 4.8 (sensitivity 61.1% and specificity 76.2%). Lactate on admission was shown to be an independent predictor of NSR on logistic regression model. Two prognostic scores had acceptable discrimination for NSR, LIU (AUC 0.73; 95% CI: 0.59-0.87; p = 0.004) and PRISM (AUC 0.71; 95% CI: 0.56-0.86; p = 0.03). In our study, the PALF delta score (PALF-ds) had lower discrimination capacity (AUC 0.63; 95% CI: 0.47-0.78; p = 0.11). Conclusions: The lactate at admission, an easily obtained parameter, had a similar capacity than the more complex scores, LIU and PRISM, to predict NSR. The prognostic value in our population of the promising dynamic score, PALF-ds, was lower than expected.
Introdução: A falência hepática aguda (FHA), apesar de rara em pediatria, é uma patologia complexa, com envolvimento multissistémico e elevada mortalidade. Têm sido estudadas variáveis isoladas ou incluídas em scores de prognóstico, com o objetivo de otimizar a alocação de órgãos. No entanto, a sua validação apresenta alguns desafios. O presente estudo tem como objetivo avaliar a precisão de vários biomarcadores e scores, como preditores de prognóstico na FHA. Métodos: Estudo observacional com método de colheita de dados retrospetivo, tendo como critérios de inclusão os casos de FHA, definida de acordo com os critérios da Pediatric Acute Liver Failure Study Group, admitidos numa Unidade de Cuidados Intensivos Pediátricos (UCIP) num período de 28 anos. Definiram-se 2 grupos: recuperação espontânea (RE) e sem recuperação espontânea (SRE) doentes submetidos a transplante hepático (TRH) ou morte na alta da UCIP. Resultados: Incluíram-se 59 doentes, com mediana de idade de 24 meses, 54% do sexo feminino. As etiologias mais frequentes foram a metabólica (25.4%) e a infeciosa (18.6%); em 32.2% foi indeterminada. Apresentaram RE 21 doentes (35.6%). No grupo SRE (N = 38, 64.4%), 25 necessitaram de TRH (42.4%) e 19 faleceram (32.2%), dos quais 6 (15.7%) tinham sido submetidos a TRH. A precisão prognóstica para a ausência de recuperação espontânea foi aceitável para o lactato na admissão (AUC 0.72; IC 95%: 0.570.86; p = 0.006), amónia máxima (AUC 0.72; IC 95%: 0.580.86; p = 0.006) e INR máximo (AUC 0.70; IC 95%: 0.560.85; p = 0.01). O valor de cut-off do lactato na admissão foi de 1.95 mmol/L (sensibilidade 78.4% e especificidade 61.9%) e da amónia máxima foi de 64 umol/L (sensibilidade 100% e especificidade 38.1%). O lactato à admissão mostrou ser um fator independente para NSR, no modelo de regressão logística. Os scores LIU e PRISM apresentaram curvas ROC com aceitável capacidade de discriminação para a ausência de recuperação espontânea, com AUC de 0.73 (IC 95%: 0.590.87; p = 0.004) e 0.71 (IC 95%: 0.560.86; p = 0.03), respetivamente. No nosso estudo, o score PALF-Delta (PALF-ds) teve uma menor capacidade de discriminação (AUC 0.63; IC 95%: 0.470.78; p = 0.11). Conclusões: O lactato na admissão, um parâmetro de fácil obtenção, teve uma capacidade semelhante aos scores mais complexos, LIU e PRISM, para predizer a ausência de recuperação espontânea. O valor prognóstico nesta série, do promissor score dinâmico PALF-ds, foi inferior ao esperado.
ABSTRACT
Parkinson's disease (PD) is recognized as the second most prevalent primary chronic neurodegenerative disorder of the central nervous system. Clinically, PD is characterized as a movement disorder, exhibiting an incidence and mortality rate that is increasing faster than any other neurological condition. In recent years, there has been a growing interest concerning the role of the gut microbiota in the etiology and pathophysiology of PD. The establishment of a brain-gut microbiota axis is now real, with evidence denoting a bidirectional communication between the brain and the gut microbiota through metabolic, immune, neuronal, and endocrine mechanisms and pathways. Among these, the vagus nerve represents the most direct form of communication between the brain and the gut. Given the potential interactions between bacteria and drugs, it has been observed that the therapies for PD can have an impact on the composition of the microbiota. Therefore, in the scope of the present review, we will discuss the current understanding of gut microbiota on PD and whether this may be a new paradigm for treating this devastating disease.
Subject(s)
Brain-Gut Axis , Brain , Gastrointestinal Microbiome , Parkinson Disease , Humans , Parkinson Disease/microbiology , Parkinson Disease/therapy , Brain/microbiology , Brain/pathology , Brain-Gut Axis/physiology , AnimalsABSTRACT
The phagocytic activity of macrophages activated with MT-II, a Lys-49 PLA2 homolog, and MT-III, an Asp-49 PLA2, from Bothrops asper snake venom, was investigated in this study using a pharmacological approach. Stimulating thioglycollate-elicited macrophages with both venom components enhanced their ability to phagocytose non-opsonized zymosan particles. MT-II and MT-III-induced phagocytosis was drastically inhibited by pretreating cells with L-NAME, aminoguanidine or L-NIL, cNOS or iNOS inhibitors, or with ODQ (sGC inhibitor) or Rp-cGMPS (PKG inhibitor). These results indicate that the NO/sGC/GMP/PKG pathway plays an essential role in the ß-glucan-mediated phagocytosis induced in macrophages by these venom-secretory PLA2s.
Subject(s)
Bothrops , Crotalid Venoms , Macrophages , Nitric Oxide , Phagocytosis , Signal Transduction , Zymosan , Animals , Phagocytosis/drug effects , Zymosan/pharmacology , Signal Transduction/drug effects , Nitric Oxide/metabolism , Macrophages/drug effects , Mice , Phospholipases A2, Secretory/metabolismABSTRACT
Disease modifying therapies (DMTs) used for treating people with relapsing-remitting multiple sclerosis (pwRRMS) target the immune system by different mechanisms of action. However, there is a lack of a comprehensive assessment of their effects on the immune system in comparison to treatment-naïve pwRRMS. Herein, we evaluated the numbers of circulating B cells, CD4+ and CD8+ T cells, regulatory T cells (Tregs), natural killer (NK) cells and NKT cells, and their subsets, in pwRRMS who were treatment-naïve or treated with different DMTs. Compared to treatment-naïve pwRRMS, common and divergent effects on immune system cells were observed on pwRRMS treated with different DMTs, with no consistent pattern across all therapies in any of the cell populations analysed. PwRRMS treated with fingolimod, dimethyl fumarate (DMF), or alemtuzumab have reduced numbers of CD4+ and CD8+ T cells, as well as Treg subsets, with fingolimod causing the most pronounced decrease in T cell subsets. In contrast, teriflunomide and interferon (IFN) ß have minimal impact on T cells, and natalizumab marginally increases the number of memory T cells in the blood. The effect of DMTs on the B cell, NKT and NK cell subsets is highly variable with alemtuzumab inducing a strong increase in the number of the most immature NK cells and its subsets. This study comprehensively evaluates the magnitude of the effect of different DMTs on blood immune cells providing a better understanding of therapy outcome. Furthermore, the lack of a discernible pattern in the effects of DMTs on blood immune cells suggests that multiple immune cells can independently modulate the disease.
Subject(s)
Multiple Sclerosis, Relapsing-Remitting , Multiple Sclerosis , Humans , Multiple Sclerosis, Relapsing-Remitting/drug therapy , Fingolimod Hydrochloride/therapeutic use , Immunosuppressive Agents , Alemtuzumab , CD8-Positive T-LymphocytesABSTRACT
BACKGROUND: Testicular germ cell tumors remain the most frequent solid malignancies in young males. Despite excellent prognosis, the fact that only 60% of patients at diagnosis have elevated serum tumor markers (dependent on stage and histology) and the poor quality of life of patients who develop resistance to chemotherapy cannot be neglected. Consequently, it is mandatory to bring out novel biomarkers. OBJECTIVES: The main goal was to evaluate EZH2 and EHMT2/G9a immunoexpression in a well-characterized patients' cohort of primary and metastatic testicular germ cell tumors, seeking associations with clinicopathological features and discovering differential immunoexpression patterns among specific subtypes. MATERIALS AND METHODS: First, an in silico analysis of the Cancer Genome Atlas database was performed regarding EZH2 and EHMT2/G9a. Then, immunohistochemistry for EZH2 and EHMT2/G9a was carried out in a cohort of testicular germ cell tumor patients, comprising 155 chemo-naïve primary tumors and 11 chemo-treated metastases. Immunoexpression was evaluated using a digital pathology analysis software. RESULTS: Higher EZH2 and EHMT2/G9a expression levels were found in non-seminoma in the in silico analysis, particularly in embryonal carcinoma. Through digital pathology analysis, non-seminomas showed significantly higher EZH2 and EHMT2/G9a immunoexpression, with embryonal carcinoma showing higher expression. Moreover, mixed tumors with 50% or more of embryonal carcinoma component revealed the highest nuclei positivity for both biomarkers. Cisplatin-exposed metastases demonstrated a higher EZH2-positive nuclei and H-score, as well as higher EHMT2/G9a-positive nuclei. DISCUSSION AND CONCLUSION: Overall, our data suggest that EZH2 and EHMT2/G9a might be associated with greater aggressiveness and, eventually, involved in the metastatic setting, paving the way for testing targeted therapies.
ABSTRACT
Phagocytosis, an essential process for host defense, requires the coordination of a variety of signaling reactions. MT-II, an enzymatically inactive Lys49 phospholipase A2 (PLA2) homolog, and MT-III, a catalytically-active Asp49 PLA2, are known to activate phagocytosis in macrophages. In this study, the signaling pathways mediating phagocytosis, focusing on protein kinases, were investigated. Macrophages from male Swiss mice peritoneum were obtained 96 h after intraperitoneal thioglycolate injection. Phagocytosis was evaluated using nonopsonized zymosan particles in the presence or absence of specific inhibitors, as well as PKC and PKC-α localization by confocal microscopy. Moreover, protein kinase C (PKC) activity was assessed by γP32 ATP in macrophages stimulated by both PLA2s. Data showed that both sPLA2s increased phagocytosis. Cytochalasin D, staurosporine/H7, wortmannin, and herbimycin, inhibitors of actin polymerization, PKC, phosphoinositide 3-kinase (PI3K), and protein tyrosine kinase (PTK), respectively, significantly reduced phagocytosis induced by both PLA2s. PKC activity was increased in macrophages stimulated by both PLA2s. Actin polymerization and talin were evidenced by immunofluorescence and talin was recruited 5 min after both PLA2s stimulation. PKC and PKC-α localization within the cell were increased after 60 min of MT-II and MT-III stimulation. These data suggest that the effect of both PLA2s depends on actin cytoskeleton rearrangements and the activation of PKC, PI3K, and PTK signaling events required for phagocytosis.
ABSTRACT
The phagocytic activity of macrophages activated with MT-II, a Lys-49 PLA2 homolog, and MT-III, an Asp-49 PLA2, from Bothrops asper snake venom, was investigated in this study using a pharmacological approach. Stimulating thioglycollate-elicited macrophages with both venom components enhanced their ability to phagocytose non-opsonized zymosan particles. MT-II and MT–III–induced phagocytosis was drastically inhibited by pretreating cells with L-NAME, aminoguanidine or L-NIL, cNOS or iNOS inhibitors, or with ODQ (sGC inhibitor) or Rp-cGMPS (PKG inhibitor). These results indicate that the NO/sGC/GMP/PKG pathway plays an essential role in the β-glucan-mediated phagocytosis induced in macrophages by these venom-secretory PLA2s.
ABSTRACT
Most anti-inflammatory drugs currently adopted to treat chronic inflammatory joint diseases can alleviate symptoms but they do not lead to remission. Therefore, new and more efficient drugs are needed to block the course of joint inflammatory diseases. Animal venoms, rich in bioactive compounds, can contribute as valuable tools in this field of research. In this study, we first demonstrate the direct action of venoms on cells that constitute the articular joints. We established a platform consisting of cell-based assays to evaluate the release of cytokines (IL-6, IL-8, TNFα, IL-1β, and IL-10) by human chondrocytes, synoviocytes and THP1 macrophages, as well as the release of neuropeptides (substance-P and β-endorphin) by differentiated sensory neuron-like cells, 24 h after stimulation of cells with 21 animal venoms from snake and arthropod species, sourced from different taxonomic families and geographic origins. Results demonstrated that at non-cytotoxic concentrations, the venoms activate at varying degrees the secretion of inflammatory mediators involved in the pathology of articular diseases, such as IL-6, IL-8, and TNF-α by chondrocytes, synoviocytes, and macrophages and of substance P by neuron-like cells. Venoms of the Viperidae snake family were more inflammatory than those of the Elapidae family, while venoms of Arthropods were less inflammatory than snake venoms. Notably, some venoms also induced the release of the anti-inflammatory IL-10 by macrophages. However, the scorpion Buthus occitanus venom induced the release of IL-10 without increasing the release of inflammatory cytokines by macrophages. Since the cell types used in the experiments are crucial elements in joint inflammatory processes, the results of this work may guide future research on the activation of receptors and inflammatory signaling pathways by selected venoms in these particular cells, aiming at discovering new targets for therapeutic intervention.
ABSTRACT
Most anti-inflammatory drugs currently adopted to treat chronic inflammatory joint diseases can alleviate symptoms but they do not lead to remission. Therefore, new and more efficient drugs are needed to block the course of joint inflammatory diseases. Animal venoms, rich in bioactive compounds, can contribute as valuable tools in this field of research. In this study, we first demonstrate the direct action of venoms on cells that constitute the articular joints. We established a platform consisting of cell-based assays to evaluate the release of cytokines (IL-6, IL-8, TNFα, IL-1ß, and IL-10) by human chondrocytes, synoviocytes and THP1 macrophages, as well as the release of neuropeptides (substance-P and ß-endorphin) by differentiated sensory neuron-like cells, 24 h after stimulation of cells with 21 animal venoms from snake and arthropod species, sourced from different taxonomic families and geographic origins. Results demonstrated that at non-cytotoxic concentrations, the venoms activate at varying degrees the secretion of inflammatory mediators involved in the pathology of articular diseases, such as IL-6, IL-8, and TNF-α by chondrocytes, synoviocytes, and macrophages and of substance P by neuron-like cells. Venoms of the Viperidae snake family were more inflammatory than those of the Elapidae family, while venoms of Arthropods were less inflammatory than snake venoms. Notably, some venoms also induced the release of the anti-inflammatory IL-10 by macrophages. However, the scorpion Buthus occitanus venom induced the release of IL-10 without increasing the release of inflammatory cytokines by macrophages. Since the cell types used in the experiments are crucial elements in joint inflammatory processes, the results of this work may guide future research on the activation of receptors and inflammatory signaling pathways by selected venoms in these particular cells, aiming at discovering new targets for therapeutic intervention.
Subject(s)
Animals, Poisonous , Arthropod Venoms , Arthropods , Joint Diseases , Scorpion Venoms , Scorpions , Viperidae , Animals , Humans , Interleukin-10 , Interleukin-6 , Interleukin-8 , Snake Venoms/chemistry , Cytokines , Tumor Necrosis Factor-alpha , Anti-Inflammatory AgentsABSTRACT
BACKGROUND: The first clinical manifestations of inherited metabolic diseases occur in the neonatal period in up to half of cases, often with nonspecific symptoms, making their recognition challenging. This study aimed to characterise inherited metabolic disease cases with neonatal presentation requiring admission to the paediatric intensive care unit in a Portuguese reference centre for inherited metabolic diseases. MATERIAL AND METHODS: An observational study with retrospective data collection was performed, including all newborns with an inherited metabolic disease admitted to the pediatric intensive care unit between June 2011 and June 2022. Three 'pathophysiological' groups were defined: cases due to small molecules, energy deficiency and complex molecules. RESULTS: Twenty newborns, with a median age at admission of 7.5 days, were included. Thirteen (65%) were female, sixteen (80%) had a small molecule disorder, and four (20%) had diseases of energy defects. Neurological manifestations were the most common, with most newborns presenting symptomatically in the first week of life. There was no difference between the groups in neurological, cardiac, and hepatic involvement and shock at presentation. A symptom-free interval was more frequent in patients with small molecule disorders than the others (p=0.01). The main metabolic changes found were altered plasma amino acids (n=13) and organic aciduria (n=10), creatine kinase elevation (n=13), hyperlactatemia (n=12), metabolic acidosis with increased anion gap (n=8) and hyperammonaemia (n=7). Newborn screening of metabolites helped make a diagnosis in 60% of cases. Five newborns died due to multiorgan failure (n=3) or refractory cardiogenic shock (n=1), and in one, therapeutic efforts were limited due to an adverse neurological prognosis. CONCLUSION: Although the symptoms and signs are often nonspecific, we should suspect inherited metabolic disease when a newborn presents with neurological symptoms after a symptom-free period, however short it might be. Newborns with suspected inherited metabolic disease should be evaluated with simple biochemical tests, and newborn screening should be urgently expanded to start specific treatment earlier, reducing mortality and morbidity.
Subject(s)
Metabolic Diseases , Child , Humans , Infant, Newborn , Female , Male , Retrospective Studies , Metabolic Diseases/diagnosis , Amino Acids , Neonatal Screening/methods , Intensive Care Units, PediatricSubject(s)
Hyperkalemia , Kidney Failure, Chronic , Renal Insufficiency, Chronic , Humans , Hyperkalemia/diagnosis , Hyperkalemia/etiology , Potassium , Kidney Failure, Chronic/diagnosis , Kidney Failure, Chronic/therapy , Kidney Failure, Chronic/complications , Disease Progression , Renal Insufficiency, Chronic/complicationsABSTRACT
Prostate cancer (PCa) is the second most common malignancy in men, and the fifth leading cause of death worldwide. Mesenchymal stromal/stem cells (MSC) have been identified in PCa, although contradictory effects in malignant transformation and tumor progression have been described. Since umbilical cord (UC) MSC and cord blood serum (CBS) are rich in numerous growth and anti-inflammatory factors, UC-MSC secretome and CBS are able to modulate tumor cell proliferation and survival as well as immunity and angiogenesis. In the present study, we address this relationship and investigate the influence of UC-MSC secretome and CBS on two human PCa cell lines (PC3 and LNCaP) and a normal epithelial prostate cell line (HPEpiC). Our results disclosed that upon exposure to UC-MSC-conditioned medium or CBS, both PC3 and LNCaP cells exhibited reduced viability, proliferation, and motility while non-malignant epithelial prostate cells were unaffected. These findings were corroborated by expression analysis of AKT/PI3K signaling pathway, p53 and interleukin genes. UC-MSC and CBS factors decreased the expression of growth-stimulating AKT and PI3K effectors and simultaneously up-regulated the expression of tumor-suppressor p53. Moreover, a more anti-inflammatory expression profile was found in both malignant PCa cell lines. Altogether, these results shed light into possible mechanisms by which UC-MSC and CBS reduce PCa progression, further reinforcing their potential use as novel therapeutic agents in PCa.
Subject(s)
Prostatic Neoplasms , Tumor Suppressor Protein p53 , Male , Humans , Prostate , Serum , Phosphatidylinositol 3-Kinases , Proto-Oncogene Proteins c-akt , Secretome , Umbilical Cord , Prostatic Neoplasms/therapy , Stem CellsABSTRACT
Bladder cancer (BlCa) is the ninth most common cancer worldwide, associated with significant morbidity and mortality. Thus, understand the biological mechanisms underlying tumour progression is of great clinical significance. Vimentin (VIM) is (over)expressed in several carcinomas, putatively in association with EMT. We have previously found that VIM promoter methylation accurately identified BlCa and VIM expression associated with unfavourable prognosis. Herein, we sought to investigate VIM expression regulation and its role in malignant transformation of BlCa. Analysis of tissue samples disclosed higher VIM transcript, protein, and methylation levels in BlCa compared with normal urothelium. VIM protein and transcript levels significantly increased from non-muscle invasive (NMIBC) to muscle-invasive (MIBC) cases and to BlCa metastases. Inverse correlation between epithelial CDH1 and VIM, and a positive correlation between mesenchymal CDH2 and VIM were also observed. In BlCa cell lines, exposure to demethylating agent increased VIM protein, with concomitant decrease in VIM methylation. Moreover, exposure to histone deacetylases pan-inhibitor increased the deposit of active post-translational marks (PTMs) across VIM promoter. In primary normal urothelium cells, lower levels of active PTMs with concomitant higher levels of repressive marks deposit were observed. Finally, VIM knockdown in UMUC3 cell line increased epithelial-like features and decreased migration and invasion in vitro, decreasing tumour size and angiogenesis in vivo. We demonstrated that VIM promoter is epigenetically regulated in normal and neoplastic urothelium, which determine a VIM switch associated with EMT and acquisition of invasive and metastatic properties. These findings might allow for development of new, epigenetic-based, therapeutic strategies for BlCa.
Subject(s)
Urinary Bladder Neoplasms , Humans , Vimentin/genetics , Vimentin/metabolism , Urinary Bladder Neoplasms/metabolism , Epigenesis, Genetic/genetics , Phenotype , Epithelial-Mesenchymal Transition/genetics , Gene Expression Regulation, Neoplastic/geneticsABSTRACT
Relatório de Estágio apresentado para obtenção do grau de Mestre em Enfermagem Comunitária na área de Enfermagem de Saúde Familiar. Tem como objetivos documentar a reflexão crítica quanto à aquisição e desenvolvimento das competências definidas pela Ordem,Enfermeiros, está dividido em duas partes. Na parte I contextualiza-se o ensino clínico, fundamenta-se teoricamente alguns conceitos de família e de enfermagem em saúde familiar e documenta-se a aquisição das competências do enfermeiro especialista em Saúde Familiar. Na parte II consiste na Revisão Sistemática Literatura (RSL) sobre as intervenções eficazes do enfermeiro família (EF) para diminuir a sobrecarga do familiar cuidador com doente oncológico. Os doentes oncológicos paliativos desejam ficar em casa, pois o meio familiar oferece a continuidade da sua vida diária pelo fato de estar rodeados das pessoas e objetos significantes. Os cuidadores familiares são cruciais nos cuidados de fim de vida, eles muitas vezes enfrentam pesadas responsabilidades e sentem-se insuficientemente preparados. Perante isto, os enfermeiros têm um papel preponderante nas intervenções de suporte em ambiente domiciliário diminuindo a sobrecarga dos cuidadores familiares. Reconhecendo esta realidade emergiu a necessidade de desenvolver esta temática, procurando responder à questão: "Quais as intervenções eficazes, do EF, na diminuição da sobrecarga do familiar cuidador, do doente oncológico paliativo em contexto domiciliário?" e ao seguinte objetivo geral: identificar as intervenções do EF eficazes na diminuição da sobrecarga da família cuidadora do doente paliativo em contexto domiciliário. A metodologia consistiu na RSL de evidência de eficácia, segundo Modelo conceptual JBI, incluindo como base de dados a EBSCO e a PUBMED. Desta pesquisa, foram identificados 31 artigos, mas somente 3 cumpriam os critérios de inclusão. A análise destes artigos evidenciam que existem três tipos de intervenções que o enfermeiro poderá empregar, uma intervenção terapêutica de conversação que reduz o stress e as preocupações que surgiam aos familiares cuidadores aquando da prestação de cuidados ao doente oncológico paliativo, uma intervenção estruturada direcionada às necessidades dos cuidadores familiares e uma intervenção psicoeducativa que melhora a capacidade para cuidar dos cuidadores familiares em cuidados paliativos a curto e a longo prazo. Os artigos selecionados reforçam a necessidade de desenvolvimento e expansão dos cuidados paliativos em contexto domiciliário, bem como a emergência da estruturação das intervenções do enfermeiro de família dirigidas ao cuidador familiar em cuidados paliativos domiciliares.
Internship Report presented to obtain the degree of Master of Community Nursing in the area of Family Health Nursing. Community Nursing in the area of Family Health Nursing. It aims to document the critical reflection on the acquisition and development of the competencies defined by the Ordem dos Enfermeiros (Portuguese Nurses Association). In part I clinical teaching is contextualized, some concepts of family and family health nursing are theoretically based, and the acquisition of competences of Family Health Nurse Specialist is documented. Part II consists of the Systematic Literature Review (SLR) on the effective interventions of the family nurse (FE) to reduce the burden of the family caregiver of cancer patients. Palliative cancer patients wish to stay at home, as the family environment provides continuity to their daily life by being surrounded by significant people and objects. Family caregivers are crucial in end-of-life care, they often face heavy responsibilities and feel insufficiently prepared. In view of this, nurses play a leading role in support interventions in the home environment to reduce the burden on family caregivers. Recognising this reality, the need emerged to develop this theme, seeking to answer the following question: "Which are the effective FE interventions in reducing the burden of the family caregiver of the palliative cancer patient at home?" and the following general objective: to identify the effective FE interventions in reducing the burden of the family caregiver of the palliative patient at home. The methodology consisted of a search for evidence of effectiveness according to the JBI conceptual model, including EBSCO and PUBMED as databases. From this search, 31 articles were identified, but only 3 met the inclusion criteria. The analysis of these articles showed that there are three types of interventions that nurses may use: a therapeutic conversation intervention that reduces the stress and concerns that arise for family caregivers when caring for the palliative cancer patient, a structured intervention aimed at the needs of family caregivers, and a psychoeducational intervention that improves the ability to care of family caregivers in palliative care in the short and long term. The selected articles reinforce the need for the development and expansion of palliative care in home settings, as well as the emergence of the structuring of the family nurse's interventions aimed at the family caregiver in home palliative care.
Subject(s)
Humans , Oncology Nursing , Family Nursing , Nurse's Role , Hospice and Palliative Care Nursing , Nursing CareABSTRACT
Coral snakes mainly cause neurotoxic symptoms in human envenomation, but experimental studies have already demonstrated several pharmacological activities in addition to these effects. This investigation was carried out with the aim of evaluating (1) non-neurogenic mechanisms involved in the inflammatory response induced by Micrurus lemniscatus venom (MLV) in rat hind paws, (2) participation of PLA2 in this response, and (3) neutralizing efficiency of commercial anti-elapid antivenom on edema. MLV promoted a rapid, significant increase in vascular permeability, influx of leukocytes, and disorganization of collagen bundles, as demonstrated by histological analysis. Several pretreatments were applied to establish the involvement of inflammatory mediators in MLV-induced edema (5 µg/paw). Treatment of animals with chlorpromazine reduced MLV-induced edema, indicating participation of TNF-α. However, the inefficiency of other pharmacological treatments suggests that eicosanoids, leukotrienes, and nitric oxide have no role in this type of edema formation. In contrast, PAF negatively modulates this venom-induced effect. MLV was recognized by anti-elapid serum, but this antivenom did not neutralize edema formation. Chemical modification of MLV with p-bromophenacyl bromide abrogated the phospholipase activity and markedly reduced edema, demonstrating PLA2 participation in MLV-induced edema. In conclusion, the non-neurogenic inflammatory profile of MLV is characterized by TNF-α-mediated edema, participation of PLA2 activity, and down-regulation by PAF. MLV induces an influx of leukocytes and destruction of collagen fibers at the site of its injection.
Subject(s)
Coral Snakes , Animals , Antivenins , Chlorpromazine/toxicity , Edema/chemically induced , Elapid Venoms/toxicity , Elapidae , Humans , Inflammation Mediators/toxicity , Nitric Oxide , Phospholipases A2/toxicity , Rats , Tumor Necrosis Factor-alpha/toxicityABSTRACT
Bothrops envenomation is a public health problem in Brazil. Despite the advances in the knowledge of the pathogenesis of systemic and local effects induced by Bothrops venom, the target tissues to this venom are not completely characterised. As preadipocytes are important cells of the adipose tissue and synthesize inflammatory mediators, we investigated the ability of B. moojeni snake venom (Bmv) to stimulate an inflammatory response in 3T3-L1 preadipocytes in vitro, focusing on (1) the release of PGE2, IL-6, TNF-α, MCP-1, KC, leptin and adiponectin; (2) the mechanisms involved in PGE2 release and (3) differentiation of these cells. Cytotoxicity of Bmv was determined by MTT assay. The concentrations of PGE2, cytokines and adipokines were quantified by EIA. Participation of the COX-1 and COX-2 enzymes, NF-κB and PGE2 receptors (EP1-4) was assessed using a pharmacological approach, and protein expression of the COX enzymes and P-NF-κB was analysed by western blotting. Preadipocyte differentiation was quantified by Oil Red O staining. Bmv (1 µg/mL) induced release of PGE2, IL-6 and KC and increased expression of COX-2 in preadipocytes. Basal levels of TNF-α, MCP-1, leptin and adiponectin were not modified. Treatment of cells with SC560 (COX-1 inhibitor) and NS398 (COX-2 inhibitor) inhibited Bmv-induced PGE2 release. Bmv induced phosphorylation of NF-κB, and treatment of the cells with TPCK and SN50, which inhibit distinct NF-κB domains, significantly reduced Bmv-induced PGE2 release, as did the treatment with an antagonist of PGE2 receptor EP1, unlike treatment with antagonists of EP2, EP3 or EP4. Bmv also induced lipid accumulation in differentiating cells. These results demonstrate that Bmv can activate an inflammatory response in preadipocytes by inducing the release of inflammatory mediators; that PGE2 production is mediated by the COX-1, COX-2 and NF-κB pathways; and that engagement of EP1 potentiates PGE2 synthesis via a positive feedback mechanism. Our findings highlight the role of the adipose tissue as another target for Bmv and suggest that it contributes to Bothrops envenomation by producing inflammatory mediators.
Subject(s)
Bothrops , Adiponectin , Animals , Bothrops/metabolism , Cyclooxygenase 2/metabolism , Dinoprostone/metabolism , Inflammation Mediators/metabolism , Interleukin-6/metabolism , Leptin , NF-kappa B , Snake Venoms , Tumor Necrosis Factor-alpha/metabolismABSTRACT
The pursuit of better therapies for disorders creating deficiencies in skeletal muscle regeneration is in progress, and several biotoxins are used in skeletal muscle research. Since recombinant proteins derived from Lonomia obliqua bristles, recombinant Lonomia obliqua Stuart-factor activator (rLosac) and recombinant Lonomia obliqua prothrombin activator protease (rLopap) act as cytoprotective agents and promote cell survival, we hypothesize that both rLosac and rLopap favour the skeletal muscle regeneration process. In the present work, we investigate the ability of these recombinant proteins rLosac and rLopap to modulate the production of key mediators of the myogenic process. The expression of myogenic regulatory factors (MRFs), cell proliferation, the production of prostaglandin E2 (PGE2) and the protein expression of cyclooxygenases COX-1 and COX-2 were evaluated in C2C12 mouse myoblasts pre-treated with rLosac and rLopap. We found an increased proliferation of myoblasts, stimulated by both recombinant proteins. Moreover, these proteins modulated PGE2 release and MRFs activities. We also found an increased expression of the EP4 receptor in the proliferative phase of C2C12 cells, suggesting the involvement of this receptor in the effects of PGE2 in these cells. Moreover, the recombinant proteins inhibited the release of IL-6 and PGE2, which is induced by an inflammatory stimulus by IL-1ß. This work reveals rLopap and rLosac as promising proteins to modulate processes involving tissue regeneration as occurs during skeletal muscle injury.