Oxidized cholesteryl ester induces exocytosis of dysfunctional lysosomes in lipidotic macrophages.

A key event in atherogenesis is the formation of lipid-loaded macrophages, lipidotic cells, which exhibit irreversible accumulation of undigested modified low-density lipoproteins (LDL) in lysosomes. This event culminates in the loss of cell homeostasis, inflammation, and cell death. Nevertheless, the exact chemical etiology of atherogenesis and the molecular and cellular mechanisms responsible for the impairment of lysosome function in plaque macrophages are still unknown. Here, we demonstrate that macrophages exposed to cholesteryl hemiazelate (ChA), one of the most prevalent products of LDL-derived cholesteryl ester oxidation, exhibit enlarged peripheral dysfunctional lysosomes full of undigested ChA and neutral lipids. Both lysosome area and accumulation of neutral lipids are partially irreversible. Interestingly, the dysfunctional peripheral lysosomes are more prone to fuse with the plasma membrane, secreting their undigested luminal content into the extracellular milieu with potential consequences for the pathology. We further demonstrate that this phenotype is mechanistically linked to the nuclear translocation of the MiT/TFE family of transcription factors. The induction of lysosome biogenesis by ChA appears to partially protect macrophages from lipid-induced cytotoxicity. In sum, our data show that ChA is involved in the etiology of lysosome dysfunction and promotes the exocytosis of these organelles. This latter event is a new mechanism that may be important in the pathogenesis of atherosclerosis.

Oxidative stress in metabolic dysfunction-associated steatotic liver disease (MASLD): How does the animal model resemble human disease?

Despite decades of research, the pathogenesis of metabolic dysfunction-associated steatotic liver disease (MASLD) is still not completely understood. Based on the evidence from preclinical models, one of the factors proposed as a main driver of disease development is oxidative stress. This study aimed to search for the resemblance between the profiles of oxidative stress and antioxidant defense in the animal model of MASLD and the group of MASLD patients. C57BL/6J mice were fed with the Western diet for up to 24 weeks and served as the animal model of MASLD. The antioxidant profile of mice hepatic tissue was determined by liquid chromatography-MS3 spectrometry (LC-MS/MS). The human cohort consisted of 20 patients, who underwent bariatric surgery, and 6 controls. Based on histological analysis, 4 bariatric patients did not have liver steatosis and as such were also classified as controls. Total antioxidant activity was measured in sera and liver biopsy samples. The hepatic levels of antioxidant enzymes and oxidative damage were determined by Western Blot. The levels of antioxidant enzymes were significantly altered in the hepatic tissue of mice with MASLD. In contrast, there were no significant changes in the antioxidant profile of hepatic tissue of MASLD patients, except for the decreased level of carbonylated proteins. Decreased protein carbonylation together with significant correlations between the thioredoxin system and parameters describing metabolic health suggest alterations in the thiol-redox signaling. Altogether, these data show that even though the phenotype of mice closely resembles human MASLD, the animal-to-human translation of cellular and molecular processes such as oxidative stress may be more challenging.

Combination immunotherapy including OncoVEXmGMCSF creates a favorable tumor immune micro-environment in transgenic BRAF murine melanoma.

Talimogene Laherparepvec (OncoVEXmGMCSF), an oncolytic virus, immune checkpoint inhibitor anti-programmed cell death protein 1 (anti-PD1), and BRAF inhibition (BRAFi), are all clinically approved for treatment of melanoma patients and are effective through diverse mechanisms of action. Individually, these therapies also have an effect on the tumor immune microenvironment (TIME). Evaluating the combination effect of these three therapies on the TIME can help determine when combination therapy is most appropriate for further study. In this study, we use a transgenic murine melanoma model (Tyr::CreER; BRAFCA/+; PTENflox/flox), to evaluate the TIME in response to combinations of BRAFi, anti-PD1, and OncoVEXmGMCSF. We find that mice treated with the triple combination BRAFi + anti-PD1 + OncoVEXmGMCSF have decreased tumor growth compared to BRAFi alone and prolonged survival compared to control. Flow cytometry shows an increase in percent CD8 + /CD3 + cytotoxic T Lymphocytes (CTLs) and a decrease in percent FOXP3 + /CD4 + T regulatory cells (Tregs) in tumors treated with OncoVEXmGMCSF compared to mice not treated with OncoVEXmGMCSF. Immunogenomic analysis at 30d post-treatment shows an increase in Th1 and interferon-related genes in mice receiving OncoVEXmGMCSF + BRAFi. In summary, treatment with combination BRAFi + anti-PD1 + OncoVEXmGMCSF is more effective than any single treatment in controlling tumor growth, and groups receiving OncoVEXmGMCSF had more tumoral infiltration of CTLs and less intratumoral Tregs in the TIME. This study provides rational basis to combine targeted agents, oncolytic viral therapy, and checkpoint inhibitors in the treatment of melanoma.

Mitochondria, oxidative stress and nonalcoholic fatty liver disease: A complex relationship.

According to the 'multiple-hit' hypothesis, several factors can act simultaneously in nonalcoholic fatty liver disease (NAFLD) progression. Increased nitro-oxidative (nitroso-oxidative) stress may be considered one of the main contributors involved in the development and risk of NAFLD progression to nonalcoholic steatohepatitis (NASH) characterized by inflammation and fibrosis. Moreover, it has been repeatedly postulated that mitochondrial abnormalities are closely related to the development and progression of liver steatosis and NAFLD pathogenesis. However, it is difficult to determine with certainty whether mitochondrial dysfunction or oxidative stress are primary events or a simple consequence of NAFLD development. On the one hand, increasing lipid accumulation in hepatocytes could cause a wide range of effects from mild to severe mitochondrial damage with a negative impact on cell fate. This can start the cascade of events, including an increase of cellular reactive nitrogen species (RNS) and reactive oxygen species (ROS) production that promotes disease progression from simple steatosis to more severe NAFLD stages. On the other hand, progressing mitochondrial bioenergetic catastrophe and oxidative stress manifestation could be considered accompanying events in the vast spectrum of abnormalities observed during the transition from NAFL to NASH and cirrhosis. This review updates our current understanding of NAFLD pathogenesis and clarifies whether mitochondrial dysfunction and ROS/RNS are culprits or bystanders of NAFLD progression.

The Alterations of Mitochondrial Function during NAFLD Progression-An Independent Effect of Mitochondrial ROS Production.

The progression of non-alcoholic fatty liver (NAFL) into non-alcoholic steatohepatitis implicates multiple mechanisms, chief of which is mitochondrial dysfunction. However, the sequence of events underlying mitochondrial failure are still poorly clarified. In this work, male C57BL/6J mice were fed with a high-fat plus high-sucrose diet for 16, 20, 22, and 24 weeks to induce NAFL. Up to the 20th week, an early mitochondrial remodeling with increased OXPHOS subunits levels and higher mitochondrial respiration occurred. Interestingly, a progressive loss of mitochondrial respiration along "Western diet" feeding was identified, accompanied by higher susceptibility to mitochondrial permeability transition pore opening. Importantly, our findings prove that mitochondrial alterations and subsequent impairment are independent of an excessive mitochondrial reactive oxygen species (ROS) generation, which was found to be progressively diminished along with disease progression. Instead, increased peroxisomal abundance and peroxisomal fatty acid oxidation-related pathway suggest that peroxisomes may contribute to hepatic ROS generation and oxidative damage, which may accelerate hepatic injury and disease progression. We show here for the first time the sequential events of mitochondrial alterations involved in non-alcoholic fatty liver disease (NAFLD) progression and demonstrate that mitochondrial ROS are not one of the first hits that cause NAFLD progression.

The Lymphocytic Scavenger Receptor CD5 Shows Therapeutic Potential in Mouse Models of Fungal Infection.

Invasive fungal diseases represent an unmet clinical need that could benefit from novel immunotherapeutic approaches. Host pattern recognition receptors (e.g., Toll-like receptors, C-type lectins, or scavenger receptors) that sense conserved fungal cell wall constituents may provide suitable immunotherapeutic antifungal agents. Thus, we explored the therapeutic potential of the lymphocyte class I scavenger receptor CD5, a nonredundant component of the antifungal host immune response that binds to fungal ß-glucans. Antifungal properties of the soluble ectodomain of human CD5 (shCD5) were assessed in vivo in experimental models of systemic fungal infection induced by pathogenic species (Candida albicans and Cryptococcus neoformans). In vitro mechanistic studies were performed by means of fungus-spleen cell cocultures. shCD5-induced survival of lethally infected mice was dose and time dependent and concomitant with reduced fungal load and increased leukocyte infiltration in the primary target organ. Additive effects were observed in vivo after shCD5 was combined with suboptimal doses of fluconazole. Ex vivo addition of shCD5 to fungus-spleen cell cocultures increased the release of proinflammatory cytokines involved in antifungal defense (tumor necrosis factor alpha and gamma interferon) and reduced the number of viable C. albicans organisms. The results prompt further exploration of the adjunctive therapeutic potential of shCD5 in severe invasive fungal diseases.

Discordant susceptibility of inbred C57BL/6 versus outbred CD1 mice to experimental fungal sepsis.

Individual susceptibility differences to fungal infection following invasive and/or immunosuppressive medical interventions are an important clinical issue. In order to explore immune response-related factors that may be linked to fungal infection susceptibility, we have compared the response of inbred C57BL/6J and outbred CD1 mouse strains to different experimental models of fungal sepsis. The challenge of animals with the zymosan-induced generalised inflammation model revealed poorer survival rates in C57BL/6J, consistent with lower Th1 cytokine interferon (IFN)-γ serum levels, compared with CD1 mice. Likewise, ex vivo exposure of C57BL/6J splenocytes to zymosan but also bacterial lipopolisaccharide or lipoteichoic acid, resulted in lower IFN-γ secretion compared with CD1 mice. C57BL/6J susceptibility could be reverted by rescue infusion of relative low IFN-γ doses (0.2 µg/kg) either alone or in combination with the ß-glucan-binding CD5 protein (0.7 mg/kg) leading to improved post zymosan-induced generalised inflammation survival. Similarly, low survival rates to systemic Candida albicans infection (2.86 × 104  CFU/gr) were ameliorated by low-dose IFN-γ infusion in C57BL/6J but not CD1 mice. Our results highlight the importance of strain choice in experimental fungal infection models and provide a susceptibility rationale for more specific antifungal immunotherapy designs.

Exploiting scavenger receptors in cancer immunotherapy: Lessons from CD5 and SR-B1.

Scavenger receptors (SRs) are structurally heterogeneous cell surface receptors characterized by their capacity to remove extraneous or modified self-macromolecules from circulation, thus avoiding the accumulation of noxious agents in the extracellular space. This scavenging activity makes SRs important molecules for host defense and homeostasis. In turn, SRs keep the activation of the steady-state immune response in check, and participate as co-receptors in the priming of the effector immune responses when the macromolecules are associated with a threat that might compromise host homeostasis. Therefore, SRs built up sophisticated sensor mechanisms controlling the immune system, which may be exploited to develop novel drugs for cancer immunotherapy. In this review, we focus on the regulation of the anti-tumor immune response by two paradigmatic SRs: the lymphocyte receptor CD5 and the more broadly distributed scavenger receptor class B type 1 (SR-B1). Cancer immunity can be boosted by blockade of SRs working as immune checkpoint inhibitors (CD5) and/or by proper engagement of SRs working as innate danger receptor (SR-B1). Thus, these receptors illustrate both the complexity of targeting SRs in cancer immunotherapy and also the opportunities offered by such an approach.

Properties that rank protein:protein docking poses with high accuracy.

The development of docking algorithms to predict near-native structures of protein:protein complexes from the structure of the isolated monomers is of paramount importance for molecular biology and drug discovery. In this study, we assessed the capacity of the interfacial area of protein:protein complexes and of Molecular Mechanics-Poisson Boltzmann Surface Area (MM-PBSA)-derived properties, to rank docking poses. We used a set of 48 protein:protein complexes, and a total of 67 docking experiments distributed among bound:bound, bound:unbound, and unbound:unbound test cases. The MM-PBSA binding free energy of protein monomers has been shown to be very convenient to predict high-quality structures with a high success rate. In fact, considering solely the top-ranked pose of more than 200 docking solutions of each of 39 protein:protein complexes, the success rate was 77% in the prediction of high-quality poses, or 90% if considering high- or medium-quality poses. If considering high- or medium-quality poses as the top-one prediction, a success rate of 87% was obtained for a scoring scheme based on computational alanine scanning mutagenesis data. Such ranking accuracy highlights the ability of these properties to predict near-native poses in protein:protein docking.

New Parameters for Higher Accuracy in the Computation of Binding Free Energy Differences upon Alanine Scanning Mutagenesis on Protein-Protein Interfaces.

Knowing how proteins make stable complexes enables the development of inhibitors to preclude protein-protein (P:P) binding. The identification of the specific interfacial residues that mostly contribute to protein binding, denominated as hot spots, is thus critical. Here, we refine an in silico alanine scanning mutagenesis protocol, based on a residue-dependent dielectric constant version of the Molecular Mechanics/Poisson-Boltzmann Surface Area method. We have used a large data set of structurally diverse P:P complexes to redefine the residue-dependent dielectric constants used in the determination of binding free energies. The accuracy of the method was validated through comparison with experimental data, considering the per-residue P:P binding free energy (ΔΔGbinding) differences upon alanine mutation. Different protocols were tested, i.e., a geometry optimization protocol and three molecular dynamics (MD) protocols: (1) one using explicit water molecules, (2) another with an implicit solvation model, and (3) a third where we have carried out an accelerated MD with explicit water molecules. Using a set of protein dielectric constants (within the range from 1 to 20) we showed that the dielectric constants of 7 for nonpolar and polar residues and 11 for charged residues (and histidine) provide optimal ΔΔGbinding predictions. An overall mean unsigned error (MUE) of 1.4 kcal mol-1 relative to the experiment was achieved in 210 mutations only with geometry optimization, which was further reduced with MD simulations (MUE of 1.1 kcal mol-1 for the MD employing explicit solvent). This recalibrated method allows for a better computational identification of hot spots, avoiding expensive and time-consuming experiments or thermodynamic integration/ free energy perturbation/ uBAR calculations, and will hopefully help new drug discovery campaigns in their quest of searching spots of interest for binding small drug-like molecules at P:P interfaces.

CD5 as a Target for Immune-Based Therapies.

CD5 was one of the first surface receptors described for mouse and human T lymphocytes. Since then, it has been found to be highly expressed by regulatory T cells and a subpopulation of regulatory B cells, to be physically associated with the T- and B-cell antigen receptors, to negatively modulate TCR- and BCR-mediated signals, and to bind certain pathogen-associated molecular patterns. These findings position CD5 as an attractive target for developing immunotherapies aimed at either boosting or dampening ongoing immune responses. Here the available data on the function of CD5 and its involvement in the regulation of immune responses in health and disease are reviewed, as well as the evidence for and future challenges in developing therapeutic strategies aimed at targeting CD5 for autoimmune diseases, cancer, and infections.

[Ectopic parathyroid adenoma]. / Adenoma paratiroideu ectópico. caso clínico.

Ectopic parathyroid adenoma is a rare cause of primary hyperparathyroidism and can become a diagnostic challenge. We present a case report of an ectopic mediastinal parathyroid adenoma leading to primary hyperparathyroidism and responsible for a persistent asymptomatic hypercalcemia. The preoperative location of the tumor assessed by imaging studies contribute to the choice of the surgical cervical approach. Tumor resection was successful and the calcemia levels promptly returned to normal levels.

Septic Sternoclavicular Arthritis: An Uncommon Manifestation in the Context of Immunosuppression Following SARS-CoV-2 Infection.

This case report discusses a rare occurrence of septic arthritis in the sternoclavicular joint (SCJ) following SARS-CoV-2 infection-induced immunosuppression in a 94-year-old patient. Despite its rarity, the case underscores the importance of recognizing unusual manifestations of COVID-19, emphasizing the need for healthcare providers to consider COVID-19-induced immunosuppression in differential diagnoses. Swift diagnosis, surgical intervention, and appropriate antibiotics led to a favorable outcome, highlighting the significance of a multidisciplinary approach.

Chilaiditi Syndrome: A Rare Case and Clinical Insights for Diagnosis and Management.

Chilaiditi syndrome is a rare medical condition characterized by the interposition of a hollow organ, usually the colon, between the liver and diaphragm, leading to abdominal pain, discomfort, bloating, constipation, or nausea; in more severe instances, respiratory symptoms may manifest due to pressure on the diaphragm. The exact cause remains unclear but is thought to present along with various factors such as anatomical anomalies (hepato-diaphragmatic interposition and intestinal malrotation) and chronic conditions (cirrhosis or chronic obstructive pulmonary disease). This case report presents a 78-year-old male with rapid deterioration, confusion, and mild abdominal discomfort. Clinical and radiological examinations confirmed Chilaiditi syndrome, highlighting the challenges in diagnosis. Management strategies range from conservative approaches to surgical interventions, emphasizing the need for increased clinical awareness among physicians to ensure accurate and timely interventions. This case report underscores the importance of recognizing this rare condition.

Bladder Perforation in the Elderly: Unraveling the Diagnostic Challenges With Magnetic Resonance Imaging.

Bladder perforation, a significant urological emergency, presents a diagnostic challenge due to its diverse etiologies and varying clinical manifestations. This paper discusses a rare case of bladder perforation in an 87-year-old woman with a history of hypertension and previous stomach and uterine cancer. The patient was admitted with a urinary tract infection and subsequently experienced mild abdominal discomfort and reduced urinary output, prompting further investigation. Imaging studies revealed bladder wall thickening and ureterohydronephrosis, raising suspicion of a bladder tumor. Intriguingly, a catheter-related bladder perforation was identified through MRI. This case emphasizes the importance of considering bladder perforation as a potential complication, especially in elderly patients with indwelling catheters. Clinicians must maintain a high index of suspicion and employ appropriate diagnostic modalities to ensure timely recognition and suitable management of this rare condition.

Perirenal Hematoma: A Rare Complication of Anticoagulant Therapy.

Hemorrhagic complications arising from anticoagulant use are a well-recognized concern in clinical practice. This case study presents an 84-year-old woman with multiple cardiovascular risk factors, including atrial fibrillation, who developed a perirenal hematoma after just five doses of enoxaparin, prescribed for stroke prevention. The patient exhibited altered mental status and abdominal pain, prompting imaging studies revealing the hematoma. This case highlights the importance of vigilance in patients at risk for bleeding complications, especially in the initial days of anticoagulant therapy. Diagnostic imaging, particularly CT scans or ultrasound, is crucial for early detection. Management strategies range from discontinuing anticoagulants to potential interventions like anticoagulation reversal, angiography, or surgery. The decision to resume anticoagulation presents a challenge and requires a personalized approach based on patient factors. This case underscores the need for continued vigilance, early diagnosis, and evidence-based decisions in managing patients on anticoagulants, emphasizing the necessity for further research to establish clear guidelines in such complex clinical scenarios.

Mitochondria-targeted anti-oxidant AntiOxCIN4 improved liver steatosis in Western diet-fed mice by preventing lipid accumulation due to upregulation of fatty acid oxidation, quality control mechanism and antioxidant defense systems.

Non-alcoholic fatty liver disease (NAFLD) is a health concern affecting 24% of the population worldwide. Although the pathophysiologic mechanisms underlying disease are not fully clarified, mitochondrial dysfunction and oxidative stress are key players in disease progression. Consequently, efforts to develop more efficient pharmacologic strategies targeting mitochondria for NAFLD prevention/treatment are underway. The conjugation of caffeic acid anti-oxidant moiety with an alkyl linker and a triphenylphosphonium cation (TPP+), guided by structure-activity relationships, led to the development of a mitochondria-targeted anti-oxidant (AntiOxCIN4) with remarkable anti-oxidant properties. Recently, we described that AntiOxCIN4 improved mitochondrial function, upregulated anti-oxidant defense systems, and cellular quality control mechanisms (mitophagy/autophagy) via activation of the Nrf2/Keap1 pathway, preventing fatty acid-induced cell damage. Despite the data obtained, AntiOxCIN4 effects on cellular and mitochondrial energy metabolism in vivo were not studied. In the present work, we proposed that AntiOxCIN4 (2.5 mg/day/animal) may prevent non-alcoholic fatty liver (NAFL) phenotype development in a C57BL/6J mice fed with 30% high-fat, 30% high-sucrose diet for 16 weeks. HepG2 cells treated with AntiOxCIN4 (100 µM, 48 h) before the exposure to supraphysiologic free fatty acids (FFAs) (250 µM, 24 h) were used for complementary studies. AntiOxCIN4 decreased body (by 43%), liver weight (by 39%), and plasma hepatocyte damage markers in WD-fed mice. Hepatic-related parameters associated with a reduction of fat liver accumulation (by 600%) and the remodeling of fatty acyl chain composition compared with the WD-fed group were improved. Data from human HepG2 cells confirmed that a reduction of lipid droplets size and number can be a result from AntiOxCIN4-induced stimulation of fatty acid oxidation and mitochondrial OXPHOS remodeling. In WD-fed mice, AntiOxCIN4 also induced a hepatic metabolism remodeling by upregulating mitochondrial OXPHOS, anti-oxidant defense system and phospholipid membrane composition, which is mediated by the PGC-1α-SIRT3 axis. AntiOxCIN4 prevented lipid accumulation-driven autophagic flux impairment, by increasing lysosomal proteolytic capacity. AntiOxCIN4 improved NAFL phenotype of WD-fed mice, via three main mechanisms: a) increase mitochondrial function (fatty acid oxidation); b) stimulation anti-oxidant defense system (enzymatic and non-enzymatic) and; c) prevent the impairment in autophagy. Together, the findings support the potential use of AntiOxCIN4 in the prevention/treatment of NAFLD.

Exploratory Data Analysis of Cell and Mitochondrial High-Fat, High-Sugar Toxicity on Human HepG2 Cells.

Non-alcoholic steatohepatitis (NASH), one of the deleterious stages of non-alcoholic fatty liver disease, remains a significant cause of liver-related morbidity and mortality worldwide. In the current work, we used an exploratory data analysis to investigate time-dependent cellular and mitochondrial effects of different supra-physiological fatty acids (FA) overload strategies, in the presence or absence of fructose (F), on human hepatoma-derived HepG2 cells. We measured intracellular neutral lipid content and reactive oxygen species (ROS) levels, mitochondrial respiration and morphology, and caspases activity and cell death. FA-treatments induced a time-dependent increase in neutral lipid content, which was paralleled by an increase in ROS. Fructose, by itself, did not increase intracellular lipid content nor aggravated the effects of palmitic acid (PA) or free fatty acids mixture (FFA), although it led to an up-expression of hepatic fructokinase. Instead, F decreased mitochondrial phospholipid content, as well as OXPHOS subunits levels. Increased lipid accumulation and ROS in FA-treatments preceded mitochondrial dysfunction, comprising altered mitochondrial membrane potential (ΔΨm) and morphology, and decreased oxygen consumption rates, especially with PA. Consequently, supra-physiological PA alone or combined with F prompted the activation of caspase pathways leading to a time-dependent decrease in cell viability. Exploratory data analysis methods support this conclusion by clearly identifying the effects of FA treatments. In fact, unsupervised learning algorithms created homogeneous and cohesive clusters, with a clear separation between PA and FFA treated samples to identify a minimal subset of critical mitochondrial markers in order to attain a feasible model to predict cell death in NAFLD or for high throughput screening of possible therapeutic agents, with particular focus in measuring mitochondrial function.