Treatment of metastatic apocrine sweat gland adenocarcinoma in a cat with association of surgical resection and angular oris reconstruction, extensive lymphadenectomy, electrochemotherapy and systemic chemotherapy.

Apocrine sweat gland adenocarcinoma (ASGAC) is a rare skin carcinoma in cats. In some cases, this tumor occurs in anatomical sites of challenging wide surgical resection, which increases the need for adjuvant therapies for residual disease. These would include radiotherapy or electrochemotherapy (ECT), local treatments for invasive tumors in companion animals often associated with surgery. However, the current literature for ASGAC treatment is limited and there are no reports of ECT as an adjuvant therapy. In this case report, we account for the case of an 11-year-old cat with a history of a non-ulcerated recurrent ASGAC, measuring 1.3 × 1.0 cm, located by the rostral mandibular region. Surgical resection was performed and included the angularis oris axial flap for facial reconstruction associated with electroporation of the surgical bed and, post-operatively, in the surgical scar. Histopathological results confirm the presence of a recurrent ASGAC. Immunostaining revealed cyclooxygenase 2 (COX-2) expression with a score of 6, 50% positivity in Ki-67 and positive for pan-cytokeratin (PCK AE-1/ AE-3). A selective COX-2 inhibitor was initiated along with systemic chemotherapy with chlorambucil. The local approach including surgery and ECT was chosen due to the unfavorable anatomical site for extensive resection and the unavailability of radiotherapy. Subsequently, carboplatin chemotherapy was required due to metastasis in the mandibular lymph node. This case report supports the effectiveness of a multimodal treatment including surgery, ECT and chemotherapy in a cat with recurrent ASGAC.

Treatment with lipoxin A4 improves influenza A infection outcome, induces macrophage reprogramming, anti-inflammatory and pro-resolutive responses.

INTRODUCTION: Influenza A is a virus from the Orthomixoviridae family responsible for high lethality rates and morbidity, despite clinically proven vaccination strategies and some anti-viral therapies. The eicosanoid Lipoxin A4 (LXA4) promotes the resolution of inflammation by decreasing cell recruitment and pro-inflammatory cytokines release, but also for inducing activation of apoptosis, efferocytosis, and macrophage reprogramming. OBJECTIVE: Here, we evaluated whether a synthetic lipoxin mimetic, designated AT-01-KG, would improve the course of influenza A infection in a murine model. METHOD: Mice were infected with influenza A/H1N1 and treated with AT-01-KG (1.7 µg/kg/day, i.p.) at day 3 post-infection. RESULTS: AT-01-KG attenuated mortality, reducing leukocyte infiltration and lung damage at day 5 and day 7 post-infection. AT-01-KG is a Formyl Peptide Receptor 2 (designated FPR2/3 in mice) agonist, and the protective responses were not observed in fpr2/3 -/- animals. In mice treated with LXA4 (50 µg/kg/day, i.p., days 3-6 post-infection), at day 7, macrophage reprogramming was observed, as seen by a decrease in classically activated macrophages and an increase in alternatively activated macrophages in the lungs. Furthermore, the number of apoptotic cells and cells undergoing efferocytosis was increased in the lavage of treated mice. Treatment also modulated the adaptive immune response, increasing the number of T helper 2 cells (Th2) and regulatory T (Tregs) cells in the lungs of the treated mice. CONCLUSION: Therefore, treatment with a lipoxin A4 analog was beneficial in a model of influenza A infection in mice. The drug decreased inflammation and promoted resolution and beneficial immune responses, suggesting it may be useful in patients with severe influenza.

Evaluation of immunophenotype and inflammation in canine mammary neoplasms with solid arrangement.

Morphological and immunohistochemical studies of solid arrangement canine mammary carcinomas have shown that the different histological types may be characterized by proliferation of epithelial and/or myoepithelial cells. However, little is known about immunophenotypes and the importance of inflammation as prognostic factors in these neoplasms. The objective of the present study was to characterize the immunophenotype and degree of inflammation in the solid type of canine mammary neoplasm and to investigate their association with metastasis, Ki-67 index, tumour size, necrosis and survival. Sixty-five carcinomas with solid pattern, basaloid carcinomas, solid papillary carcinomas, malignant adenomyoepitheliomas (MAMEs) or malignant myoepitheliomas (MMEs) were investigated. Luminal A, luminal B HER2 negative and HER2 positive, HER2 overexpressed and triple negative immunophenotypes were immunolabelled as were Ki-67 protein and cyclooxygenase-2 (Cox-2). Histological peritumoural and intratumoural inflammatory infiltrates were graded (distribution × intensity) and the presence of necrosis identified. We found a statistical difference between histological types and immunophenotypes, with MME and MAME having a higher occurrence of luminal A, whereas most neoplasms had the luminal B HER-negative immunophenotype. There was no correlation between immunophenotype and degree of peri- and intratumoural inflammation, nodal metastasis, necrosis or tumour size. An increased degree of peri- and intratumoural inflammation was significantly associated with lymph node metastasis, and more severe intratumoural inflammation was associated with the presence of tumour necrosis. Tumour size, Ki-67 index and Cox-2 score were not associated with inflammation in either peri- or intratumoural regions. No difference was observed in survival in relation to immunophenotype or degree of inflammation, but the Cox regression model revealed that nodal metastasis influenced the risk of death.

Hybrid Nanosystem Formed by DOX-Loaded Liposomes and Extracellular Vesicles from MDA-MB-231 Is Effective against Breast Cancer Cells with Different Molecular Profiles.

Drug delivery selectivity is a challenge for cancer treatment. A hybrid pegylated pH-sensitive liposome-extracellular vesicle isolated from human breast cancer cell MDA-MB-231 was developed to investigate its in vitro activity against breast cancer cells of different molecular profiles to overcome this inconvenience. The hybrid nanosystem was produced by film hydration, and doxorubicin (DOX) was encapsulated in this system using the ammonium sulfate gradient method. The characterization of this hybrid nanosystem revealed a mean diameter of 140.20 ± 2.70 nm, a polydispersity index of 0.102 ± 0.033, an encapsulation efficiency of doxorubicin of 88.9% ± 2.4, and a great storage stability for 90 days at 4 °C. The fusion of extracellular vesicles with liposomes was confirmed by nanoflow cytometry using PE-conjugated human anti-CD63. This hybrid nanosystem demonstrated cytotoxicity against human breast cancer cell lines with different molecular subtypes, enhanced anti-migration properties, and exhibited similar cellular uptake to the free DOX treatment. Preliminary acute toxicity assessments using Balb/C female mice indicated a median lethal dose of 15-17.5 mg/kg, with no evidence of splenic, liver, heart, bone marrow, and renal damage at a dose of 15 mg/kg. These findings suggest the hybrid formulation as a versatile nanocarrier for the treatment of various breast cancer subtypes.

Canine Visceral Leishmaniasis: A Histological and Immunohistochemical Study of Fibropoiesis in Chronic Interstitial Pneumonitis.

We studied some fibrotic aspects of chronic interstitial pneumonitis in the lungs of dogs infected with Leishmania infantum. The lungs of eleven naturally infected dogs, twelve experimentally infected with two distinct strains of L. infantum (BH401 and BH46), and six uninfected (controls) dogs, were analyzed by histological, parasitological, and immunohistochemical studies. Conventional histology (HE), collagen deposition (Gomori's silver staining for reticulin collagen fibers), and immunohistochemistry for myofibroblast characterization were carried out based on the cellular expression of alpha-smooth muscle actin, vimentin, cytokeratin, E-cadherin, snail antigen homologue 1 (SNAI1) (Snail), and the cytokine expression of transforming growth factor-beta (TGF-ß). Parasitological screening was carried out using conventional polymerase chain reaction (PCR) and the immunohistochemical reaction of streptavidin-peroxidase for visualizing Leishmania amastigotes. Dogs naturally infected with L. infantum and experimentally infected with L. infantum BH401 strains showed intense interstitial pneumonitis characterized by thickening of the alveolar septa as a consequence of an intense diffuse and focal (plaques) chronic exudate of mononuclear cells associated with fibrogenesis. The expression of alpha-actin, vimentin, and TGF-ß was higher in the lung interstitium of all infected dogs than in the other two groups (BH46 strain and controls). Moreover, in both the naturally and experimentally infected dog (BH401 strain) groups, the expression of Snail was moderate to intense in contrast to the other groups. Based on these immunohistochemical results, we concluded that mesenchymal cells are active in promoting changes in the extracellular matrix in the lungs of dogs naturally and experimentally infected with L. infantum, but it depends on the virulence of the parasite.

Collagen modifications predictive of lymph node metastasis in dogs with carcinoma in mixed tumours.

Introduction: Mixed tumours in the canine mammary gland are the most common histological type in routine diagnosis. In general, these neoplasms have a favourable prognosis that does not evolve into metastatic disease. However, some cases develop into lymph node metastases and are associated with worse patient survival rates. Methods: Here is a retrospective study of 46 samples of primary mixed tumours of the canine mammary gland: 15 cases of benign mixed tumours (BMT), 16 cases of carcinoma in mixed tumours without lymph node metastasis (CMT), and 15 cases of carcinomas in mixed tumours with lymph node metastasis (CMTM). In addition, we selected 23 cases of normal mammary glands (NMT) for comparison. The samples were collected from biopsies performed during nodulectomy, simple mastectomy, regional mastectomy, or unilateral/bilateral radical mastectomy. We used multiphoton microscopy, second harmonic generation, and two-photon excited fluorescence, to evaluate the characteristics of collagen fibres and cellular components in biopsies stained with haematoxylin and eosin. We performed Ki67, ER, PR, and HER-2 immunostaining to define the immunophenotype and COX-2. We showed that carcinomas that evolved into metastatic disease (CMTM) present shorter and wavier collagen fibres as compared to CMT. Results and discussion: When compared to NMT and BMT the carcinomas present a smaller area of fibre coverage, a larger area of cellular coverage, and a larger number of individual fibres. Furthermore, we observed a correlation between the strong expression of COX-2 and a high rate of cell proliferation in carcinomas with a smaller area covered by cell fibres and a larger number of individual fibres. These findings highlight the fundamental role of collagen during tumour progression, especially in invasion and metastatic dissemination.

The role of lymph nodes and their drainage in canine mammary gland tumours: Systematic review.

Mammary gland tumours are the most common neoplasms in intact bitches. Over the last decades, veterinary oncology has evolved in detecting and determining the lymph nodes to be removed in these patients for an accurate staging and prognosis, as well as to achieve better disease control and higher overall survival time. Our objective was to describe recent advances related to lymphatic drainage in bitches with mammary gland tumours, focusing on surgery, diagnosis, and prognosis. Through a systematic review using PubMed as the database, a thorough multi-step search reduced 316 studies to 30 for analysis. Vital dyes appear to be crucial in reducing the overall surgery time through transoperative staining of the lymph nodes. Imaging contrasts provide information regarding specific tumour drainage; however, there is still little evidence for their use. The axillary and superficial inguinal lymph nodes are well-established as regional lymph nodes of the cranial and caudal mammary glands. In sequence, accessory axillary, medial iliac, popliteal, and sternal lymph nodes should receive attention if they demonstrate contrast drainage, even considering that the literature has not shown a relationship between drainage and metastasis in these cases. In conclusion, recent studies have provided us with more support in regional lymph node excision regarding the TNM staging system. Studies are highly heterogeneous and method comparisons do not fit due to the non-uniformity of samples, materials, and procedures. We suggest further studies with a larger sample size, complete follow-up of patients, contrast use, and lymph node morphological and immunohistochemical analysis.

Mammary fibromatosis in a female dog: case report.

Fibromatosis, or desmoid tumour, is characterized by excessive and infiltrative proliferation of connective tissue originating from aponeurotic muscle structures. Mammary fibromatosis is rare in humans and animals and its precise aetiology is unknown. A 10-year-old mixed-breed female dog developed a mass in the right cranial thoracic mammary gland (M1) and underwent lumpectomy. The mass was firm, with an irregular surface and distinct limits. Microscopically, it was a neoplastic proliferation of fusiform cells with low atypia, interspersed with abundant dense collagenous tissue, confirmed by histochemical staining with Gomori's trichrome and Masson's trichrome and immunopositivity for vimentin and smooth muscle actin, confirming mammary fibromatosis. Mammary fibromatosis in dogs needs further studies to elucidate its clinical, epidemiological and aetiopathogenic aspects.

Preclinical evaluation of L-fucoside from lapachol-loaded nanoemulsion as a strategy to breast cancer treatment.

Breast cancer prevails as the most common cancer in women, underscoring an urgent need for more effective therapies. This study explores the potential of our newly developed nanoemulsion containing a novel fucoside derivative of lapachol (NE-F-LapA) as an intravenous treatment strategy. We sought to overcome the solubility issues associated with fucoside with this improved drug delivery strategy that enhances tumor delivery and mitigates other dose-limiting toxicities. Nanoemulsion was prepared and characterized by DLS, zeta potential, encapsulation efficiency, and storage stability. Cytotoxicity against breast cancer cell lines (4T1 and MDA-MB-231) and non-tumor human fibroblasts (NTHF) were evaluated. In vivo assays included antitumoral activity performance and acute systemic toxicity in mice models. NE-F-LapA was synthesized and optimized to 200 nm size, - 20 mV zeta potential, and near-complete (>98%) drug encapsulation. Stability exceeded 6 months, and biological fluid exposure maintained suitable properties for administration. In vitro, NE-F-LapA showed high toxicity (3 µM) against 4T1 and MDA-MB-231, enhanced five times the breast cancer cell uptake and three times the selectivity when compared to normal cells. Systemic toxicity assessment in mice revealed no concerning hematological or biochemical changes. Finally, in a 4T1 breast tumor model, NE-F-LapA significantly inhibited growth by 50% of the subcutaneous 4T1 tumor and reduced lung metastases 5-fold versus control. Overall, tailored nanoemulsification of the lapachol derivative enabled effective intravenous administration and improved efficacy over the free drug, indicating promise for enhanced breast cancer therapy pending further optimization.

Platelet-activating factor and protease-activated receptor 2 cooperate to promote neutrophil recruitment and lung inflammation through nuclear factor-kappa B transactivation.

Although it is well established that platelet-activated receptor (PAF) and protease-activated receptor 2 (PAR2) play a pivotal role in the pathophysiology of lung and airway inflammatory diseases, a role for a PAR2-PAFR cooperation in lung inflammation has not been investigated. Here, we investigated the role of PAR2 in PAF-induced lung inflammation and neutrophil recruitment in lungs of BALB/c mice. Mice were pretreated with the PAR2 antagonist ENMD1068, PAF receptor (PAFR) antagonist WEB2086, or aprotinin prior to intranasal instillation of carbamyl-PAF (C-PAF) or the PAR2 agonist peptide SLIGRL-NH2 (PAR2-AP). Leukocyte infiltration in bronchoalveolar lavage fluid (BALF), C-X-C motif ligand 1 (CXCL)1 and CXCL2 chemokines, myeloperoxidase (MPO), and N-acetyl-glycosaminidase (NAG) levels in BALF, or lung inflammation were evaluated. Intracellular calcium signaling, PAFR/PAR2 physical interaction, and the expression of PAR2 and nuclear factor-kappa B (NF-КB, p65) transcription factor were investigated in RAW 264.7 cells stimulated with C-PAF in the presence or absence of ENMD1068. C-PAF- or PAR2-AP-induced neutrophil recruitment into lungs was inhibited in mice pretreated with ENMD1068 and aprotinin or WEB2086, respectively. PAR2 blockade impaired C-PAF-induced neutrophil rolling and adhesion, lung inflammation, and production of MPO, NAG, CXCL1, and CXCL2 production in lungs of mice. PAFR activation reduced PAR2 expression and physical interaction of PAR2 and PAFR; co-activation is required for PAFR/PAR2 physical interaction. PAR2 blockade impaired C-PAF-induced calcium signal and NF-κB p65 translocation in RAW 264.7 murine macrophages. This study provides the first evidence for a cooperation between PAFR and PAR2 mediating neutrophil recruitment, lung inflammation, and macrophage activation.

Gut microbiota from patients with COVID-19 cause alterations in mice that resemble post-COVID symptoms.

Long-term sequelae of coronavirus disease (COVID)-19 are frequent and of major concern. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection affects the host gut microbiota, which is linked to disease severity in patients with COVID-19. Here, we report that the gut microbiota of post-COVID subjects had a remarkable predominance of Enterobacteriaceae strains with an antibiotic-resistant phenotype compared to healthy controls. Additionally, short-chain fatty acid (SCFA) levels were reduced in feces. Fecal transplantation from post-COVID subjects to germ-free mice led to lung inflammation and worse outcomes during pulmonary infection by multidrug-resistant Klebsiella pneumoniae. transplanted mice also exhibited poor cognitive performance. Overall, we show prolonged impacts of SARS-CoV-2 infection on the gut microbiota that persist after subjects have cleared the virus. Together, these data demonstrate that the gut microbiota can directly contribute to post-COVID sequelae, suggesting that it may be a potential therapeutic target.

Dietary lipids management: are there any benefits from the prevention and treatment of 4T1 murine breast carcinoma?

This study investigated the effect of vegetable and fish oils with different n-3 / n-6 PUFAS ratios on the lipoprotein profile and on the development of murine breast cancer 4T1. Female Balb/c mice (6-7 weeks) received diets containing 4.0% fat during seven weeks. On the fourth week, animals were inoculated into the posterior left flank with 2.5 × 106 4T1 cells. Body weight and food intake were registered and the profile serum lipoproteins was determined. Tumor volume, histopathological and immunohistochemical studies, myeloperoxidase and N-acetylglucosaminidase activities, TNF-α, hemoglobin and VEGF levels were analysed. The highest n-3 / n-6 ratio was found in fish oil (15.8:1), followed by linseed (2.4:1), canola (1:2.1) and soybean (1:9.4) oils. Body weight, food and caloric intake, lipoprotein profile, tumor weight, tumor evolution and histopathological analysis were not different. Canola oil increased cell proliferation when compared to soybean oil, and fish oil changed the inflammatory response and increased VEGF in tumors compared to other groups. The type of fatty acid and the high ratio of n-3 / n-6 PUFAs in the diet influenced cell proliferation and inflammation in the tumor differentially, highlighting the increase of neutrophils and VEGF levels in animals fed on fish oil.

Evaluation of acute toxicity and in vitro antitumor activity of a novel doxorubicin-loaded folate-coated pH-sensitive liposome.

Doxorubicin (DOX) loaded liposomes have been used and studied in the last decades due to the significant decrease in DOX induced cardiac and systemic toxicity relative to administration of free drug. Therefore, new strategies are sought to improve DOX delivery and antitumor activity, while avoiding side effects. Recently, folate-coated pH-sensitive liposomes (SpHL-Fol) have been studied as a tool to enhance cellular uptake and antitumor activity of paclitaxel and DOX in breast cancer cells expressing folate receptor (FR+). However, the elucidation of folate functionalization relevance in DOX-loaded SpHL (SpHL-DOX-Fol) in different cell types (MDA-MB-231, MCF-7, and A549), as well as, the complete safety evaluation, is necessary. To achieve these objectives, SpHL-DOX-Fol was prepared and characterized as previously described. Antitumor activity and acute toxicity were evaluated in vivo through direct comparison of free DOX verses SpHL-DOX, a well-known formulation to reduce DOX cardiotoxicity. The obtained data are crucial to support future translational research. Liposomes showed long-term stability, suitable for biological use. Cellular uptake, cytotoxicity, and percentage of migration inhibition were significantly higher for MDA-MB-231 (FR+) treated with SpHL-DOX-Fol. In addition, SpHL-DOX-Fol demonstrated a decrease in the systemic toxic effects of DOX, mainly in renal and cardiac parameters evaluation, even using a higher dose (20 mg/kg). Collectively these data build the foundation of support demonstrating that SpHL-DOX-Fol could be considered a promising drug delivery strategy for the treatment of FR+ breast tumors.

GATA-3 expression and its correlation with prognostic factors and survival in canine mammary tumors.

Introduction: The transcription factor GATA-3 plays a significant role in mammary gland development and differentiation. Recent studies on human oncology have demonstrated its association with favorable pathologic factors in breast cancer. Canine mammary tumours, proposed as comparative and translational study models, have epidemiological, clinical, biological, and genetic characteristics similar to those of human breast cancers. Methods: Here, we evaluated the frequency of GATA-3 expression in mammary tumors of dogs and its relationship with prognostic factors and survival. Tumor samples were obtained from 40 female dogs and grouped according to histological type into benign tumors (n = 10), carcinoma in mixed tumors (CMTs) (n = 20), and aggressive tumors (n = 10). CMTs were further separated according to histological grade, and data on clinical staging and diagnosis, histopathological grading, and survival rate were collected. Results: GATA-3 and estrogen receptor (ER) expression were higher in benign and well-differentiated carcinomas than in aggressive tumors, which showed greater Ki-67 expression. The expression rate of ER in the studied groups was equivalent to that of GATA-3. We identified a strong positive correlation between GATA-3 and ER expression frequencies and a negative correlation between those of GATA-3 and Ki-67. There were associations between GATA-3 (p < 0.001), Ki-67 (p = 0.003), tumor size (p < 0.001), clinical stage (p = 0.002), lymph node metastasis (p < 0.001), and histological grade (p < 0.001) by univariate survival analysis. The parameters ER (p = 0.015) and GATA-3 (p = 0.005) also influenced survival in a multifactorial manner. Discussion: Kaplan-Meier analysis of survival curves validated our previous findings that dogs with GATA-3 expression in ≥79.4% of cells had significantly higher survival rates (p < 0.001). The performance analysis showed that the expression of GATA-3 in ≥79.4% of cells effectively predicted survival or death in dogs with mammary tumors. Collectively, these results suggest that GATA-3 can be a relevant marker in the study of mammary tumor progression and has potential as a prognosis marker for predicting outcomes in canine mammary tumors.

Metastatic exocrine pancreatic adenocarcinoma in a giant otter (Pteronura brasiliensis).

Neoplasms of the exocrine pancreas are uncommon in domestic animals and rarely occur in wildlife. This article describes the clinical and pathological findings of one case of metastatic exocrine pancreatic adenocarcinoma in an 18-year-old giant otter (Pteronura brasiliensis) in captivity with a history of inappetence and apathy. Abdominal ultrasonography was inconclusive, and tomography revealed a neoplasm affecting the urinary bladder and hydroureter. During the anaesthesia recovery, the animal presented a cardiorespiratory arrest and died. Grossly, there were neoplastic nodules in the pancreas, urinary bladder, spleen, adrenal glands, and mediastinal lymph node. Microscopically, all nodules were composed of a malignant hypercellular proliferation of epithelial cells with acinar or solid disposition, supported by a sparse fibrovascular stroma. Neoplastic cells were immunolabeled with antibodies to Pan-CK, CK7, CK20, PPP and chromogranin A. Approximately 25% of the cells were positive for the presence of Ki-67 too. Pathological and immunohistochemical findings confirmed the diagnosis of metastatic exocrine pancreatic adenocarcinoma.

Emetic Tartar-Loaded Liposomes as a New Strategy for Leishmaniasis Treatment.

Emetic tartar (ET), was used in the treatment of leishmaniasis but its use was discontinued due to its low therapeutic index. Liposomes have been shown to be a promising strategy for delivery of bioactive substances in the region of interest, in order to reduce and/or eliminate undesirable effects. In the present study, liposomes containing ET were prepared and characterized to evaluate acute toxicity as well as their leishmanicidal action using BALB/c mice with an inoculum of Leishmania (Leishmania) infantum. Liposomes were composed of egg phosphatidylcholine and 3ß-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol, with an average diameter of 200 nm, zeta potential of +18 mV, and ET encapsulated into liposomes at a concentration near 2 g/L. Healthy mice were treated with ET or liposome containing ET (Lip-ET) in a single dose of 16 mg/kg of Sb3+ intravenously and observed for 14 days. The death of two animals in the ET-treated group and no deaths in the Lip-ET-treated group was observed. Higher hepatic and cardiac toxicity were observed in animals treated with ET when compared to animals treated with Lip-ET, blank liposomes (Blank-Lip) and PBS. The study of antileishmanial efficacy was conducted by intraperitoneal administration of Lip-ET, for ten consecutive days. It was observed by limiting dilution that treatments with liposomal formulations containing ET, as well as Glucantime®, led to a significant reduction in parasitic load in spleen and liver (p < 0.05) when compared to the untreated control group.

Cisplatin-Loaded Thermosensitive Liposomes Functionalized with Hyaluronic Acid: Cytotoxicity and In Vivo Acute Toxicity Evaluation.

Cisplatin (CDDP) is a potent antitumor drug used in first-line chemotherapy against several solid tumors, including breast cancer. However, toxicities and drug resistance limit its clinical application. Thermosensitive liposome (TSL) functionalized with hyaluronic acid (HA) containing cisplatin (TSL-CDDP-HA) was developed by our research group aiming to promote the release of CDDP in the tumor region under hyperthermia conditions, as well as to decrease toxicity. Thus, this study aimed to evaluate this new formulation (HA-coated TSL-CDDP) concerning in vitro behavior and in vivo toxicity compared to non-coated TSL-CDDP and free CDDP. Cytotoxicity assays and nuclear morphology were carried out against triple-negative breast cancer cells (MDA-MB-231), while an in vivo toxicity study was performed using healthy Swiss mice. The results showed an increase (around 3-fold) in cytotoxicity of the cationic formulation (non-coated TSL-CDDP) compared to free CDDP. On the other hand, TSL-CDDP treatment induced the appearance of 2.5-fold more senescent cells with alteration of nuclear morphology than the free drug after hyperthermia condition. Furthermore, the association of liposomal formulations treatment with hyperthermia increased the percentage of apoptotic cells compared to those without heating. The percentage of apoptotic cells was 1.7-fold higher for TSL-CDDP-HA than for TSL-CDDP. For the in vivo toxicity data, the TSL-CDDP treatment was also toxic to healthy cells, inducing nephrotoxicity with a significant increase in urea levels compared to the saline control group (73.1 ± 2.4 vs. 49.2 ± 2.8 mg/mL). On the other hand, the HA-coated TSL-CDDP eliminated the damages related to the use of CDDP since the animals did not show changes in hematological and biochemical examinations and histological analyses. Thus, data suggest that this new formulation is a potential candidate for the intravenous therapy of solid tumors.

High SARS-CoV-2 tropism and activation of immune cells in the testes of non-vaccinated deceased COVID-19 patients.

BACKGROUND: Cellular entry of SARS-CoV-2 has been shown to rely on angiotensin-converting enzyme 2 (ACE2) receptors, whose expression in the testis is among the highest in the body. Additionally, the risk of mortality seems higher among male COVID-19 patients, and though much has been published since the first cases of COVID-19, there remain unanswered questions regarding SARS-CoV-2 impact on testes and potential consequences for reproductive health. We investigated testicular alterations in non-vaccinated deceased COVID-19-patients, the precise location of the virus, its replicative activity, and the immune, vascular, and molecular fluctuations involved in the pathogenesis. RESULTS: We found that SARS-CoV-2 testicular tropism is higher than previously thought and that reliable viral detection in the testis requires sensitive nanosensors or RT-qPCR using a specific methodology. Through an in vitro experiment exposing VERO cells to testicular macerates, we observed viral content in all samples, and the subgenomic RNA's presence reinforced the replicative activity of SARS-CoV-2 in testes of the severe COVID-19 patients. The cellular structures and viral particles, observed by transmission electron microscopy, indicated that macrophages and spermatogonial cells are the main SARS-CoV-2 lodging sites, where new virions form inside the endoplasmic reticulum Golgi intermediate complex. Moreover, we showed infiltrative infected monocytes migrating into the testicular parenchyma. SARS-CoV-2 maintains its replicative and infective abilities long after the patient's infection. Further, we demonstrated high levels of angiotensin II and activated immune cells in the testes of deceased patients. The infected testes show thickening of the tunica propria, germ cell apoptosis, Sertoli cell barrier loss, evident hemorrhage, angiogenesis, Leydig cell inhibition, inflammation, and fibrosis. CONCLUSIONS: Our findings indicate that high angiotensin II levels and activation of mast cells and macrophages may be critical for testicular pathogenesis. Importantly, our findings suggest that patients who become critically ill may exhibit severe alterations and harbor the active virus in the testes.

Association of Fructo-oligosaccharides and Arginine Improves Severity of Mucositis and Modulate the Intestinal Microbiota.

Mucositis is defined as inflammatory and ulcerative lesions along of the gastrointestinal tract that leads to the imbalance of the intestinal microbiota. The use of compounds with action on the integrity of the intestinal epithelium and their microbiota may be a beneficial alternative for the prevention and/or treatment of mucositis. So, the aim of this study was to evaluate the effectiveness of the association of fructo-oligosaccharides (FOS) and arginine on intestinal damage in experimental mucositis. BALB/c mice were randomized into five groups: CTL (without mucositis + saline), MUC (mucositis + saline), MUC + FOS (mucositis + supplementation with FOS-1st until 10th day), MUC + ARG (mucositis + supplementation with arginine-1st until 10th day), and MUC + FOS + ARG (mucositis + supplementation with FOS and arginine-1st until 10th day). On the 7th day, mucositis was induced with an intraperitoneal injection of 300 mg/kg 5-fluorouracil (5-FU), and after 72 h, the animals were euthanized. The results showed that association of FOS and arginine reduced weight loss and oxidative stress (P < 0.05) and maintained intestinal permeability and histological score at physiological levels. The supplementation with FOS and arginine also increased the number of goblet cells, collagen area, and GPR41 and GPR43 gene expression (P < 0.05). Besides these, the association of FOS and arginine modulated intestinal microbiota, leading to an increase in the abundance of the genera Bacteroides, Anaerostipes, and Lactobacillus (P < 0.05) in relation to increased concentration of propionate and acetate. In conclusion, the present results show that the association of FOS and arginine could be important adjuvants in the prevention of intestinal mucositis probably due to modulated intestinal microbiota.

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

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