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INTRODUCTION: The etiology of brain aneurysms remains poorly understood. Finnish research suggests that oral bacteria might contribute to the development and rupture of brain aneurysms. Previous studies by our team have not confirmed these findings, likely due to methodological differences. We aimed to replicate the Finnish study with a French population, using the same primers and comparing the results to internal controls. METHODS: We used RT-qPCR to retrospectively analyze the expression of oral bacterial genes in eight patients. During surgical procedures, four tissue types were consistently sampled from each patient: the aneurysmal wall, the superficial temporal artery (STA), the middle meningeal artery (MMA), and the dura mater (DM). Results were expressed as fold differences employing the 2-∆∆Ct method, and statistical analyses were performed accordingly. RESULTS: Our cohort included eight patients, evenly split into ruptured and unruptured groups. The sex distribution was balanced (4 females, 4 males). We observed DNA expression from oral bacteria in all sampled tissues; however, there were no significant differences between the ruptured and unruptured groups. CONCLUSION: We detected oral bacterial gene expression in the aneurysmal wall, STA, MMA, and DM in a sample of French patients. Although limited by the small sample size, our results suggest a potential role for bacterial involvement in vascular invasiveness related to bacteremia. These findings do not definitively link oral bacteria to the pathogenesis of aneurysm development and rupture.
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Aneurisma Intracraneal , Humanos , Femenino , Masculino , Aneurisma Intracraneal/microbiología , Persona de Mediana Edad , Francia , Anciano , Estudios Retrospectivos , Boca/microbiología , Adulto , Aneurisma Roto/microbiología , Arterias Temporales , Duramadre , Bacterias/genética , Bacterias/aislamiento & purificación , Arterias MeníngeasRESUMEN
Ovarian cancer (OC) is one of the most lethal cancers among women. Frequent recurrence in the peritoneum due to the presence of microscopic tumor residues justifies the development of new therapies. Indeed, our main objective is to develop a targeted photodynamic therapy (PDT) treatment of peritoneal carcinomatosis from OC to improve the life expectancy of cancer patients. Herein, we propose a targeted-PDT using a vectorized photosensitizer (PS) coupled with a newly folic acid analog (FAA), named PSFAA, in order to target folate receptor alpha (FRα) overexpressed on peritoneal metastasis. This PSFAA was the result of the coupling of pyropheophorbide-a (Pyro-a), as the PS, to a newly synthesized FAA via a polyethylene glycol (PEG) spacer. The selectivity and the PDT efficacy of PSFAA was evaluated on two human OC cell lines overexpressing FRα compared to fibrosarcoma cells underexpressing FRα. Final PSFAA, including the synthesis of a newly FAA and its conjugation to Pyro-a, was obtained after 10 synthesis steps, with an overall yield of 19%. Photophysical properties of PSFAA in EtOH were performed and showed similarity with those of free Pyro-a, such as the fluorescence and singlet oxygen quantum yields (Φf = 0.39 and ΦΔ = 0.53 for free Pyro-a, and Φf = 0.26 and ΦΔ = 0.41 for PSFAA). Any toxicity of PSFAA was noticed. After light illumination, a dose-dependent effect on PS concentration and light dose was shown. Furthermore, a PDT efficacy of PSFAA on OC cell secretome was detected inducing a decrease of a pro-inflammatory cytokine secretion (IL-6). This new PSFAA has shown promising biological properties highlighting the selectivity of the therapy opening new perspectives in the treatment of a cancer in a therapeutic impasse.
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Clorofila , Ácido Fólico , Interleucina-6 , Neoplasias Ováricas , Fotoquimioterapia , Fármacos Fotosensibilizantes , Fotoquimioterapia/métodos , Humanos , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/patología , Femenino , Fármacos Fotosensibilizantes/farmacología , Fármacos Fotosensibilizantes/administración & dosificación , Ácido Fólico/química , Línea Celular Tumoral , Clorofila/análogos & derivados , Clorofila/farmacología , Clorofila/administración & dosificación , Clorofila/uso terapéutico , Clorofila/química , Interleucina-6/metabolismo , Muerte Celular/efectos de los fármacos , Receptor 1 de Folato/metabolismo , Inflamación/tratamiento farmacológico , Supervivencia Celular/efectos de los fármacosRESUMEN
Background: Pancreatic adenocarcinoma (PDAC) is a devastating disease with an urgent need for therapeutic innovation. Immune checkpoint inhibition has shown promise in a variety of solid tumors, but most clinical trials have failed to demonstrate clinical efficacy in PDAC. This low efficacy is partly explained by a highly immunosuppressive microenvironment, which dampens anti-tumor immunity through the recruitment or induction of immunosuppressive cells, particularly regulatory T cells (Tregs). In this context, our laboratory has developed a novel immunotherapeutic strategy aimed at inhibiting the suppressive activity of Tregs, based on a patented (EP3152234B1) monoclonal antibody (mAb) targeting galectin-9 (LGALS9). Materials and methods: CD4+ conventional T cells (TCD4 or Tconv), Treg ratio, and LGALS9 expression were analyzed by immunohistochemistry (IHC) and cytometry in blood and pancreas of K-rasLSL.G12D/+;Pdx-1-Cre (KC) and K-rasWildType (WT);Pdx1-Cre (WT) mice aged 4-13 months. Pancreatic intraepithelial neoplasm (PanIN) progression and grade were quantified using FIJI software and validated by pathologists. The anti-galectin-9 mAb was validated for its use in mice on isolated murine C57BL/6 Treg by immunofluorescence staining and cytometry. Its specificity and functionality were validated in proliferation assays on rLGALS9-immunosuppressed murine Tconv and in suppression assays between murine Treg and Tconv. Finally, 2-month-old KC mice were treated with anti-LGALS9 and compared to WT mice for peripheral and infiltrating TCD4, Treg, and PanIN progression. Results: IHC and cytometry revealed a significant increase in LGALS9 expression and Treg levels in the blood and pancreas of KC mice proportional to the stages of precancerous lesions. Although present in WT mice, LGALS9 is expressed at a basal level with low and restricted expression that increases slightly over time, while Treg cells are few in number in their circulation and even absent from the pancreas over time. Using our anti-LGALS9 mAb in mice, it is shown that (i) murine Treg express LGALS9, (ii) the mAb could target and inhibit recombinant murine LGALS9, and (iii) neutralize murine Treg suppressive activity. Finally, the anti-LGALS9 mAb in KC mice reduced (i) LGALS9 expression in pancreatic cancer cells, (ii) the Treg ratio, and (iii) the total surface area and grade of PanIN. Conclusion: We demonstrate for the first time that an anti-LGALS9 antibody, by specifically targeting endogenous LGALS9 tumor and exogenous LGALS9 produced by Treg, was able to limit the progression of pancreatic neoplastic lesions in mice, opening up new prospects for its use as an immunotherapeutic tool in PDAC.
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Adenocarcinoma , Carcinoma in Situ , Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Ratones , Animales , Ratones Transgénicos , Ratones Endogámicos C57BL , Carcinoma in Situ/metabolismo , Carcinoma in Situ/patología , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/uso terapéutico , Galectinas , Inmunoterapia , Microambiente TumoralRESUMEN
Nasopharyngeal carcinoma-derived small extracellular vesicles (NPCSEVs) have an immunosuppressive impact on the tumour microenvironment. In this study, we investigated their influence on the generation of tolerogenic dendritic cells and the potential involvement of the galectin-9 (Gal9) they carry in this process. We analysed the phenotype and immunosuppressive properties of NPCSEVs and explored the ability of DCs exposed to NPCSEVs (NPCSEV-DCs) to regulate T cell proliferation. To assess their impact at the pathophysiological level, we performed real-time fluorescent chemoattraction assays. Finally, we analysed phenotype and immunosuppressive functions of NPCSEV-DCs using a proprietary anti-Gal9 neutralising antibody to assess the role of Gal9 in this effect. We described that NPCSEV-DCs were able to inhibit T cell proliferation despite their mature phenotype. These mature regulatory DCs (mregDCs) have a specific oxidative metabolism and secrete high levels of IL-4. Chemoattraction assays revealed that NPCSEVs could preferentially recruit NPCSEV-DCs. Finally, and very interestingly, the reduction of the immunosuppressive function of NPCSEV-DCs using an anti-Gal9 antibody clearly suggested an important role for vesicular Gal9 in the induction of mregDCs. These results revealed for the first time that NPCSEVs promote the emergence of mregDCs using a galectin-9 dependent mechanism and open new perspectives for antitumour immunotherapy targeting NPCSEVs.
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Vesículas Extracelulares , Neoplasias Nasofaríngeas , Humanos , Carcinoma Nasofaríngeo , Células Dendríticas , Galectinas/metabolismo , Neoplasias Nasofaríngeas/metabolismo , Microambiente TumoralRESUMEN
Photodynamic therapy (PDT) has shown improvements in cancer treatment and in the induction of a proper anti-tumor immune response. However, current photosensitizers (PS) lack tumor specificity, resulting in reduced efficacy and side effects in patients with intraperitoneal ovarian cancer (OC). In order to target peritoneal metastases of OC, which overexpress folate receptor (FRα) in 80% of cases, we proposed a targeted PDT using a PS coupled with folic acid. Herein, we applied this targeted PDT in an in vivo mouse model of peritoneal ovarian carcinomatosis. The efficacy of the treatment was evaluated in mice without and with human peripheral blood mononuclear cell (PBMC) reconstitution. When mice were reconstituted, using a fractionized PDT protocol led to a significantly higher decrease in the tumor growth than that obtained in the non-reconstituted mice (p = 0.0469). Simultaneously, an immune response was reflected by an increase in NK cells, and both CD4+ and CD8+ T cells were activated. A promotion in cytokines IFNγ and TNFα and an inhibition in cytokines TGFß, IL-8, and IL-10 was also noticed. Our work showed that a fractionized FRα-targeted PDT protocol is effective for the treatment of OC and goes beyond local induction of tumor cell death, with the promotion of a subsequent anti-tumor response.
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Neoplasias Ováricas , Neoplasias Peritoneales , Fotoquimioterapia , Humanos , Ratones , Animales , Femenino , Leucocitos Mononucleares/metabolismo , Fotoquimioterapia/métodos , Neoplasias Ováricas/metabolismo , Fármacos Fotosensibilizantes/farmacología , Fármacos Fotosensibilizantes/uso terapéutico , Neoplasias Peritoneales/secundario , Citocinas/metabolismo , Ácido Fólico/uso terapéutico , Línea Celular Tumoral , Receptor 1 de Folato/metabolismo , Receptor 1 de Folato/uso terapéuticoRESUMEN
Photodynamic therapy (PDT) is a two-stage treatment relying on cytotoxicity induced by photoexcitation of a nontoxic dye, called photosensitizer (PS). Using 5-aminolevulinic acid (5-ALA), the pro-drug of PS protoporphyrin IX, we investigated the impact of PDT on hepatocellular carcinoma (HCC). Optimal 5-ALA PDT dose was determined on three HCC cell lines by analyzing cell death after treatment with varying doses. HCC-patient-derived tumor hepatocytes and healthy donor liver myofibroblasts were treated with optimal 5-ALA PDT doses. The proliferation of cancer cells and healthy donor immune cells cultured with 5-ALA-PDT-treated conditioned media was analyzed. Finally, therapy efficacy on humanized SCID mice model of HCC was investigated. 5-ALA PDT induced a dose-dependent decrease in viability, with an up-to-four-fold reduction in viability of patient tumor hepatocytes. The 5-ALA PDT treated conditioned media induced immune cell clonal expansion. 5-ALA PDT has no impact on myofibroblasts in terms of viability, while their activation decreased cancer cell proliferation and reduced the tumor growth rate of the in vivo model. For the first time, 5-ALA PDT has been validated on primary patient tumor hepatocytes and donor healthy liver myofibroblasts. 5-ALA PDT may be an effective anti-HCC therapy, which might induce an anti-tumor immune response.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Trasplante de Hígado , Fotoquimioterapia , Ratones , Animales , Humanos , Ácido Aminolevulínico/farmacología , Ácido Aminolevulínico/uso terapéutico , Carcinoma Hepatocelular/tratamiento farmacológico , Medios de Cultivo Condicionados/farmacología , Neoplasias Hepáticas/tratamiento farmacológico , Ratones SCID , Donadores Vivos , Fármacos Fotosensibilizantes/farmacología , Fármacos Fotosensibilizantes/uso terapéutico , Protoporfirinas/metabolismo , Línea Celular TumoralRESUMEN
Photodynamic Therapy (PDT) relies on local or systemic administration of a light-sensitive dye, called photosensitizer, to accumulate into the target site followed by excitation with light of appropriate wavelength and fluence. This photo-activated molecule reacts with the intracellular oxygen to induce selective cytotoxicity of targeted cells by the generation of reactive oxygen species. Hepatocellular carcinoma (HCC), one of the leading causes of cancer-associated mortality worldwide, has insufficient treatment options available. In this review, we discuss the mechanism and merits of PDT along with its recent developments as an anti-cancerous therapy. We also highlight the application of this novel therapy for diagnosis, visualization, and treatment of HCC. We examine the underlying challenges, some pre-clinical and clinical studies, and possibilities of future studies associated with PDT. Finally, we discuss the mechanism of an active immune response by PDT and thereafter explored the role of PDT in the generation of anti-tumor immune response in the context of HCC, with an emphasis on checkpoint inhibitor-based immunotherapy. The objective of this review is to propose PDT as a plausible adjuvant to existing therapies for HCC, highlighting a feasible combinatorial approach for HCC treatment.
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There has been very limited use of computer assisted semen analysis (CASA) to evaluate reptile sperm. The aim of this study was to examine sperm kinematic variables in American crocodile (Crocodylus acutus) semen samples and to assess whether sperm subpopulations could be characterized. Eight ejaculates (two ejaculates/male) from four sexually mature captive crocodiles were obtained. An ISAS®v1 CASA-Mot system, with an image acquisition rate of 50 Hz, and ISAS®D4C20 counting chambers were used for sperm analyses. The percentages of motile and progressively motile spermatozoa did not differ among animals (P > 0.05) but there was a significant animal effect with regards to kinematic variables (P < 0.05). Principal component (PC) analysis revealed that kinematic variables grouped into three components: PC1, related to velocity; PC2 to progressiveness and PC3 to oscillation. Subpopulation structure analysis identified four groups (P < 0.05), which represented, on average, 9.8%, 32.1%, 26.8%, and 31.3% of the total sperm population. Males differed in the proportion of sperm in each of the kinematic subpopulations. This new approach for the analysis of reptile sperm kinematic subpopulations, reflecting quantifiable parameters generated by CASA system technology, opens up possibilities for future assessments of crocodile sperm and will be useful in the future development of assisted reproduction for these species.
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Caimanes y Cocodrilos/genética , Linaje de la Célula/genética , Reproducción/genética , Espermatozoides/citología , Animales , Fenómenos Biomecánicos , Masculino , Semen/citología , Semen/fisiología , Análisis de Semen , Preservación de Semen , Motilidad Espermática/genética , Espermatozoides/fisiología , Estados UnidosRESUMEN
The development of immunotherapy has recently modified the anti-tumor therapeutic arsenal; particularly, immune checkpoint inhibitors have led to a significant increase in overall survival. The current challenge is now to select good responder patients by identifying early biomarkers to propose therapeutic combinations that potentiate the efficacy of the therapy. Here we report the case of a 60-year-old man with superficial melanoma treated with high-dose hypo fractionated radiotherapy (H-SRT) combined with a single dose of anti-PD1 immunotherapy (Nivolumab) for a metastatic lymph node recurrence due to cancer progression. In this study, we present the results obtained regarding the activation of the Th1 immune response after H-SRT treatment followed by anti PD-1 therapeutic protocol. These results were correlated with clinical data to identify potential immunological biomarkers of treatment efficacy. This exceptional case report shows that a combination of H-SRT with a single dose of anti-PD1 immunotherapy may allow a better activation of the immune response in favor of a complete clinical response.
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Antineoplásicos Inmunológicos/uso terapéutico , Melanoma/radioterapia , Melanoma/terapia , Nivolumab/uso terapéutico , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Células TH1/efectos de los fármacos , Humanos , Inmunoterapia/métodos , Masculino , Melanoma/inmunología , Persona de Mediana EdadRESUMEN
Nasopharyngeal carcinoma (NPC) is a malignant tumour of the head and neck affecting localised regions of the world, with the highest rates described in Southeast Asia, Northern Africa, and Greenland. Its high morbidity rate is linked to both late-stage diagnosis and unresponsiveness to conventional anti-cancer treatments. Multiple aetiological factors have been described including environmental factors, genetics, and viral factors (Epstein Barr Virus, EBV), making NPC treatment that much more complex. The most common forms of NPCs are those that originate from the epithelial tissue lining the nasopharynx and are often linked to EBV infection. Indeed, they represent 75-95% of NPCs in the low-risk populations and almost 100% of NPCs in high-risk populations. Although conventional surgery has been improved with nasopharyngectomy's being carried out using more sophisticated surgical equipment for better tumour resection, recent findings in the tumour microenvironment have led to novel treatment options including immunotherapies and photodynamic therapy, able to target the tumour and improve the immune system. This review provides an update on the disease's aetiology and the future of NPC treatments with a focus on therapies activating T cell immunity.
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Infecciones por Virus de Epstein-Barr/terapia , Carcinoma Nasofaríngeo/virología , Neoplasias Nasofaríngeas/virología , Linfocitos T/metabolismo , Terapia Combinada , Infecciones por Virus de Epstein-Barr/inmunología , Herpesvirus Humano 4/inmunología , Humanos , Inmunoterapia , Carcinoma Nasofaríngeo/inmunología , Carcinoma Nasofaríngeo/terapia , Neoplasias Nasofaríngeas/inmunología , Neoplasias Nasofaríngeas/terapia , Faringectomía , Fotoquimioterapia , Microambiente TumoralRESUMEN
Often discovered at an advanced stage, ovarian cancer progresses to peritoneal carcinoma, which corresponds to the invasion of the serosa by multiple tumor implants. The current treatment is based on the combination of chemotherapy and tumor cytoreduction surgery. Despite the progress and standardization of surgical techniques combined with effective chemotherapy, post-treatment recurrences affect more than 60% of women in remission. Photodynamic therapy (PDT) has been particularly indicated for the treatment of superficial lesions on large surfaces and appears to be a relevant candidate for the treatment of microscopic intraperitoneal lesions and non-visible lesions. However, the impact of this therapy on immune cells remains unclear. Hence, the objective of this study is to validate the efficacy of a new photosensitizer [pyropheophorbide a-polyethylene glycol-folic acid (PS)] on human ovarian cancer cells and to assess the impact of the secretome of PDT-treated cells on human peripheral blood mononuclear cells (PBMC). We show that PS, upon illumination, can induce cell death of different ovarian tumor cells. Furthermore, PDT using this new PS seems to favor activation of the immune response by inducing the secretion of effective cytokines and inhibiting the pro-inflammatory and immunosuppressive ones, as well as releasing extracellular vesicles (EVs) prone to activating immune cells. Finally, we show that PDT can activate CD4+ and CD8+ T cells, resulting in a potential immunostimulating process. The results of this pilot study therefore indicate that PS-PDT treatment may not only be effective in rapidly and directly destroying target tumor cells but also promote the activation of an effective immune response; notably, by EVs. These data thus open up good prospects for the treatment of micrometastases of intraperitoneal ovarian carcinosis which are currently inoperable.
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Surgical management of peritoneal metastases raises the problem of the theoretical spread of the entire peritoneal surface. Intraperitoneal photodynamic therapy (IntraPDT) has been limited by the lack of specificity of photosensitizers (PS) and difficulties to bring light into the abdominal cavity. Recent data in ovarian cancer may give development opportunities for IntraPDT. Intraperitoneal PDT could be an option but the level of evidence of research in this topic must increase. Our opinion is that the most important is to have a realistic idea of what we can objectively expect from PDT and the feasibility of its daily application. At the time of personalized medicine, it is mandatory to select population eligible for a targeted PS administration and who could benefit from the process. The design of a specific PS for each subtype of cancers seems essential to avoid side effects on healthy tissue. On the contrary, our progress on lighting solutions can be beneficial for all patients with an indication of IntraPDT regardless of the origin of PM. A common lighting system developed for all cancers eligible for IntraPDT could be adapted with light source of specific wavelength to activate dedicated PS.
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Neoplasias Ováricas , Neoplasias Peritoneales , Fotoquimioterapia , Carcinoma Epitelial de Ovario/tratamiento farmacológico , Femenino , Humanos , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Peritoneales/tratamiento farmacológico , Fotoquimioterapia/métodos , Fármacos Fotosensibilizantes/uso terapéuticoRESUMEN
To date, pancreatic adenocarcinoma (ADKP) is a devastating disease for which the incidence rate is close to the mortality rate. The survival rate has evolved only 2-5% in 45 years, highlighting the failure of current therapies. Otherwise, the use of photodynamic therapy (PDT), based on the use of an adapted photosensitizer (PS) has already proved its worth and has prompted a growing interest in the field of oncology. We have developed a new photosensitizer (PS-FOL/PS2), protected by a recently published patent (WO2019 016397-A1, 24 January 2019). This photosensitizer is associated with an addressing molecule (folic acid) targeting the folate receptor 1 (FOLR1) with a high affinity. Folate binds to FOLR1, in a specific way, expressed in 100% of ADKP or over-expressed in 30% of cases. The first objective of this study is to evaluate the effectiveness of this PS2-PDT in four ADKP cell lines: Capan-1, Capan-2, MiapaCa-2, and Panc-1. For this purpose, we first evaluated the gene and protein expression of FOLR1 on four ADKP cell lines. Subsequently, we evaluated PS2's efficacy in our cell lines and we assessed the impact of PDT on the secretome of cancer cells and its impact on the immune system. Finally, we evaluate the PDT efficacy on a humanized SCID mouse model of pancreatic cancer. In a very interesting way, we observed a significant increase in the proliferation of activated-human PBMC when cultured with conditioned media of ADKP cancer cells subjected to PDT. Furthermore, to evaluate in vivo the impact of this new PS, we analyzed the tumor growth in a humanized SCID mice model of pancreatic cancer. Four conditions were tested: Untreated, mice (nontreated), mice with PS (PS2), mice subjected to illumination (Light only), and mice subjected to illumination in the presence of PS (PDT). We noticed that the mice subjected to PDT presented a strong decrease in the growth of the tumor over time after illumination. Our investigations have not only suggested that PS2-PDT is an effective therapy in the treatment of PDAC but also that it activates the immune system and could be considered as a real adjuvant for anti-cancer vaccination. Thus, this new study provides new treatment options for patients in a therapeutic impasse and will provide a new arsenal in the fight against PDAC.
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La valoración subjetiva de la calidad seminal ha dado paso al uso de las técnicas objetivas de valoración por medio de la tecnología CASA (computer-assisted semen analysis). Se pueden aplicar una serie de pruebas cinéticas, morfométricas, morfológicas o de integridad del ADN para caracterizar y entender la biología reproductiva del espermatozoide. En los últimos años, se ha dado un cambio significativo de paradigma conceptual respecto de qué es un eyaculado. Cabe decir que, hasta tiempos recientes, se consideró que una población representativa (miles de millones) de espermatozoides estaba formada por células "equivalentes" con un objetivo común: ser el que finalmente pudiese fecundar el ovocito. La comprobación de que el conjunto de espermatozoides se agrupa en subpoblaciones bien definidas de acuerdo con sus características cinéticas y/o morfométricas, ha abierto el camino hacia una visión más cooperativa. Además, se ha visto que la distribución subpoblacional es diferente entre individuos, lo que parece indicar diferentes estrategias que se pueden entender dentro de otro paradigma: el de la competencia espermática entre diferentes eyaculados. A pesar de que aún no se conoce el papel de las diferentes subpoblaciones, se deben continuar los trabajos en esa dirección. En esta revisión, se caracterizó la evaluación de la fertilidad con la tecnología CASA y se examinaron los principales métodos multivariados en la evaluación de subpoblaciones de espermatozoides analizados mediante un sistema CASA.
In recent years, there has been a substantive change in the conceptual paradigm regarding what an ejaculate is. Until recently, it was considered that a representative population (billions) of sperm was made up of "equivalent" cells with a common goal: to be the one that could finally fertilize the oocyte. New data suggesting that a set of spermatozoa is grouped in distinct subpopulations, according to their kinematic and morphometric characteristics, has opened the way towards a more cooperative vision. In addition, recently, it has been established that the subpopulation distribution is different among males, which seems to indicate that there are different strategies that can be understood within another paradigm: sperm competition between different ejaculates. These heterogeneous subpopulations of spermatozoa in the ejaculate, that show kinematics and morphometric patterns, has been widely known for a while, but the biological meaning of these different sperm subpopulations is still not clear. For this reason, the subjective evaluation of seminal quality is being displaced by objective assessment techniques using CASA technology (computer-assisted semen analysis). Also, the application of principal components (PC) and clustering methods to reveal subpopulations of spermatozoa are complementary tools used to characterize raw semen and processed cell suspensions. Despite the advances in such powerful tools, researchers are not well versed on such techniques and its advantages. For instance, PC analysis is a multivariate statistical method that reduces the number of variables used in subsequent calculations for describing the data. By integrating the original data into new complex mathematical variables, homogenous subpopulations of spermatozoa can define clearly. Furthermore, kinematic, morphometric, morphological or DNA integrity tests can be also applied to characterize and understand the reproductive biology of the spermatozoon. Although the role of the different subpopulations of spermatozoa remains unknown, the work should continue by implementing novel technologies and coordinated tools. In this review, we characterize the fertility evaluation with CASA technology and examine the main multivariate methods in the assessment of sperm subpopulations analyzed by CASA systems.
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BACKGROUND: The role of regulatory T cells (Tregs) is now well established in the progression of hepatocellular carcinoma (HCC) linked to Hepatitis C virus (HCV) infection. However, nothing is known about the potential interplay between Tregs and HCV. In this pilot study, we have investigated the ability of Tregs to hang HCV on and the subsequent effect on their suppressive function and phenotype. Moreover, we have evaluated how HCV could promote the recruitment of Tregs by infected primary human hepatocytes. METHODS: Tregs of healthy donors were incubated with JFH-1/HCVcc. Viral inoculation was assessed using adapted assays (RT-qPCR, Flow Citometry (FACS) and Western Blot (WB). Expression of Tregs phenotypic (CD4, CD25, CD127 and Foxp3) and functional (IL-10, GZMB, TGF-ß1 and IL-2) markers was monitored by RT-qPCR, FACS and ELISA. Suppressive activity was validated by suppressive assays. Tregs recruitment by infected primary hepatic cells was evaluated using Boyden Chamber. RESULTS: Tregs express the classical HCV receptors (CD81, CLDN1 and LDLR) and some co-receptors (CD5). HCV inoculation significantly increases the suppressive phenotype and activity of Tregs, and raises their anergy by inducing an unexpected IL-2 production. Moreover, HCV infection induces the expression of chemokines (CCL17, CXCL16, and CCL20) by primary hepatic human hepatocytes and chemokine receptors (CCR4, CXCR6 and CCR6) by Tregs. Finally, infected hepatocytes have a significantly higher potential to recruit Tregs in a seemingly CCL20-dependent manner. CONCLUSIONS: Direct interaction between HCV and Tregs represents a newly defined mechanism that could potentiate HCV immune evasion and favor intratumoral recruitment contributing to HCC progression.
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Hepacivirus/inmunología , Hepatitis C/inmunología , Evasión Inmune , Hígado/inmunología , Linfocitos T Reguladores/inmunología , Antígenos de Diferenciación/inmunología , Regulación de la Expresión Génica/inmunología , Hepatitis C/patología , Humanos , Hígado/patología , Hígado/virología , Linfocitos T Reguladores/patologíaRESUMEN
The development of analytical methods for the evaluation of crocodilian semen is an important component for the assessment of male breeding soundness and the development of assisted breeding technology in this taxon. Computer-Assisted Semen Analysis (CASA) technology is becoming an increasingly common technique in seminal evaluations for animals but there has been no application of this technique for reptilian spermatozoa. The aim of this study was to analyse sperm kinematic and morphometric variables in Caiman crocodilus fuscus semen samples and to determine whether there were sperm subpopulations. Four ejaculates from four sexually mature captive caimans were used for this study. A CASA-Mot and CASA-Morph system was used with an image acquisition rate of 50â¯Hz for 2â¯s of capture. The ISAS®D4C20 counting chambers were used and spermatozoa incubated at 25⯰C. Total and progressive motilities did not differ among animals (Pâ¯>â¯0.05). There was a significant animal effect in the model with respect to sperm morphometry, and kinematic indices including linearity (LIN) and straightness (STR) (Pâ¯<â¯0.05). Results for principal component (PC) analysis indicated variables were grouped into four components: PC1 related to velocity, PC2 to progressivity, PC3 to oscillation and PC4 to sperm path cross-linking. Subpopulation (SP) structure analysis indicated there were four groups, namely, rapid non-progressive (SP1), slow non-progressive (SP2), rapid progressive (SP3) and medium progressive (SP4), representing 14.5%, 45.4%, 18.7%, and 21.4% respectively. Findings in the present study indicate the importance of continuing development of reliable protocols regarding the standardisation of computer-based semen analyses in reptilian species.
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Caimanes y Cocodrilos , Análisis de Semen/veterinaria , Animales , Fenómenos Biomecánicos/fisiología , Masculino , Reproducción/fisiología , Cabeza del Espermatozoide/fisiología , EspermatozoidesRESUMEN
Colorectal cancer (CRC) remains the most cancer type related to chronic inflammation; however, the mechanisms that link inflammation to CRC development and progression are still poorly understood. Our study aimed to investigate one of the prominent inflammatory response in cancers, iNOS/NO system. In this regard, we evaluated the link between the iNOS/NO system and CRC progression, its relation with the host immune responses and its response to cetuximab combined with chemotherapy. We found that the nitrite levels were nearly twice as high in metastatic CRC plasma and culture supernatants from PBMCs and tumor explants compared with those without metastases and healthy controls. Interestingly, we showed that the highest iNOS expression and NO levels are present in the damaged CRC tissues that have highest leukocyte infiltration. Our findings highlight the implication of iNOS/NO system in tissue alteration and leukocyte invasion. Thus, we observed imbalance between effector/memory T cell markers and Treg transcription factor (Foxp3). Accordingly, we detected higher IFNγ and T-bet expression levels in colorectal tumor tissues at early stage. In contrast, consistent with iNOS and Foxp3 expression, TGFß, CTLA-4 and IL-10 were significantly related to the tumor stage progression. Furthermore, our study revealed that Cetuximab combined with chemotherapy treatment markedly down-regulates iNOS/NO system as well as IL-10 and TGFß levels. Altogether, we conclude that cetuximab can potentiate the efficacy of chemotherapy, particularly by iNOS/NO system and immunosuppressive cytokines modulation. Thus, we suggest that iNOS/NO system may represent an attractive candidate biomarker for monitoring CRC progression, malignity and response to therapy.
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Biomarcadores de Tumor/metabolismo , Neoplasias Colorrectales/metabolismo , Factores de Transcripción Forkhead/metabolismo , Factores Inmunológicos/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Óxido Nítrico/metabolismo , Regulación hacia Arriba/fisiología , Adulto , Anciano , Anciano de 80 o más Años , Cetuximab/uso terapéutico , Neoplasias Colorrectales/tratamiento farmacológico , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/fisiología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Linfocitos T Reguladores/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Extra-cellular galectin-9 (gal-9) is an immuno-modulatory protein with predominant immunosuppressive effects. Inappropriate production of gal-9 has been reported in several human malignancies and viral diseases like nasopharyngeal, pancreatic and renal carcinomas, metastatic melanomas and chronic active viral hepatitis. Therefore therapeutic antibodies neutralizing extra-cellular gal-9 are expected to contribute to immune restoration in these pathological conditions. Two novel monoclonal antibodies targeting gal-9 -Gal-Nab 1 and 2-have been produced and characterized in this study. We report a protective effect of Gal-Nab1 and Gal-Nab2 on the apoptotic cell death induced by gal-9 in primary T cells. In addition, they inhibit late phenotypic changes observed in peripheral T cells that survive gal-9-induced apoptosis. Gal-Nab1 and Gal-Nab2 bind nearly identical, overlapping linear epitopes contained in the 213-224 amino-acid segments of gal-9. Nevertheless, they have some distinct functional characteristics suggesting that their three-dimensional epitopes are distinct. These differences are best demonstrated when gal-9 is applied on Jurkat cells where Gal-Nab1 is less efficient than Gal-Nab2 in the prevention of apoptotic cell death. In addition, Gal-Nab1 stimulates non-lethal phosphatidylserine translocation at the plasma membrane and calcium mobilization triggered by gal-9 in these cells. Both Gal-Nab1 and 2 cross-react with murine gal-9. They bind its natural as well as its recombinant form. This cross-species recognition will be an advantage for their assessment in pre-clinical tumor models.
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Anticuerpos Monoclonales/farmacología , Anticuerpos Neutralizantes/farmacología , Epítopos/inmunología , Galectinas/química , Linfocitos T/citología , Animales , Apoptosis/efectos de los fármacos , Transporte Biológico , Calcio/metabolismo , Galectinas/efectos adversos , Galectinas/inmunología , Humanos , Inmunización , Células Jurkat , Ratones , Fosfatidilserinas/metabolismo , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismoRESUMEN
Background: We analyzed telomere maintenance mechanisms (TMMs) in lymph node samples from HL patients treated with standard therapy. The TMMs correlated with clinical outcomes of patients. Materials and Methods: Lymph node biopsies obtained from 38 HL patients and 24 patients with lymphadenitis were included in this study. Seven HL cell lines were used as in vitro models. Telomerase activity (TA) was assessed by TRAP assay and verified through hTERT immunofluorescence expression; alternative telomere lengthening (ALT) was also assessed, along with EBV status. Results: Both TA and ALT mechanisms were present in HL lymph nodes. Our findings were reproduced in HL cell lines. The highest levels of TA were expressed in CD30-/CD15- cells. Small cells were identified with ALT and TA. Hodgkin and Reed Sternberg cells contained high levels of PML bodies, but had very low hTERT expression. There was a significant correlation between overall survival (p < 10-3), event-free survival (p < 10-4), and freedom from progression (p < 10-3) and the presence of an ALT profile in lymph nodes of EBV+ patients. Conclusion: The presence of both types of TMMs in HL lymph nodes and in HL cell lines has not previously been reported. TMMs correlate with the treatment outcome of EBV+ HL patients.
RESUMEN
Colitis associated cancer (CAC) is the colorectal cancer (CRC) subtype that is associated with bowel disease such as ulcerative colitis (UC). The data on role of NF-κB signaling in development and progression of CAC were derived from preclinical studies, whereas data from human are rare. The aim of this work was to study the contribution of NF-κB pathway during UC and CAC, as well as the immunomodulatory effect of all-trans retinoic acid (AtRA). We analyzed the expression of NOS2, TNF-α, TLR4, and NF-κB, in colonic mucosa. We also studied NO/TNF-α modulation by LPS in colonic mucosa pretreated with AtRA. A marked increase in TLR4, NF-κB, TNF-α, and NOS2 expression was reported in colonic mucosa. The relationship between LPS/TLR4 and TNF-α/NO production, as well as the role of NF-κB signaling, was confirmed by ex vivo experiments and the role of LPS/TLR4 in NOS2/TNF-α induction through NF-κB pathway was suggested. AtRA downregulates NOS2 and TNF-α expression. Collectively, our study indicates that AtRA modulates in situ LPS/TLR4/NF-κB signaling pathway targeting NOS2 and TNF-α expression. Therefore, we suggest that AtRA has a potential value in new strategies to improve the current therapy, as well as in the clinical prevention of CAC development and progression.