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Invasive fungal disease accounts for about 3.8â million deaths annually, an unacceptable rate that urgently prompts the discovery of new knowledge-driven treatments. We report the use of camelid single-domain nanobodies (Nbs) against fungal ß-1,3-glucanosyltransferases (Gel) involved in ß-1,3-glucan transglycosylation. Crystal structures of two Nbs with Gel4 from Aspergillus fumigatus revealed binding to a dissimilar CBM43 domain and a highly conserved catalytic domain across fungal species, respectively. Anti-Gel4 active site Nb3 showed significant antifungal efficacy in vitro and in vivo prophylactically and therapeutically against different A. fumigatus and Cryptococcus neoformans isolates, reducing the fungal burden and disease severity, thus significantly improving immunocompromised animal survival. Notably, C. deneoformans (serotypeâ D) strains were more susceptible to Nb3 and genetic Gel deletion than C. neoformans (serotype A) strains, indicating a key role for ß-1,3-glucan remodelling in C. deneoformans survival. These findings add new insight about the role of ß-1,3-glucan in fungal biology and demonstrate the potential of nanobodies in targeting fungal enzymes to combat invasive fungal diseases.
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Aspergillus fumigatus , Dominio Catalítico , Anticuerpos de Dominio Único , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/inmunología , Anticuerpos de Dominio Único/farmacología , Aspergillus fumigatus/inmunología , Aspergillus fumigatus/enzimología , Cryptococcus neoformans/enzimología , Cryptococcus neoformans/inmunología , Antifúngicos/química , Antifúngicos/farmacología , Animales , Ratones , Glucano Endo-1,3-beta-D-GlucosidasaRESUMEN
Granzymes are serine proteases previously believed to play exclusive and somewhat redundant roles in lymphocyte-mediated target cell death. However, recent studies have challenged this paradigm. Distinct substrate profiles and functions have since emerged for each granzyme while their dysregulated proteolytic activities have been linked to diverse pathologies.
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Granzimas , Humanos , Granzimas/metabolismo , Cicatrización de Heridas , Serina Proteasas , InflamaciónRESUMEN
AIM: endoscopy identifies inflammatory activity, however, it is an unpleasant test and is not always accessible. The aim of the study was to compare the usefulness of quantitative fecal immunochemical test (FIT) versus fecal calprotectin (FC) to determine endoscopic activity in patients with inflammatory bowel disease (IBD). METHODS: cross-sectional prospective observational study. The stool samples were collected within three days before starting the preparation for the colonoscopy. We used the Mayo index for ulcerative colitis (UC) and the simplified endoscopic index for Crohn's disease (CD). Mucosal healing (MH) was defined as the score 0 points in each of the endoscopic indices. RESULTS: eighty-four patients were included, 40 (47.6 %) with UC. In patients with IBD, FIT and FC showed a significant correlation with the presence of inflammatory activity/MH on endoscopy, with no statistically significant differences between the two receiver-operating characteristic (ROC) curves. Both tests improved their diagnostic performance when assessing patients with UC; the Spearman correlations between FIT and FC and endoscopic inflammatory activity were r = 0.6 (p = 0.0001) and r = 0.7 (p = 0.0001), respectively. In Crohn's disease, the diagnostic utility of both tests was lower. CONCLUSIONS: FIT is an alternative to monitor endoscopic activity among ulcerative colitis patients. In Crohn's disease, more studies are needed to determine the role of fecal biomarkers.
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Cytotoxic immune cells, including T lymphocytes (CTLs) and natural killer (NK) cells, are essential components of the host response against tumors. CTLs and NK cells secrete granzyme A (GzmA) upon recognition of cancer cells; however, there are very few tools that can detect physiological levels of active GzmA with high spatiotemporal resolution. Herein, we report the rational design of the near-infrared fluorogenic substrates for human GzmA and mouse GzmA. These activity-based probes display very high catalytic efficiency and selectivity over other granzymes, as shown in tissue lysates from wild-type and GzmA knock-out mice. Furthermore, we demonstrate that the probes can image how adaptive immune cells respond to antigen-driven recognition of cancer cells in real time.
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Colorantes Fluorescentes , Linfocitos T Citotóxicos , Animales , Humanos , Ratones , Granzimas , Células Asesinas Naturales , Ratones NoqueadosRESUMEN
Granzyme A (gzmA), a serine protease involved in the modulation of the inflammatory immune response, is found at an elevated level in the serum from ALS patients. However, the influence of gzmA on the progression of ALS remains unclear. The aim of our work was to assess whether the absence of gzmA in an ALS murine model could help slow down the progression of the disease. Homozygous and hemizygous gzmA-deficient mice expressing the hSOD1G93A transgene were generated, and survival of these mice was monitored. Subsequently, gene and protein expression of inflammatory and oxidative stress markers was measured in the spinal cord and quadriceps of these mice. We observed the longest lifespan in gzmA+/- mice. GzmA gene and protein expression was downregulated in the spinal cord and serum from gmzA+/- mice, confirming that the increased survival of hemizygous mice is correlated with lower levels of gzmA. In addition, mRNA and protein levels of glutathione reductase (GSR), involved in oxidative stress, were found downregulated in the spinal cord and quadriceps of gmzA+/- mice, together with lower IL-1ß and IL-6 mRNA levels in hemyzigous mice. In summary, our findings indicate for the first time that reduced levels, but not the absence, of gzmA could slightly ameliorate the disease progression in this animal model.
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Esclerosis Amiotrófica Lateral , Ratones , Animales , Granzimas/metabolismo , Esclerosis Amiotrófica Lateral/genética , Longevidad/genética , Médula Espinal/metabolismo , Modelos Animales de Enfermedad , Transgenes , ARN Mensajero , Ratones Transgénicos , Ratones Endogámicos C57BL , Superóxido Dismutasa/genéticaRESUMEN
Immunotherapy has become a new paradigm in oncology, improving outcomes for several types of cancer. However, there are some aspects about its management that remain uncertain. One of the key points that needs better understanding is the interaction between immunotherapy and gut microbiome and how modulation of the microbiome might modify the efficacy of immunotherapy. Consequently, the negative impact of systemic antibiotics and corticosteroids on the efficacy of immunotherapy needs to be clarified.
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Corticoesteroides/farmacología , Antibacterianos/farmacología , Interacciones Microbiota-Huesped , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Microbiota , Neoplasias/tratamiento farmacológico , Probióticos , Corticoesteroides/uso terapéutico , Animales , Antibacterianos/uso terapéutico , Interacciones Microbiota-Huesped/efectos de los fármacos , Interacciones Microbiota-Huesped/inmunología , Humanos , Inhibidores de Puntos de Control Inmunológico/farmacología , Inmunomodulación/efectos de los fármacos , Interacciones Microbianas/efectos de los fármacos , Interacciones Microbianas/inmunología , Microbiota/efectos de los fármacos , Neoplasias/etiología , Resultado del TratamientoRESUMEN
Granule-mediated cytotoxicity is the main effector mechanism of cytotoxic CD8+ T cells. We report that CD8+ T cells from acid sphingomyelinase (ASMase)-deficient (ASMase-KO) mice are defective in exocytosis of cytolytic effector molecules; this defect resulted in attenuated cytotoxic activity of ASMase-KO CD8+ T cells and delayed elimination of lymphocytic choriomeningitis virus from ASMase-KO mice. Cytolytic granules of ASMase-KO and wild-type CD8+ T cells were equally loaded with granzymes and perforin, and correctly directed to the immunological synapse. In wild-type CD8+ T cells, secretory granules underwent shrinkage by 82% after fusion with the plasma membrane. In ASMase-KO CD8+ T cells, the contraction of secretory granules was markedly impaired. Thus, ASMase is required for contraction of secretory granules and expulsion of cytotoxic effector molecules.
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Citotoxicidad Inmunológica/inmunología , Vesículas Secretoras/metabolismo , Esfingomielina Fosfodiesterasa/metabolismo , Linfocitos T/inmunología , Animales , Infecciones por Arenaviridae/inmunología , Infecciones por Arenaviridae/metabolismo , Infecciones por Arenaviridae/virología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Quimiocina CCL5/metabolismo , Femenino , Granzimas/genética , Granzimas/metabolismo , Immunoblotting , Sinapsis Inmunológicas/inmunología , Virus de la Coriomeningitis Linfocítica/fisiología , Masculino , Ratones , Ratones Noqueados , Microscopía Fluorescente , Perforina/genética , Perforina/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Vesículas Secretoras/enzimología , Esfingomielina Fosfodiesterasa/genética , Linfocitos T/citología , Linfocitos T/metabolismo , Linfocitos T Citotóxicos/citología , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/metabolismoRESUMEN
Granzyme A (GzmA) is considered a major proapoptotic protease. We have discovered that GzmA-induced cell death involves rapid membrane damage that depends on the synergy between micromolar concentrations of GzmA and sublytic perforin (PFN). Ironically, GzmA and GzmB, independent of their catalytic activity, both mediated this swift necrosis. Even without PFN, lower concentrations of human GzmA stimulated monocytic cells to secrete proinflammatory cytokines (interleukin-1beta [IL-1beta], TNFalpha, and IL-6) that were blocked by a caspase-1 inhibitor. Moreover, murine GzmA and GzmA(+) cytotoxic T lymphocytes (CTLs) induce IL-1beta from primary mouse macrophages, and GzmA(-/-) mice resist lipopolysaccharide-induced toxicity. Thus, the granule secretory pathway plays an unexpected role in inflammation, with GzmA acting as an endogenous modulator.
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Granzimas/inmunología , Interleucina-1beta/inmunología , Interleucina-6/inmunología , Leucocitos Mononucleares/inmunología , Perforina/inmunología , Linfocitos T Citotóxicos/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Adenoviridae/inmunología , Animales , Adhesión Celular , Muerte Celular , Línea Celular Tumoral , Citotoxicidad Inmunológica , Técnicas de Silenciamiento del Gen , Granzimas/metabolismo , Células HeLa , Humanos , Inflamación/inmunología , Inflamación/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Células Jurkat , Macrófagos/inmunología , Ratones , Perforina/metabolismo , Linfocitos T Citotóxicos/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Células U937RESUMEN
BACKGROUND: The special physicochemical properties of gold nanoprisms make them very useful for biomedical applications including biosensing and cancer therapy. However, it is not clear how gold nanoprisms may affect cellular physiology including viability and other critical functions. We report a multiparametric investigation on the impact of gold-nanoprisms on mice and human, transformed and primary cells as well as tissue distribution and toxicity in vivo after parental injection. METHODS: Cellular uptake of the gold-nanoprisms (NPRs) and the most crucial parameters of cell fitness such as generation of reactive oxygen species (ROS), mitochondria membrane potential, cell morphology and apoptosis were systematically assayed in cells. Organ distribution and toxicity including inflammatory response were analysed in vivo in mice at 3 days or 4 months after parental administration. RESULTS: Internalized gold-nanoprisms have a significant impact in cell morphology, mitochondrial function and ROS production, which however do not affect the potential of cells to proliferate and form colonies. In vivo NPRs were only detected in spleen and liver at 3 days and 4 months after administration, which correlated with some changes in tissue architecture. However, the main serum biochemical markers of organ damage and inflammation (TNFα and IFNγ) remained unaltered even after 4 months. In addition, animals did not show any macroscopic sign of toxicity and remained healthy during all the study period. CONCLUSION: Our data indicate that these gold-nanoprisms are neither cytotoxic nor cytostatic in transformed and primary cells, and suggest that extensive parameters should be analysed in different cell types to draw useful conclusions on nanomaterials safety. Moreover, although there is a tendency for the NPRs to accumulate in liver and spleen, there is no observable negative impact on animal health.
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Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Oro/toxicidad , Nanopartículas del Metal/toxicidad , Células A549 , Animales , Línea Celular Transformada , Forma de la Célula/efectos de los fármacos , Femenino , Oro/administración & dosificación , Oro/farmacocinética , Células HeLa , Humanos , Mediadores de Inflamación/sangre , Inyecciones Intravenosas , Interferón gamma/sangre , Masculino , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Nanopartículas del Metal/administración & dosificación , Ratones Endogámicos C57BL , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/patología , Cultivo Primario de Células , Especies Reactivas de Oxígeno/metabolismo , Medición de Riesgo , Distribución Tisular , Factor de Necrosis Tumoral alfa/sangreRESUMEN
Cytotoxic T cells (Tc) use perforin and granzyme B (gzmB) to kill virus-infected cells and cancer cells. Recent evidence suggests that human gzmB primarily induces apoptosis via the intrinsic mitochondrial pathway by either cleaving Bid or activating Bim leading to the activation of Bak/Bax and subsequent generation of active caspase-3. In contrast, mouse gzmB is thought to predominantly induce apoptosis by directly processing pro-caspase-3. However, in certain mouse cell types gzmB-mediated apoptosis mainly occurs via the mitochondrial pathway. To investigate whether Bim is involved under the latter conditions, we have now employed ex vivo virus-immune mouse Tc that selectively kill by using perforin and gzmB (gzmB(+)Tc) as effector cells and wild type as well as Bim- or Bak/Bax-deficient spontaneously (3T9) or virus-(SV40) transformed mouse embryonic fibroblast cells as targets. We show that gzmB(+)Tc-mediated apoptosis (phosphatidylserine translocation, mitochondrial depolarization, cytochrome c release, and caspase-3 activation) was severely reduced in 3T9 cells lacking either Bim or both Bak and Bax. This outcome was related to the ability of Tc cells to induce the degradation of Mcl-1 and Bcl-XL, the anti-apoptotic counterparts of Bim. In contrast, gzmB(+)Tc-mediated apoptosis was not affected in SV40-transformed mouse embryonic fibroblast cells lacking Bak/Bax. The data provide evidence that Bim participates in mouse gzmB(+)Tc-mediated apoptosis of certain targets by activating the mitochondrial pathway and suggest that the mode of cell death depends on the target cell. Our results suggest that the various molecular events leading to transformation and/or immortalization of cells have an impact on their relative resistance to the multiple gzmB(+)Tc-induced death pathways.
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Proteínas Reguladoras de la Apoptosis/inmunología , Apoptosis , Granzimas/inmunología , Proteínas de la Membrana/inmunología , Mitocondrias/inmunología , Proteínas Proto-Oncogénicas/inmunología , Linfocitos T Citotóxicos/enzimología , Animales , Proteínas Reguladoras de la Apoptosis/genética , Proteína 11 Similar a Bcl2 , Caspasa 3/inmunología , Línea Celular , Fibroblastos/citología , Fibroblastos/inmunología , Fibroblastos/metabolismo , Eliminación de Gen , Inmunoterapia , Proteínas de la Membrana/genética , Ratones , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/inmunología , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Neoplasias/terapia , Proteínas Citotóxicas Formadoras de Poros/inmunología , Proteolisis , Proteínas Proto-Oncogénicas/genética , Linfocitos T Citotóxicos/inmunologíaRESUMEN
Early and accurate diagnosis of invasive aspergillosis (IA) is one of the most critical steps needed to efficiently treat the infection and reduce the high mortality rates that can occur. We have previously found that the Aspergillus spp. secondary metabolite, bis(methylthio)gliotoxin (bmGT), can be detected in the serum from patients with possible/probable IA. Thus, it could be used as a diagnosis marker of the infection. However, there is no data available concerning the sensitivity, specificity and performance of bmGT to detect the infection. Here, we have performed a prospective study comparing bmGT detection with galactomannan (GM), the most frequently used and adopted approach for IA diagnosis, in 357 sera from 90 episodes of patients at risk of IA. Our results, involving 79 patients that finally met inclusion criteria, suggest that bmGT presents higher sensitivity and positive predictive value (PPV) than GM and similar specificity and negative predictive value (NPV). Importantly, the combination of GM and bmGT increased the PPV (100 %) and NPV (97.5 %) of the individual biomarkers, demonstrating its potential utility in empirical antifungal treatment guidance and withdrawal. These results indicate that bmGT could be a good biomarker candidate for IA diagnosis and, in combination with GM, could result in highly specific diagnosis of IA and management of patients at risk of infection.
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Biomarcadores/sangre , Gliotoxina/análogos & derivados , Aspergilosis Pulmonar Invasiva/diagnóstico , Anciano , Anciano de 80 o más Años , Femenino , Galactosa/análogos & derivados , Gliotoxina/sangre , Humanos , Masculino , Mananos/sangre , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Estudios Prospectivos , Sensibilidad y EspecificidadRESUMEN
BACKGROUND: Recently, novel atypical Brucella strains isolated from humans and wild rodents have been reported. They are phenotypically close to Ochrobactrum species but belong to the genus Brucella, based on genetic relatedness, although genetic diversity is higher among the atypical Brucella strains than between the classic species. They were classified within or close to the novel species Brucella inopinata. However, with the exception of Brucella microti, the virulence of these novel strains has not been investigated in experimental models of infection. METHODS: The type species B. inopinata strain BO1 (isolated from a human) and Brucella species strain 83-210 (isolated from a wild Australian rodent) were investigated. A classic infectious Brucella reference strain, B. suis 1330, was also used. BALB/c, C57BL/6, and CD1 mice models and C57BL/6 mouse bone-marrow-derived macrophages (BMDMs) were used as infection models. RESULTS: Strains BO1 and 83-210 behaved similarly to reference strain 1330 in all mouse infection models: there were similar growth curves in spleens and livers of mice and similar intracellular replication rates in BMDMs. However, unlike strain 1330, strains BO1 and 83-210 showed lethality in the 3 mouse models. CONCLUSIONS: The novel atypical Brucella strains of this study behave like classic intracellular Brucella pathogens. In addition, they cause death in murine models of infection, as previously published for B. microti, another recently described environmental and wildlife species.
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Brucella/clasificación , Brucella/patogenicidad , Brucelosis/microbiología , Brucelosis/mortalidad , Animales , Células Cultivadas , Macrófagos/microbiología , Ratones , Ratones Endogámicos , VirulenciaRESUMEN
In this study, we report that cytoplasmic granules from in vivo and in vitro derived mouse mast cells (MCs) contain active granzyme B (gzmB) and caspase-3, which is consistent with recent findings. Studying WT and gzmB-deficient mice, we observed that BM-derived MCs (BMMCs) from both strains contain cytosolic pro-caspase-3, but only WT BMMCs expressed active caspase-3 limited to their secretory lysosomes. Confocal microscopy revealed colocalization of active caspase-3 and gzmB in these cytoplasmic granules. The combined data demonstrate that the generation and storage of active caspase-3 is gzmB-dependent. The finding that BMMCs secrete caspase-3 and gzmB after Ag stimulation suggests that both proteases contribute to extracellular MC-mediated proteolytic events. Although the extracellular function of MC-derived caspase-3 remains unclear, we show that BMMC-secreted caspase-3 cleaves IL-33, a cytokine that contributes to the development of asthma and arthritis. We also show that an in vitro propagated cytolytic T-lymphocyte line constitutively expresses gzmB together with active caspase-3, suggesting a novel interaction of these proteases in the execution of multiple innate and adaptive immune responses.
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Células de la Médula Ósea/inmunología , Caspasa 3/inmunología , Exocitosis/inmunología , Granzimas/inmunología , Lisosomas/inmunología , Mastocitos/inmunología , Inmunidad Adaptativa/fisiología , Animales , Antígenos/inmunología , Células de la Médula Ósea/citología , Células de la Médula Ósea/enzimología , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/enzimología , Linfocitos T CD8-positivos/inmunología , Caspasa 3/biosíntesis , Caspasa 3/genética , Línea Celular , Exocitosis/genética , Regulación Enzimológica de la Expresión Génica/genética , Regulación Enzimológica de la Expresión Génica/inmunología , Granzimas/biosíntesis , Granzimas/genética , Inmunidad Innata/fisiología , Interleucina-33 , Interleucinas/genética , Interleucinas/inmunología , Interleucinas/metabolismo , Lisosomas/enzimología , Lisosomas/genética , Mastocitos/citología , Mastocitos/enzimología , Ratones , Ratones Noqueados , ProteolisisRESUMEN
Hypericin is a natural photosensitizer used in photodynamic therapy (PDT), which has shown in vitro antifungal effect against Candida spp. The aim of this study was to evaluate the in vitro fungicidal effect of hypericin-PDT on dermatophytes. Trichophyton rubrum and Trichophyton mentagrophytes strains were incubated with different concentrations of hypericin for different times and exposed to light-emitting diode lamp (602 ± 10 nm, 10.3 mW cm(-2), and fluence 37 J cm(-2)). Using the optimal incubation time, 60 min, a 3-log fungicidal effect was achieved with hypericin concentration ranges of 10-20 µM for T. rubrum and 20-50 µM for T. mentagrophytes (p = 0.95). Confocal fluorescence microscopy showed the localization of hypericin inside the dermatophytes diffusely distributed in the cytoplasm of conidia and hyphae and outside the nucleus. In conclusion, hypericin-PDT has a fungicidal effect in vitro on dermatophytes. Hypericin seems to be a promising photosensitizer to treat localized dermatophytic infections such as tinea pedis and onychomycosis.
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Antifúngicos/farmacología , Luz , Perileno/análogos & derivados , Fármacos Fotosensibilizantes/farmacología , Trichophyton/efectos de los fármacos , Trichophyton/efectos de la radiación , Antracenos , Recuento de Colonia Microbiana , Humanos , Viabilidad Microbiana/efectos de los fármacos , Viabilidad Microbiana/efectos de la radiación , Perileno/farmacología , Factores de TiempoRESUMEN
Granzymes (granule-secreted enzymes) are a family of serine proteases that have been viewed as redundant cytotoxic enzymes since their discovery more than 30 years ago. Predominantly produced by cytotoxic lymphocytes and natural killer cells, granzymes are delivered into the cytoplasm of target cells through immunological synapses in cooperation with the pore-forming protein perforin. After internalization, granzymes can initiate cell death through the cleavage of intracellular substrates. However, evidence now also demonstrates the existence of non-cytotoxic, pro-inflammatory, intracellular and extracellular functions that are granzyme specific. Under pathological conditions, granzymes can be produced and secreted extracellularly by immune cells as well as by non-immune cells. Depending on the granzyme, accumulation in the extracellular milieu might contribute to inflammation, tissue injury, impaired wound healing, barrier dysfunction, osteoclastogenesis and/or autoantigen generation.
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Granzimas , Inflamación , Enfermedades Reumáticas , Granzimas/metabolismo , Humanos , Inflamación/inmunología , Enfermedades Reumáticas/inmunología , Enfermedades Reumáticas/enzimología , AnimalesRESUMEN
Natural killer (NK) cells play a crucial role in antitumoral and antiviral responses. Yet, cancer cells can alter themselves or the microenvironment through the secretion of cytokines or other factors, hindering NK cell activation and promoting a less cytotoxic phenotype. These resistance mechanisms, often referred to as the "hallmarks of cancer" are significantly influenced by the activation of oncogenes, impacting most, if not all, of the described hallmarks. Along with oncogenes, other types of genes, the tumor suppressor genes are frequently mutated or modified during cancer. Traditionally, these genes have been associated with uncontrollable tumor growth and apoptosis resistance. Recent evidence suggests oncogenic mutations extend beyond modulating cell death/proliferation programs, influencing cancer immunosurveillance. While T cells have been more studied, the results obtained highlight NK cells as emerging key protagonists for enhancing tumor cell elimination by modulating oncogenic activity. A few recent studies highlight the crucial role of oncogenic mutations in NK cell-mediated cancer recognition, impacting angiogenesis, stress ligands, and signaling balance within the tumor microenvironment. This review will critically examine recent discoveries correlating oncogenic mutations to NK cell-mediated cancer immunosurveillance, a relatively underexplored area, particularly in the era dominated by immune checkpoint inhibitors and CAR-T cells. Building on these insights, we will explore opportunities to improve NK cell-based immunotherapies, which are increasingly recognized as promising alternatives for treating low-antigenic tumors, offering significant advantages in terms of safety and manufacturing suitability.
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Vigilancia Inmunológica , Células Asesinas Naturales , Mutación , Neoplasias , Microambiente Tumoral , Humanos , Células Asesinas Naturales/inmunología , Neoplasias/inmunología , Neoplasias/genética , Microambiente Tumoral/inmunología , Microambiente Tumoral/genética , Animales , Oncogenes/genéticaRESUMEN
BACKGROUND AND OBJECTIVE: Immune cell migration is one of the key features that enable immune cells to find invading pathogens, control tissue damage, and eliminate primary developing tumors. Chimeric antigen receptor (CAR) T-cell therapy is a novel strategy in the battle against various cancers. It has been successful in treating hematological tumors, yet it still faces many challenges in the case of solid tumors. In this work, we evaluate the three-dimensional (3D) migration capacity of T and CAR-T cells within dense collagen-based hydrogels. Quantifying three-dimensional (3D) cell migration requires microscopy techniques that may not be readily accessible. Thus, we introduce a straightforward mathematical model designed to infer 3D trajectories of cells from two-dimensional (2D) cell trajectories. METHODS: We develop a 3D agent-based model (ABM) that simulates the temporal changes in the direction of migration with an inverse transform sampling method. Then, we propose an optimization procedure to accurately orient cell migration over time to reproduce cell migration from 2D experimental cell trajectories. With this model, we simulate cell migration assays of T and CAR-T cells in microfluidic devices conducted under hydrogels with different concentrations of type I collagen and validate our 3D cell migration predictions with light-sheet microscopy. RESULTS: Our findings indicate that CAR-T cell migration is more sensitive to collagen concentration increases than T cells, resulting in a more pronounced reduction in their invasiveness. Moreover, our computational model reveals significant differences in 3D movement patterns between T and CAR-T cells. T cells exhibit migratory behavior in 3D whereas that CAR-T cells predominantly move within the XY plane, with limited movement in the Z direction. However, upon the introduction of a CXCL12 chemical gradient, CAR-T cells present migration patterns that closely resemble those of T cells. CONCLUSIONS: This framework demonstrates that 2D projections of 3D trajectories may not accurately represent real migration patterns. Moreover, it offers a tool to estimate 3D migration patterns from 2D experimental data, which can be easily obtained with automatic quantification algorithms. This approach helps reduce the need for sophisticated and expensive microscopy equipment required in laboratories, as well as the computational burden involved in producing and analyzing 3D experimental data.
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Influenza A is an emerging zoonotic virus with worldwide distribution. To our knowledge, no studies have been conducted to assess influenza A exposure in stray cats in regions with positive cases of wild birds. This study aimed to determine the seroprevalence of anti-influenza A antibodies in feral cats from a region in Spain with cases of positive wild birds. A cross-sectional study of stray cats (n = 183) was conducted between March 2022 and March 2023. The presence of antibodies against the influenza A virus was tested using a commercial enzyme-linked immunosorbent assay kit adapted for this study and confirmed by competitive enzyme-linked immunosorbent assay for the detection of antibodies against the haemagglutinin H5. During sample collection, none of the cats exhibited clinical signs of illness. Four of the 183 animals tested showed anti-influenza A antibodies by ELISA, and the seroprevalence of influenza A was 2.19% (95% confidence interval 0.85%-5.48%). Due to the low number of positive cases detected, it appears that cats did not have an important epidemiological role in influenza A transmission during this period.
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Enfermedades de los Gatos , Gripe Aviar , Gripe Humana , Animales , Gatos , Humanos , Gripe Aviar/epidemiología , Estudios Seroepidemiológicos , Estudios Transversales , Anticuerpos Antivirales , Animales Salvajes , Aves , Ensayo de Inmunoadsorción Enzimática/veterinaria , Enfermedades de los Gatos/epidemiologíaRESUMEN
SARS-CoV-2 is the causal agent of Coronavirus Disease 2019 (COVID-19) in humans that emerged in late 2019. This virus is able to infect humans and different animal species. Among pets, cats and ferrets are more susceptible to be infected by the SARS-CoV-2. Epidemiological studies are an important tool to provide information under natural conditions of exposure to SARS-CoV-2 virus. In comparison to cats, limited epidemiological studies have been performed in domestic ferrets (Mustela putorius furo) reporting the presence of antibodies in this species. This study analysed the presence of anti-SARS-CoV-2 antibodies in 432 cliend-owned ferrets from different geographical areas of Spain during the different waves of COVID-19 outbreaks from December 2019 to May 2023 (42 months). For this purpose, anti-SARS-CoV-2 antibodies were detected by an enzyme-linked immunosorbent method (ELISA) using the receptor binding domain (RBD) of Spike antigen and confirmed by serum virus neutralization assay. Eighteen of the 432 ferrets included were seroreactive by the in-house ELISA (4.17%, 95% Confidence Interval (CI): 2.65-6.49). In this sense, the wave of COVID-19 with the higher number of seropositive ferrets occurred during the seventh wave when the different Omicron subvariants were the dominant virus variants. Our results suggest that the risk of SARS-CoV-2 transmission in domestic ferrets in natural conditions is low. Further research is need to evaluate the potential risk of transmission of SARS-CoV-2 from human to pets.
Asunto(s)
COVID-19 , Hurones , Animales , Humanos , COVID-19/epidemiología , COVID-19/veterinaria , SARS-CoV-2 , Estudios Seroepidemiológicos , España/epidemiología , Anticuerpos AntiviralesRESUMEN
Self-assembling peptides (SAPs) have gained significant attention in biomedicine because of their unique properties and ability to undergo molecular self-assembly driven by non-covalent interactions. By manipulating their composition and structure, SAPs can form well-ordered nanostructures with enhanced selectivity, stability and biocompatibility. SAPs offer advantages such as high chemical and biological diversity and the potential for functionalization. However, studies concerning its potentially toxic effects are very scarce, a limitation that compromises its potential translation to humans. This study investigates the potentially toxic effects of six different SAP formulations composed of natural amino acids designed for nervous tissue engineering and amenable to ready cross-linking boosting their biomechanical properties. All methods were performed in accordance with the relevant guidelines and regulations. A wound-healing assay was performed to evaluate how SAPs modify cell migration. The results in vitro demonstrated that SAPs did not induce genotoxicity neither skin sensitization. In vivo, SAPs were well-tolerated without any signs of acute systemic toxicity. Interestingly, SAPs were found to promote the migration of endothelial, macrophage, fibroblast, and neuronal-like cells in vitro, supporting a high potential for tissue regeneration. These findings contribute to the development and translation of SAP-based biomaterials for biomedical applications.