Production and Immunogenicity of FeLV Gag-Based VLPs Exposing a Stabilized FeLV Envelope Glycoprotein.

The envelope glycoprotein (Env) of retroviruses, such as the Feline leukemia virus (FeLV), is the main target of neutralizing humoral response, and therefore, a promising vaccine candidate, despite its reported poor immunogenicity. The incorporation of mutations that stabilize analogous proteins from other viruses in their prefusion conformation (e.g., HIV Env, SARS-CoV-2 S, or RSV F glycoproteins) has improved their capability to induce neutralizing protective immune responses. Therefore, we have stabilized the FeLV Env protein following a strategy based on the incorporation of a disulfide bond and an Ile/Pro mutation (SOSIP) previously used to generate soluble HIV Env trimers. We have characterized this SOSIP-FeLV Env in its soluble form and as a transmembrane protein present at high density on the surface of FeLV Gag-based VLPs. Furthermore, we have tested its immunogenicity in DNA-immunization assays in C57BL/6 mice. Low anti-FeLV Env responses were detected in SOSIP-FeLV soluble protein-immunized animals; however, unexpectedly no responses were detected in the animals immunized with SOSIP-FeLV Gag-based VLPs. In contrast, high humoral response against FeLV Gag was observed in the animals immunized with control Gag VLPs lacking SOSIP-FeLV Env, while this response was significantly impaired when the VLPs incorporated SOSIP-FeLV Env. Our data suggest that FeLV Env can be stabilized as a soluble protein and can be expressed in high-density VLPs. However, when formulated as a DNA vaccine, SOSIP-FeLV Env remains poorly immunogenic, a limitation that must be overcome to develop an effective FeLV vaccine.

VLPs generated by the fusion of RSV-F or hMPV-F glycoprotein to HIV-Gag show improved immunogenicity and neutralizing response in mice.

Respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) vaccines have been long overdue. Structure-based vaccine design created a new momentum in the last decade, and the first RSV vaccines have finally been approved in older adults and pregnant individuals. These vaccines are based on recombinant stabilized pre-fusion F glycoproteins administered as soluble proteins. Multimeric antigenic display could markedly improve immunogenicity and should be evaluated in the next generations of vaccines. Here we tested a new virus like particles-based vaccine platform which utilizes the direct fusion of an immunogen of interest to the structural human immunodeficient virus (HIV) protein Gag to increase its surface density and immunogenicity. We compared, in mice, the immunogenicity of RSV-F or hMPV-F based immunogens delivered either as soluble proteins or displayed on the surface of our VLPs. VLP associated F-proteins showed better immunogenicity and induced superior neutralizing responses. Moreover, when combining both VLP associated and soluble immunogens in a heterologous regimen, VLP-associated immunogens provided added benefits when administered as the prime immunization.

Prostate Cancer and the Mevalonate Pathway.

Antineoplastic therapies for prostate cancer (PCa) have traditionally centered around the androgen receptor (AR) pathway, which has demonstrated a significant role in oncogenesis. Nevertheless, it is becoming progressively apparent that therapeutic strategies must diversify their focus due to the emergence of resistance mechanisms that the tumor employs when subjected to monomolecular treatments. This review illustrates how the dysregulation of the lipid metabolic pathway constitutes a survival strategy adopted by tumors to evade eradication efforts. Integrating this aspect into oncological management could prove valuable in combating PCa.

Virus-like particle-mediated delivery of structure-selected neoantigens demonstrates immunogenicity and antitumoral activity in mice.

BACKGROUND: Neoantigens are patient- and tumor-specific peptides that arise from somatic mutations. They stand as promising targets for personalized therapeutic cancer vaccines. The identification process for neoantigens has evolved with the use of next-generation sequencing technologies and bioinformatic tools in tumor genomics. However, in-silico strategies for selecting immunogenic neoantigens still have very low accuracy rates, since they mainly focus on predicting peptide binding to Major Histocompatibility Complex (MHC) molecules, which is key but not the sole determinant for immunogenicity. Moreover, the therapeutic potential of neoantigen-based vaccines may be enhanced using an optimal delivery platform that elicits robust de novo immune responses. METHODS: We developed a novel neoantigen selection pipeline based on existing software combined with a novel prediction method, the Neoantigen Optimization Algorithm (NOAH), which takes into account structural features of the peptide/MHC-I interaction, as well as the interaction between the peptide/MHC-I complex and the TCR, in its prediction strategy. Moreover, to maximize neoantigens' therapeutic potential, neoantigen-based vaccines should be manufactured in an optimal delivery platform that elicits robust de novo immune responses and bypasses central and peripheral tolerance. RESULTS: We generated a highly immunogenic vaccine platform based on engineered HIV-1 Gag-based Virus-Like Particles (VLPs) expressing a high copy number of each in silico selected neoantigen. We tested different neoantigen-loaded VLPs (neoVLPs) in a B16-F10 melanoma mouse model to evaluate their capability to generate new immunogenic specificities. NeoVLPs were used in in vivo immunogenicity and tumor challenge experiments. CONCLUSIONS: Our results indicate the relevance of incorporating other immunogenic determinants beyond the binding of neoantigens to MHC-I. Thus, neoVLPs loaded with neoantigens enhancing the interaction with the TCR can promote the generation of de novo antitumor-specific immune responses, resulting in a delay in tumor growth. Vaccination with the neoVLP platform is a robust alternative to current therapeutic vaccine approaches and a promising candidate for future personalized immunotherapy.

Exploring FeLV-Gag-Based VLPs as a New Vaccine Platform-Analysis of Production and Immunogenicity.

Feline leukemia virus (FeLV) is one of the most prevalent infectious diseases in domestic cats. Although different commercial vaccines are available, none of them provides full protection. Thus, efforts to design a more efficient vaccine are needed. Our group has successfully engineered HIV-1 Gag-based VLPs that induce a potent and functional immune response against the HIV-1 transmembrane protein gp41. Here, we propose to use this concept to generate FeLV-Gag-based VLPs as a novel vaccine strategy against this retrovirus. By analogy to our HIV-1 platform, a fragment of the FeLV transmembrane p15E protein was exposed on FeLV-Gag-based VLPs. After optimization of Gag sequences, the immunogenicity of the selected candidates was evaluated in C57BL/6 and BALB/c mice, showing strong cellular and humoral responses to Gag but failing to generate anti-p15E antibodies. Altogether, this study not only tests the versatility of the enveloped VLP-based vaccine platform but also sheds light on FeLV vaccine research.

An engineered HIV-1 Gag-based VLP displaying high antigen density induces strong antibody-dependent functional immune responses.

Antigen display on the surface of Virus-Like Particles (VLPs) improves immunogenicity compared to soluble proteins. We hypothesised that immune responses can be further improved by increasing the antigen density on the surface of VLPs. In this work, we report an HIV-1 Gag-based VLP platform engineered to maximise the presence of antigen on the VLP surface. An HIV-1 gp41-derived protein (Min), including the C-terminal part of gp41 and the transmembrane domain, was fused to HIV-1 Gag. This resulted in high-density MinGag-VLPs. These VLPs demonstrated to be highly immunogenic in animal models using either a homologous (VLP) or heterologous (DNA/VLP) vaccination regimen, with the latter yielding 10-fold higher anti-Gag and anti-Min antibody titres. Despite these strong humoral responses, immunisation with MinGag-VLPs did not induce neutralising antibodies. Nevertheless, antibodies were predominantly of an IgG2b/IgG2c profile and could efficiently bind CD16-2. Furthermore, we demonstrated that MinGag-VLP vaccination could mediate a functional effect and halt the progression of a Min-expressing tumour cell line in an in vivo mouse model.

Alanine-based spacers promote an efficient antigen processing and presentation in neoantigen polypeptide vaccines.

Neoantigens are tumor-specific antigens that are mostly particular for each patient. Since the immune system is able to mount a specific immune response against these neoantigens, they are a promising tool for the development of therapeutic personalized cancer vaccines. Neoantigens must be presented to T cells by antigen presenting cells (APC) in the context of MHC-I or MHC-II molecules. Therefore, the strategy of vaccine delivery may have a major impact on the magnitude and quality of T cell responses. Neoantigen-based vaccines are frequently administered as a pool of individual synthetic peptides that induce mainly CD4+ T cell responses. MHC-I-mediated presentation and the elicitation of CD8+ T cell responses may be improved using DNA or RNA sequences that code for a unique long polypeptide that concatenates the different neoantigens spaced by linker sequences. When administered this way, the selection of the spacer between neoantigens is of special interest, as it might influence the processing and presentation of the right peptides by APCs. Here, we evaluate the impact of such linker regions on the MHC-I-dependent antigen presentation using an in vitro assay that assesses the MHC-I presentation of SIINFEKL, a H-2 Kb-restricted OVA peptide. Our results show that spacers used to generate epitope concatenates have a large impact on the efficiency of neoantigen processing and presentation by MHC-I molecules; in contrast, the peptide position and the flanking regions have a minimal impact. Moreover, linkers based on alanine residues promote a more efficient peptide presentation than the commonly used GGGS linker.

A genomics approach identifies selective effects of trans-resveratrol in cerebral cortex neuron and glia gene expression.

The mode of action of trans-resveratrol, a promising lead compound for the development of neuroprotective drugs, is unknown. Data from a functional genomics study were retrieved with the aim to find differentially expressed genes that may be involved in the benefits provided by trans-resveratrol. Genes that showed a significantly different expression (p<0.05, cut-off of a two-fold change) in mice fed with a control diet or a control diet containing trans-resveratrol were different in cortex, heart and skeletal muscle. In neocortex, we identified 4 up-regulated (Strap, Pkp4, Rab2a, Cpne3) and 22 down-regulated (Actn1, Arf3, Atp6v01, Atp1a3, Atp1b2, Cacng7, Crtc1, Dbn1, Dnm1, Epn1, Gfap, Hap, Mark41, Rab5b, Nrxn2, Ogt, Palm, Ptprn2, Ptprs, Syn2, Timp2, Vamp2) genes upon trans-resveratrol consumption. Network analysis of gene products provided evidence of plakophilin 4 up-regulation as a triggering factor for down-regulation of events related to synaptic vesicle transport and neurotransmitter release via underexpression of dynamin1 and Vamp2 (synaptobrevin 2) as node-gene drivers. Analysis by RT-qPCR of some of the selected genes in a glioma cell line showed that dynamin 1 mRNA was down-regulated even in acute trans-resveratrol treatments. Taken all together, these results give insight on the glial-neuronal networks involved in the neuroprotective role of trans-resveratrol.

Neumoperitoneo masivo: el reto de no operar / Conservative management of pneumoperitoneum secondary to pneumatosis intestinalis

Background: In Cystic Pneumatosis Intestinalis, intramural gas filled cysts are formed in the gastrointestinal wall. Its pathogenesis is unknown and its clinical manifestations are variable. Case report: We report a 33 years old HIV positive woman in retroviral treatment presenting with abdominal pain and signs of peritoneal irritation. An abdominal CT scan showed intra and retroperitoneal gas and pneumatosis intestinalis in the right and transverse colon. The patient was managed conservatively with nasogastric aspiration with a favorable evolution and was discharged ten days after admission.

Introducción: Se denomina neumatosis quística intestinal (NQI) a la formación de quistes intramurales rellenos de gas ubicados en la pared del sistema gastrointestinal, de patogénesis desconocida y de manifestaciones clínicas muy variables. Caso clínico: Presentamos un caso de neumoperitoneo masivo por NQI intestinal en una paciente VIH+ que acude a urgencias por dolor y distensión abdominal y que, a pesar de las espectaculares imágenes de la radiografía simple de abdomen y la tomografía computarizada (TC), se trató conservadoramente. Este caso pone de manifiesto el reto diagnóstico y la duda que supone para el cirujano acostumbrado a "operar para curar", no operar un cuadro que hasta hace poco tiempo inexcusablemente suponía una laparotomía exploradora.

Association between serum tissue inhibitor of matrix metalloproteinase-1 levels and mortality in patients with severe brain trauma injury.

OBJECTIVE: Matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) play a role in neuroinflammation after brain trauma injury (TBI). Previous studies with small sample size have reported higher circulating MMP-2 and MMP-9 levels in patients with TBI, but no association between those levels and mortality. Thus, the aim of this study was to determine whether serum TIMP-1 and MMP-9 levels are associated with mortality in patients with severe TBI. METHODS: This was a multicenter, observational and prospective study carried out in six Spanish Intensive Care Units. Patients with severe TBI defined as Glasgow Coma Scale (GCS) lower than 9 were included, while those with Injury Severity Score (ISS) in non-cranial aspects higher than 9 were excluded. Serum levels of TIMP-1, MMP-9 and tumor necrosis factor (TNF)-alpha, and plasma levels of tissue factor (TF) and plasminogen activator inhibitor (PAI)-1 plasma were measured in 100 patients with severe TBI at admission. Endpoint was 30-day mortality. RESULTS: Non-surviving TBI patients (n = 27) showed higher serum TIMP-1 levels than survivor ones (n = 73). We did not find differences in MMP-9 serum levels. Logistic regression analysis showed that serum TIMP-1 levels were associated 30-day mortality (OR = 1.01; 95% CI = 1.001-1.013; P = 0.03). Survival analysis showed that patients with serum TIMP-1 higher than 220 ng/mL presented increased 30-day mortality than patients with lower levels (Chi-square = 5.50; P = 0.02). The area under the curve (AUC) for TIMP-1 as predictor of 30-day mortality was 0.73 (95% CI = 0.624-0.844; P<0.001). An association between TIMP-1 levels and APACHE-II score, TNF- alpha and TF was found. CONCLUSIONS: The most relevant and new findings of our study, the largest series reporting data on TIMP-1 and MMP-9 levels in patients with severe TBI, were that serum TIMP-1 levels were associated with TBI mortality and could be used as a prognostic biomarker of mortality in TBI patients.

High throughput phenotypic selection of Mycobacterium tuberculosis mutants with impaired resistance to reactive oxygen species identifies genes important for intracellular growth.

Mycobacterium tuberculosis has the remarkable capacity to survive within the hostile environment of the macrophage, and to resist potent antibacterial molecules such as reactive oxygen species (ROS). Thus, understanding mycobacterial resistance mechanisms against ROS may contribute to the development of new anti-tuberculosis therapies. Here we identified genes involved in such mechanisms by screening a high-density transposon mutant library, and we show that several of them are involved in the intracellular lifestyle of the pathogen. Many of these genes were found to play a part in cell envelope functions, further strengthening the important role of the mycobacterial cell envelope in protection against aggressions such as the ones caused by ROS inside host cells.

In situ analysis of lung antigen-presenting cells during murine pulmonary infection with virulent Mycobacterium tuberculosis.

Scarce information exists about the role of lung antigen-presenting cells (APCs) in vivo during pulmonary tuberculosis. As APCs activate cellular immunity, following intratracheal inoculation with virulent Mycobacterium tuberculosis, we assessed in situ lung APC recruitment, distribution, granuloma involvement, morphology and mycobacterial burden by using MHC-CII, CD14, scavenger receptor class A (SRA), the murine dendritic cell (DC)-restricted marker CD11c and Ziehl-Neelsen staining. CD11c(+) DC and CD14(+) cell recruitment into lungs appeared by day 14, continuing until day 60. MHC-CII(+) cells increased since day 7, persisting until day 60. Thus, virulent mycobacteria delays (14-21 days) lung APC recruitment compared to model antigens and nonvirulent bacilli (24-48 h). Regarding granuloma constitution, highly bacillary CD14(+) and SRA(+) cells were centrally located. MHC-CII(+) cells were more peripheral, with less mycobacteria. CD11c(+) cells were heterogeneously distributed within granulomas, with scarce bacilli. When labelling lung suspensions for MHC-CII and classifying cells as macrophages or DC, then staining for Ziehl-Neelsen, a remarkable segregation was found regarding bacillary burden. Most macrophage-like cells contained numerous bacilli, while DC had no or scarce mycobacteria. This implies differential APC contributions in situ during pulmonary tuberculosis regarding mycobacterial uptake, granuloma involvement and perhaps bacillary growth.

Morbimortalidad en anestesia general para cirugía electiva / Morbimortality in general anesthesia for elective surgery

Introducción. En la cirugía electiva, gracias a la mejor preparación de los pacientes, la morbimortalidad se reduce, no obstante, el estudio de la misma, contribuye en mayor cuantía a su disminución. Objetivos. En este trabajo nos propusimos determinar la incicencia de complicaciones anestésicas y su relación con algunos factores de riesgo. Material y método. Se realizó un estudio prospectivo seleccionando una muestra de 350 pacientes intervenidos en electivo bajo anestesia general durante los años 1993-1994 en nuestro hospital. La información recogida en encuestas se procesó haciendo un análisis univariado. Resultados. La incidencia total de pacientes complicados fue del 26.9 por ciento. Las complicaciones más frecuentes fueron las cardiovasculares (12.6 por ciento) y las digestivas (8.6 por ciento). Los factores de riesgo que tuvieron una relación significativa (p<0.01) con la aparición de complicaciones fueron: Estado Físico (ASA) mayor de II, la presencia de enfermedades crónicas no transmisible y el número de ellas (dos o más), el uso de la anestesia general endotraqueal. No sucedió así con la edad y región intervenida. Conclusiones. La incidencia de complicaciones fue relativamente baja. Se demostró la existencia de factores de riesgo que repercuten en la aparición de complicaciones anestésicas

Estudio de la morbimortalidad anestésica en cirugía abdominal de urgencia / Anesthetic morbimortality in emergency abdominal surgical procedures

Introducción. El presente trabajo se basa en el estudio de la morbimortalidad anestésica en pacientes intervenidos de urgencia por patologías intrabdominales, donde se conoce que el riesgo aumenta considerablemente. Objetivo. Determinar la incidencia de complicaciones anestésicas, el momento en el que ocurren con mayor frecuencia y su relación con algunos factores de riesgo. Material y método. Se realizó un estudio prospectivo, con una muestra de 300 pacientes que fueron intervenidos por abdomen agudo y en los que se utilizó anestesia general, durante los años 1993-1994 en nuestro hospital. La información recogida en encuestas se procesó haciendo un análisis univariado. Resultados. La incidencia total de pacientes complicados fue del 34.0 por ciento. Las complicaciones más frecuentes fueron las cardiovasculares (22.7 por ciento) y las digestivas (7.3 por ciento). El momento en que ocurrieron con mayor frecuencia fue el postoperatorio inmediato (22.7 por ciento). Los factores de riesgo que tuvieron una relación significativa (p<0.01) con la aparición de complicaciones fueron: pacientes mayores de 60 años, estado físico (ASA) mayor de II, la presencia de alguna alteración aguda previa a la cirugía y el tiempo quirúrgico mayor de dos horas. No fue significaiva la relación con el sexo. Solo encontramos un fallecido (0.3 por ciento) de causa no anestésica. Conclusiones. La incidencia de complicaciones fue relativamente baja, y ocurrieron principalmente en el postoperatorio inmediato. Se demostró la existencia de factores de riesgo que repercuten en la aparición de complicaciones anestésicas

Determinación de residuos de halothane en el ambiente de los salones de operaciones / Determination of halothane residues in the environment of the operating rooms

Con el objetivo de demostrar la presencia de contaminantes ambientales en la atmósfera quirúrgica, factor que contribuye a los riesgos profesionales de dicha área, se tomaron muestras de aire de 4 salones y 2 de ellas en la sala de posoperatorio de 1 de los hospitales, siguiendo el método de aspiración con absorbedores de vidrio. La muestra obtenida se analizó por un método espectrofotométrico. Los puntos donde hubo mayor concentración del anestésico estudiado fueron en los más cercanos a la máquina de anestesia y en la sala de posoperatorio en la cabecera del enfermo. Se observó que los niveles más elevados ocurrían cuando no había un buen funcionamiento del aire acondicionado. Las concentraciones encontradas fueron muy superiores a las descritas como aceptables, que son de 0,5 p.p.m.; además, no existía mecanismo de evacuación para los residuos anestésicos

Efectos de la anestesia por inhalación con halothane en la espermatogenesis de las ratas / Effects of halothane inhalation anesthesia on rat spermatogenesis

Se realiza un estudio de las alteraciones en la espermatogénesis en ratas a las que se les administró halothane y oxígeno. Para ello se utiliza un total de 60 ratas machos, de la raza blanca o albina, de 8 a 10 semanas de edad, y con un peso promedio de 200 a 250 g, distribuidas en 3 grupos de 20 ratas cada uno. El grupo control recibió una mezcla de aire enriquecido con oxígeno; uno de los grupos experimentales fue expuesto a una atmófera de oxígeno y halothane, y el otro, a oxígeno, óxido nitroso y halothane, ambos por un período de 8 horas diarias hasta completar 100 horas de exposición. A los 28 días se sacrificaron por dislocación cervical, se extrajeron los epidídimos y se encontró que existe un índice significativo de anormalidades espermáticas en los 2 grupos de animales expuestos a los agentes inhalatorios utilizados al compararlos con el grupo control