The Herpes Simplex Virus Latency-Associated Transcript Gene Is Associated with a Broader Repertoire of Virus-Specific Exhausted CD8+ T Cells Retained within the Trigeminal Ganglia of Latently Infected HLA Transgenic Rabbits.

UNLABELLED: Persistent pathogens, such as herpes simplex virus 1 (HSV-1), have evolved a variety of immune evasion strategies to avoid being detected and destroyed by the host's immune system. A dynamic cross talk appears to occur between the HSV-1 latency-associated transcript (LAT), the only viral gene that is abundantly transcribed during latency, and the CD8(+)T cells that reside in HSV-1 latently infected human and rabbit trigeminal ganglia (TG). The reactivation phenotype of TG that are latently infected with wild-type HSV-1 or with LAT-rescued mutant (i.e., LAT(+)TG) is significantly higher than TG latently infected with LAT-null mutant (i.e., LAT(-)TG). Whether LAT promotes virus reactivation by selectively shaping a unique repertoire of HSV-specific CD8(+)T cells in LAT(+)TG is unknown. In the present study, we assessed the frequency, function, and exhaustion status of TG-resident CD8(+)T cells specific to 40 epitopes derived from HSV-1 gB, gD, VP11/12, and VP13/14 proteins, in human leukocyte antigen (HLA-A*0201) transgenic rabbits infected ocularly with LAT(+)versus LAT(-)virus. Compared to CD8(+)T cells from LAT(-)TG, CD8(+)T cells from LAT(+)TG (i) recognized a broader selection of nonoverlapping HSV-1 epitopes, (ii) expressed higher levels of PD-1, TIM-3, and CTLA-4 markers of exhaustion, and (iii) produced less tumor necrosis factor alpha, gamma interferon, and granzyme B. These results suggest a novel immune evasion mechanism by which the HSV-1 LAT may contribute to the shaping of a broader repertoire of exhausted HSV-specific CD8(+)T cells in latently infected TG, thus allowing for increased viral reactivation. IMPORTANCE: A significantly larger repertoire of dysfunctional (exhausted) HSV-specific CD8(+)T cells were found in the TG of HLA transgenic rabbits latently infected with wild-type HSV-1 or with LAT-rescued mutant (i.e., LAT(+)TG) than in a more restricted repertoire of functional HSV-specific CD8(+)T cells in the TG of HLA transgenic rabbits latently infected with LAT-null mutant (i.e., LAT(-)TG). These findings suggest that the HSV-1 LAT locus interferes with the host cellular immune response by shaping a broader repertoire of exhausted HSV-specific CD8(+)T cells within the latency/reactivation TG site.

Therapeutic immunization with a mixture of herpes simplex virus 1 glycoprotein D-derived "asymptomatic" human CD8+ T-cell epitopes decreases spontaneous ocular shedding in latently infected HLA transgenic rabbits: association with low frequency of local PD-1+ TIM-3+ CD8+ exhausted T cells.

UNLABELLED: Most blinding ocular herpetic disease is due to reactivation of herpes simplex virus 1 (HSV-1) from latency rather than to primary acute infection. No herpes simplex vaccine is currently available for use in humans. In this study, we used the HLA-A*02:01 transgenic (HLA Tg) rabbit model of ocular herpes to assess the efficacy of a therapeutic vaccine based on HSV-1 gD epitopes that are recognized mainly by CD8(+) T cells from "naturally" protected HLA-A*02:01-positive, HSV-1-seropositive healthy asymptomatic (ASYMP) individuals (who have never had clinical herpes disease). Three ASYMP CD8(+) T-cell epitopes (gD(53-61), gD(70-78), and gD(278-286)) were linked with a promiscuous CD4(+) T-cell epitope (gD(287-317)) to create 3 separate pairs of CD4-CD8 peptides, which were then each covalently coupled to an Nε-palmitoyl-lysine moiety, a Toll-like receptor 2 (TLR-2) ligand. This resulted in the construction of 3 CD4-CD8 lipopeptide vaccines. Latently infected HLA Tg rabbits were immunized with a mixture of these 3 ASYMP lipopeptide vaccines, delivered as eye drops in sterile phosphate-buffered saline (PBS). The ASYMP therapeutic vaccination (i) induced HSV-specific CD8(+) T cells that prevent HSV-1 reactivation ex vivo from latently infected explanted trigeminal ganglia (TG), (ii) significantly reduced HSV-1 shedding detected in tears, (iii) boosted the number and function of HSV-1 gD epitope-specific CD8(+) T cells in draining lymph nodes (DLN), conjunctiva, and TG, and (iv) was associated with fewer exhausted HSV-1 gD-specific PD-1(+) TIM-3+ CD8(+) T cells. The results underscore the potential of an ASYMP CD8(+) T-cell epitope-based therapeutic vaccine strategy against recurrent ocular herpes. IMPORTANCE: Seventy percent to 90% of adults harbor herpes simplex virus 1 (HSV-1), which establishes lifelong latency in sensory neurons of the trigeminal ganglia. This latent state sporadically switches to spontaneous reactivation, resulting in viral shedding in tears. Most blinding herpetic disease in humans is due to reactivation of HSV-1 from latency rather than to primary acute infection. To date, there is no licensed therapeutic vaccine that can effectively stop or reduce HSV-1 reactivation from latently infected sensory ganglia and the subsequent shedding in tears. In the present study, we demonstrated that topical ocular therapeutic vaccination of latently infected HLA transgenic rabbits with a lipopeptide vaccine that contains exclusively human "asymptomatic" CD8(+) T-cell epitopes successfully decreased spontaneous HSV-1 reactivation, as judged by a significant reduction in spontaneous shedding in tears. The findings should guide the clinical development of a safe and effective T-cell-based therapeutic herpes vaccine.

Increased neurovirulence and reactivation of the herpes simplex virus type 1 latency-associated transcript (LAT)-negative mutant dLAT2903 with a disrupted LAT miR-H2.

At least six microRNAs (miRNAs) appear to be encoded by the latency-associated transcript (LAT) of herpes simplex virus type 1 (HSV-1). The gene for ICP0, an important immediate early (IE) viral protein, is anti-sense to, and overlaps with, the region of LAT from which miRNA H2 (miR-H2) is derived. We recently reported that a mutant (McK-ΔH2) disrupted for miR-H2 on the wild-type HSV-1 strain McKrae genomic background has increased ICP0 expression, increased neurovirulence, and slightly more rapid reactivation. We report here that HSV-1 mutants deleted for the LAT promoter nonetheless make significant amounts of miR-H2 during lytic tissue culture infection, presumably via readthrough transcription from an upstream promoter. To determine if miR-H2 might also play a role in the HSV-1 latency/reactivation cycle of a LAT-negative mutant, we constructed dLAT-ΔH2, in which miR-H2 is disrupted in dLAT2903 without altering the predicted amino acid sequence of the overlapping ICP0 open reading frame. Similar to McK-ΔH2, dLAT-ΔH2 expressed more ICP0, was more neurovirulent, and had increased reactivation in the mouse TG explant-induced reactivation model of HSV-1 compared with its parental virus. Interestingly, although the increased reactivation of McK-ΔH2 compared with its parental wild-type (wt) virus was subtle and only detected at very early times after explant TG induced reactivation, the increased reactivation of dLAT-ΔH2 compared with its dLAT2903 parental virus appeared more robust and was significantly increased even at late times after induction. These results confirm that miR-H2 plays a role in modulating the HSV-1 reactivation phenotype.

The herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) protects cells against cold-shock-induced apoptosis by maintaining phosphorylation of protein kinase B (AKT).

The herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) blocks apoptosis and inhibits caspase-3 activation. We previously showed that serum starvation (removal of serum from tissue culture media), which takes several days to induce apoptosis, results in decreased levels of both AKT (protein kinase B) and phosphorylated AKT (pAKT) in cells not expressing LAT. In contrast in mouse neuroblastoma cells expressing LAT, AKT, and pAKT levels remained high. AKT is a serine/threonine protein kinase that promotes cell survival. To examine the effect of LAT on AKT-pAKT using a different and more rapid method of inducing apoptosis, a stable cell line expressing LAT was compared to non-LAT expressing cells as soon as 15 min following recovery from cold-shock-induced apoptosis. Expression of LAT appeared to inhibit dephosphorylation of pAKT. This protection correlated with blocking numerous pro-apoptotic events that are inhibited by pAKT. These results support the hypothesis that inhibiting dephosphorylation of pAKT may be one of the pathways by which LAT protects cells against apoptosis.

Decreased reactivation of a herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) mutant using the in vivo mouse UV-B model of induced reactivation.

Blinding ocular herpetic disease in humans is due to herpes simplex virus type 1 (HSV-1) reactivations from latency, rather than to primary acute infection. The cellular and molecular immune mechanisms that control the HSV-1 latency-reactivation cycle remain to be fully elucidated. The aim of this study was to determine if reactivation of the HSV-1 latency-associated transcript (LAT) deletion mutant (dLAT2903) was impaired in this model, as it is in the rabbit model of induced and spontaneous reactivation and in the trigeminal ganglia (TG) explant-induced reactivation model in mice. The eyes of mice latently infected with wild-type HSV-1 strain McKrae (LAT((+)) virus) or dLAT2903 (LAT((-)) virus) were irradiated with UV-B, and reactivation was determined. We found that compared to LAT((-)) virus, LAT((+)) virus reactivated at a higher rate as determined by shedding of virus in tears on days 3 to 7 after UV-B treatment. Thus, the UV-B-induced reactivation mouse model of HSV-1 appears to be a useful small animal model for studying the mechanisms involved in how LAT enhances the HSV-1 reactivation phenotype. The utility of the model for investigating the immune evasion mechanisms regulating the HSV-1 latency/reactivation cycle and for testing the protective efficacy of candidate therapeutic vaccines and drugs is discussed.

A herpes simplex virus type 1 mutant disrupted for microRNA H2 with increased neurovirulence and rate of reactivation.

The herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) encodes several microRNAs. One of these, miR-H2, overlaps and is antisense to the ICP0 gene and appears to decrease expression of the ICP0 protein. To determine if miR-H2 plays a role in the HSV-1 latency-reactivation cycle, we constructed a mutant, McK-ΔH2, in which this microRNA has been disrupted without altering the predicted amino acid sequence of ICP0. McK-ΔH2 produced increased amounts of ICP0. Although replication of McK-ΔH2 was similar to that of its wild-type (wt) McKrae parental virus in RS cells and mouse eyes, McK-ΔH2 was more neurovirulent in Swiss-Webster mice than McKrae based on the percent of mice that died from herpes encephalitis following ocular infection. In addition, using a mouse trigeminal ganglia (TG) explant model of induced reactivation, we show here for the first time that miR-H2 appears to play a role in modulating HSV-1 reactivation. Although the percent of TG from which virus reactivated by day 10 after explant was similar for McK-ΔH2, wt McKrae, and the marker-rescued virus McK-ΔH2Res, at earlier times, significantly more reactivation was seen with McK-ΔH2. Our results suggest that in the context of the virus, miR-H2 downregulates ICP0 and this moderates both HSV-1 neurovirulence and reactivation.

Role of litter turnover in soil quality in tropical degraded lands of Colombia.

Land degradation is the result of soil mismanagement that reduces soil productivity and environmental services. An alternative to improve degraded soils through reactivation of biogeochemical nutrient cycles (via litter production and decomposition) is the establishment of active restoration models using new forestry plantations, agroforestry, and silvopastoral systems. On the other hand, passive models of restoration consist of promoting natural successional processes with native plants. The objective in this review is to discuss the role of litter production and decomposition as a key strategy to reactivate biogeochemical nutrient cycles and thus improve soil quality in degraded land of the tropics. For this purpose the results of different projects of land restoration in Colombia are presented based on the dynamics of litter production, nutrient content, and decomposition. The results indicate that in only 6-13 years it is possible to detect soil properties improvements due to litter fall and decomposition. Despite that, low soil nutrient availability, particularly of N and P, seems to be major constraint to reclamation of these fragile ecosystems.

The herpes simplex virus type 1 latency-associated transcript can protect neuron-derived C1300 and Neuro2A cells from granzyme B-induced apoptosis and CD8 T-cell killing.

The herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) is the only HSV-1 gene transcript abundantly expressed throughout latency. LAT null mutants have a significantly reduced reactivation phenotype. LAT's antiapoptosis activity is the major LAT factor involved in supporting the wild-type reactivation phenotype. During HSV-1 latency, some ganglionic neurons are surrounded by CD8 T cells, and it has been proposed that these CD8 T cells help maintain HSV-1 latency by suppressing viral reactivations. Surprisingly, despite injection of cytotoxic lytic granules by these CD8 T cells into latently infected neurons, neither apoptosis nor neuronal cell death appears to occur. We hypothesized that protection of latently infected neurons against cytotoxic CD8 T-cell killing is due to LAT's antiapoptosis activity. Since CD8 T-cell cytotoxic lytic granule-mediated apoptosis is critically dependent on granzyme B (GrB), we examined LAT's ability to block GrB-induced apoptosis. We report here that (i) LAT can interfere with GrB-induced apoptosis in cell cultures, (ii) LAT can block GrB-induced cleavage (activation) of caspase-3 both in cell culture and in a cell-free in vitro cell extract assay, and (iii) LAT can protect C1300 and Neuro2A cells from cytotoxic CD8 T-cell killing in vitro. These findings support the hypothesis that LAT's antiapoptosis activity can protect latently infected neurons from being killed by CD8 T-cell lytic granules in vivo.

The herpes simplex virus 1 latency-associated transcript promotes functional exhaustion of virus-specific CD8+ T cells in latently infected trigeminal ganglia: a novel immune evasion mechanism.

Following ocular herpes simplex virus 1 (HSV-1) infection of C57BL/6 mice, HSV-specific (HSV-gB(498-505) tetramer(+)) CD8(+) T cells are induced, selectively retained in latently infected trigeminal ganglia (TG), and appear to decrease HSV-1 reactivation. The HSV-1 latency-associated transcript (LAT) gene, the only viral gene that is abundantly transcribed during latency, increases reactivation. Previously we found that during latency with HSV-1 strain McKrae-derived viruses, more of the total TG resident CD8 T cells expressed markers of exhaustion with LAT(+) virus compared to LAT(-) virus. Here we extend these findings to HSV-1 strain 17syn+-derived LAT(+) and LAT(-) viruses and to a virus expressing just the first 20% of LAT. Thus, the previous findings were not an artifact of HSV-1 strain McKrae, and the LAT function involved mapped to the first 1.5 kb of LAT. Importantly, to our knowledge, we show here for the first time that during LAT(+) virus latency, most of the HSV-1-specific TG resident CD8 T cells were functionally exhausted, as judged by low cytotoxic function and decreased gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) production. This resulted in LAT(-) TG having more functional HSV-gB(498-505) tetramer(+) CD8(+) T cells compared to LAT(+) TG. In addition, LAT expression, in the absence of other HSV-1 gene products, appeared to be able to directly or indirectly upregulate both PD-L1 and major histocompatibility complex class I (MHC-I) on mouse neuroblastoma cells (Neuro2A). These findings may constitute a novel immune evasion mechanism whereby the HSV-1 LAT directly or indirectly promotes functional exhaustion (i.e., dysfunction) of HSV-specific CD8(+) T cells in latently infected TG, resulting in increased virus reactivation.

Cellular FLIP can substitute for the herpes simplex virus type 1 latency-associated transcript gene to support a wild-type virus reactivation phenotype in mice.

Latency-associated transcript (LAT) deletion mutants of herpes simplex virus type 1 (HSV-1) have reduced reactivation phenotypes. Thus, LAT plays an essential role in the latency-reactivation cycle of HSV-1. We have shown that LAT has antiapoptosis activity and demonstrated that the chimeric virus, dLAT-cpIAP, resulting from replacing LAT with the baculovirus antiapoptosis gene cpIAP, has a wild-type HSV-1 reactivation phenotype in mice and rabbits. Thus, LAT can be replaced by an alternative antiapoptosis gene, confirming that LAT's antiapoptosis activity plays an important role in the mechanism by which LAT enhances the virus' reactivation phenotype. However, because cpIAP interferes with both of the major apoptosis pathways, these studies did not address whether LAT's proreactivation phenotype function was due to blocking the extrinsic (Fas-ligand-, caspase-8-, or caspase-10-dependent pathway) or the intrinsic (mitochondria-, caspase-9-dependent pathway) pathway, or whether both pathways must be blocked. Here we constructed an HSV-1 LAT(-) mutant that expresses cellular FLIP (cellular FLICE-like inhibitory protein) under control of the LAT promoter and in place of LAT nucleotides 76 to 1667. Mice were ocularly infected with this mutant, designated dLAT-FLIP, and the reactivation phenotype was determined using the trigeminal ganglia explant model. dLAT-FLIP had a reactivation phenotype similar to wild-type virus and significantly higher than the LAT(-) mutant dLAT2903. Thus, the LAT function responsible for enhancing the reactivation phenotype could be replaced with an antiapoptosis gene that primarily blocks the extrinsic signaling apoptosis pathway.

A speculated ribozyme site in the herpes simplex virus type 1 latency-associated transcript gene is not essential for a wild-type reactivation phenotype.

During herpes simplex virus-1 (HSV-1) latency in sensory neurons, LAT (latency-associated transcript) is the only abundantly expressed viral gene. LAT plays an important role in the HSV-1 latency-reactivation cycle, because LAT deletion mutants have a significantly decreased reactivation phenotype. Based solely on sequence analysis, it was speculated that LAT encodes a ribozyme that plays an important role in how LAT enhances the virus' reactivation phenotype. Because LAT ribozyme activity has never been reported, we decided to test the converse hypothesis, namely, that this region of LAT does not encode a ribozyme function important for LAT's ability to enhance the reactivation phenotype. We constructed a viral mutant (LAT-Rz) in which the speculated ribozyme consensus sequence was altered such that no ribozyme was encoded. We report here that LAT-Rz had a wild-type reactivation phenotype in mice, confirming the hypothesis that the speculated LAT ribozyme is not a dominant factor in stimulating the latency-reactivation cycle in mice.

Validación de cuatro reglas de predicción clínica de la nefropatía inducida por contraste en pacientes llevados a intervención coronaria percutánea / Validation of Four Clinical Risk Scores for Predicting Contrast-Induced Nephropathy in Patients Undergoing Percutaneous Coronary Intervention

RESUMEN Introducción: La nefropatía inducida por contraste (NIC) es el empeoramiento agudo de la función renal tras administrarse medio de contraste endovenoso, y conlleva una importante carga de morbilidad y mortalidad. Actualmente se cuenta con múltiples reglas clínicas para predecir su desarrollo. El objetivo del presente trabajo es validar cuatro reglas para la predicción de la nefropatía inducida por contraste en pacientes llevados a procedimiento intervencionista coronario percutáneo (ICP) . Material y métodos: Estudio de cohorte retrospectiva unicéntrico, que incluyó adultos llevados a ICP entre enero de 2014 y diciembre de 2018. Se excluyeron pacientes en diálisis, los que murieron durante el procedimiento o aquellos de los que no se dispusiera de los datos necesarios para el análisis. Se aplicaron las cuatro reglas de predicción, se obtuvo la puntuación de cada una para cada uno de los pacientes y se calculó el área bajo la curva ROC para el desarrollo de NIC. Resultados: En 785 pacientes se pudo calcular las cuatro reglas; 109 (13,8%) desarrollaron NIC y 14 (1,7%) requirieron diálisis. La media de edad fue 65 años y el 36,1% fueron mujeres. La media de tasa de filtración glomerular fue 69,1 mL/min. La regla de Mehran obtuvo un área bajo la curva de 0,574 para NIC y 0,881 para diálisis; Gao, 0,487 para NIC y 0,831 para diálisis; Lin, 0,572 para NIC y 0,854 para diálisis; y Bartholomew, 0,506 para NIC y 0,754 para diálisis. Conclusiones: La aplicación de las reglas de predicción clínica de Mehran, Gao, Lin y Bartholomew en pacientes llevados a ICP mostró una pobre capacidad de discriminación para la NIC aunque su desempeño fue excelente para predecir la necesidad de diálisis.

ABSTRACT Background: Contrast-induced nephropathy (CIN) is the acute deterioration of kidney function after the administration of intravenous contrast media and is associated with significant morbidity and mortality. Several clinical risk scores to predict CIN are currently available. The aim of the present study is to validate four risk scores for predicting CIN in patients undergoing percutaneous coronary intervention (PCI). Methods: We conducted a retrospective single-center cohort study including adult patients undergoing PCI between January 2014 and December 2018. Patients on dialysis, those who died during the procedure or lack of necessary data for the analysis were excluded. The four risk scores were estimated for each patient and the area under the ROC curve for the development of CIN was calculated. Results: The four risk scores were calculated in 785 patients; 109 (13.8%) developed CIN and 14 (1.7%) required dialysis. Mean age was 65 years and 36.1% were women. Mean glomerular filtration rate was 69.1 mL/min. The areas under the curve for each risk score to predict CIN and dialysis were: Mehran 0.574 and 0.881, respectively; Gao, 0.487 and 0.831; Lin, 0.572 and 0.854; and Bartholomew, 0.506 and 0.754. Conclusions: The use of the Mehran, Gao, Lin, and Bartholomew risk scores in patients undergoing PCI showed poor discriminatory ability for CIN, although their performance was excellent for predicting the need for dialysis.

Pérdida de biodiversidad y vulnerabilidad humana frente a enfermedades infecciosas zoonóticas: humanos, relatos y ambiente

Propósito/Contexto. El crecimiento exponencial de la transmisión de enfermedades de tipo zoonótico, en los últimos años, ha generado grandes afectaciones en la población mundial. A su vez, una eventual pérdida de biodiversidad aumenta el nivel de la vulnerabilidad humana frente a enfermedades infecciosas zoonóticas. El presente artículo pretende aportar algunos elementos para comprender la manera en que la pérdida de biodiversidad generada por la actividad económica humana favorece la transmisión de enfermedades infecciosas de tipo zoonótico y aumenta, como consecuencia, el nivel de vulnerabilidad de nuestra especie frente a este tipo de amenazas. Metodología/Enfoque. Se realizó un análisis crítico de las intervenciones realizadas por los ponentes que participaron en cada uno de los cuatro eventos de la Cátedra Abierta de Bioética Versión Especial 2020-1, titulada "Ambiente, Humanos y Pandemia", organizada por el Departamento de Bioética de la Universidad El Bosque. También se hizo una recopilación sistemática de diferentes artículos, en tres de las más importantes bases de datos de bioética en el mundo: Bioethics Research Library, Bioethics y German Reference Centre for Ethics in the Life Science. Resultados/Hallazgos. Se logró identificar la urgente necesidad de restaurar las relaciones entre la naturaleza y los seres humanos en torno a la vida, a partir de cambios en el relacionamiento intersubjetivo, interespecífico (humanos con otras especies) y con el ambiente. Discusión/Conclusiones/Contribuciones. A modo de conclusión, las acciones de los seres humanos en contra del equilibrio dinámico ecosistémico son perjudiciales para sí mismos y afectan a las demás especies, generando así una mayor exposición humana a las enfermedades infecciosas de tipo zoonótico, como la COVID-19.

Purpose/Context. The exponential growth of zoonotic diseases has dramatically affected the world population in recent years. In turn, an eventual loss of biodiversity will increase the level of human vulnerability to zoonotic infectious diseases. This article aims to provide some inputs for understanding how the loss of biodiversity due to human economic activity favors the transmission of zoonotic infectious diseases and thus increases the vulnerability level of our species against this threat. Methodology/Approach. We performed a critical analysis of the presentations in four events of the Open Lecture in Bioethics, Special Version 2020-1, entitled "Environment, Humans, and Pandemic," organized by the Bioethics Department, Universidad El Bosque. Besides, we systematically compiled several articles from three of the most critical bioethics databases worldwide: Bioethics Research Library, Bioethics, and German Reference Center for Ethics in the Life Science. Results/Findings. We could identify the pressing need to restore the relationships between nature and human beings around life, based on changes in the intersubjective, interspecific (humans with other species), and environmental relationships. Discussion/Conclusions/Contributions. The actions of human beings against the dynamic ecosystem balance are harmful to themselves and adversely affect other species, thus worsening human exposure to zoonotic infectious diseases such as COVID-19.

Objetivo/Contexto. O crescimento exponencial da transmissão de doenças de tipo zoonótico nos últimos anos, tem gerado grandes afetações na população mundial. Por sua vez, uma eventual perda de biodiversidade aumenta o nível de vulnerabilidade humana perante doenças infecciosas zoonóticas. O presente artigo destina-se a fornecer alguns elementos para a compreensão da forma como a perda de biodiversidade gerada pela atividade económica humana favorece a transmissão de doenças infecciosas zoonóticas e aumenta, como consequência, o nível de vulnerabilidade da nossa espécie perante este tipo de ameaças. Metodologia/Abordagem. Realizou-se uma análise crítica das intervenções realizadas pelos relatores que participaram em cada um dos quatro eventos da Cátedra Aberta de Bioética Versão Especial 2020-1, intitulada "Ambiente, Humanos e Pandemia" organizada pelo Departamento de Bioética da Universidade El Bosque. Também se fez uma recolha sistemática de diferentes artigos, em três das mais importantes bases de dados de bioética do mundo: Bioethics Research Library, Bioethics e German Reference Centre for Ethics in the Life Science. Resultados/Descobertas. Conseguiu-se identificar a urgente necessidade de restaurar as relações entre a natureza e os seres humanos em torno da vida, a partir de mudanças no relacionamento intersubjetivo, interespecífico (humanos com outras espécies) e com o ambiente. Discussão/Conclusões/Contribuições. A título de conclusão, as ações dos seres humanos contra o equilíbrio dinâmico ecossistêmico são prejudiciais para si próprios e afetam as outras espécies, gerando assim uma maior exposição humana às doenças infecciosas de tipo zoonótico, como o COVID-19.

Confocal Microscopic Analysis of a Rabbit Eye Model of High-Incidence Recurrent Herpes Stromal Keratitis.

PURPOSE: Using CJLAT, a chimeric herpes simplex virus (HSV-1) that produces a high incidence of herpes stromal keratitis (HSK) in latently infected rabbits, and in vivo confocal microscopy (CM), we characterized the cellular events that precede the development of HSK. METHODS: Thirty days after infection, in vivo CM was performed daily for 10 days and then weekly for up to 80 days after infection. RESULTS: We detected 3 types of subclinical corneal lesions before HSK was clinically apparent: (1) small epithelial erosions; (2) regenerating epithelium overlying small cell infiltrates within the basal epithelial cell layer; and (3) dendritic-like cells within the basal epithelial layer overlying stromal foci containing infiltrating cells. Sequential in vivo CM observations suggested that subclinical foci resolved over time but were larger and more abundant with CJLAT than with wild-type HSV-1 McKrae. Active HSK was observed only with CJLAT and was initially associated with a large epithelial lesion overlying stromal immune cell infiltrates. CONCLUSIONS: These results suggest that replication in the cornea of reactivated virus from the trigeminal ganglia produces epithelial lesions, which recruit immune cell infiltrates into the basal epithelial layer and anterior stroma. The virus is usually cleared rapidly eliminating viral antigens before the arrival of the immune cells, which disperse. However, if the virus is not cleared rapidly, or if an additional reactivation results in an additional round of virus at the same site before the immune cells disperse, then the immune cells are stimulated and may induce an immunopathological response leading to the development of HSK.

Prior Corneal Scarification and Injection of Immune Serum are Not Required Before Ocular HSV-1 Infection for UV-B-Induced Virus Reactivation and Recurrent Herpetic Corneal Disease in Latently Infected Mice.

PURPOSE: Blinding ocular herpetic disease in humans is due to spontaneous reactivation of herpes simplex virus type 1 (HSV-1) from latency, rather than to primary acute infection. Mice latently infected with HSV-1 undergo little or no in vivo spontaneous reactivation with accompanying virus shedding in tears. HSV-1 reactivation can be induced in latently infected mice by several in vivo procedures, with UV-B-induced reactivation being one commonly used method. In the UV-B model, corneas are scarified (lightly scratched) just prior to ocular infection to increase efficiency of the primary infection and immune serum containing HSV-1 neutralizing antibodies is injected intraperitoneally (i.p.) to increase survival and decrease acute corneal damage. Since scarification can significantly alter host gene transcription in the cornea and in the trigeminal ganglia (TG; the site of HSV-1 latency) and since injection of immune serum likely modulates innate and adaptive herpes immunity, we investigated eliminating both treatments. MATERIAL AND METHODS: Mice were infected with HSV-1 with or without corneal scarification and immune serum. HSV-1 reactivation and recurrent disease were induced by UV-B irradiation. RESULTS: When corneal scarification and immune serum were both eliminated, UV-B irradiation still induced both HSV-1 reactivation, as measured by shedding of reactivated virus in tears and herpetic eye disease, albeit at reduced levels compared to the original procedure. CONCLUSION: Despite the reduced reactivation and disease, avoidance of both corneal scarification and immune serum should improve the clinical relevance of the UV-B mouse model.

Large Amounts of Reactivated Virus in Tears Precedes Recurrent Herpes Stromal Keratitis in Stressed Rabbits Latently Infected with Herpes Simplex Virus.

AIM: Recurrent herpetic stromal keratitis (rHSK), due to an immune response to reactivation of herpes simplex virus (HSV-1), can cause corneal blindness. The development of therapeutic interventions such as drugs and vaccines to decrease rHSK have been hampered by the lack of a small and reliable animal model in which rHSK occurs at a high frequency during HSV-1 latency. The aim of this study is to develop a rabbit model of rHSK in which stress from elevated temperatures increases the frequency of HSV-1 reactivations and rHSK. MATERIALS AND METHODS: Rabbits latently infected with HSV-1 were subjected to elevated temperatures and the frequency of viral reactivations and rHSK were determined. RESULTS: In an experiment in which rabbits latently infected with HSV-1 were subjected to ill-defined stress as a result of failure of the vivarium air conditioning system, reactivation of HSV-1 occurred at over twice the normal frequency. In addition, 60% of eyes developed severe rHSK compared to <1% of eyes normally. All episodes of rHSK were preceded four to five days prior by an unusually large amount of reactivated virus in the tears of that eye and whenever this unusually large amount of reactivated virus was detected in tears, rHSK always appeared 4-5 days later. In subsequent experiments using well defined heat stress the reactivation frequency was similarly increased, but no eyes developed rHSK. CONCLUSIONS: The results reported here support the hypothesis that rHSK is associated not simply with elevated reactivation frequency, but rather with rare episodes of very high levels of reactivated virus in tears 4-5 days earlier.

Identification of a protein encoded in the herpes simplex virus type 1 latency associated transcript promoter region.

Herpes simplex virus type 1 (HSV-1) establishes a lifelong latency in the neurons of its host. Sporadically, the latent virus reactivates and spreads back to the original site of infection and causes recrudescent diseases. The only gene actively transcribed during neuronal latency is the latency associated transcript (LAT) gene. Several transcripts have been detected in the important LAT promoter region. However, no polypeptides coded by these transcripts are known. In this communication, we reported the cloning, sequencing, and characterization of a transcript immediately upstream of LAT. We designated this gene UOL (Upstream of LAT). The UOL RNA is polyadenylated, expressed as a late gene in infected cells, transcribed in the same direction as LAT, and contains an open reading frame (ORF) capable of encoding a protein of 96 amino acids with a predicted molecular mass of 11 kDa. The UOL transcript contains 466 nucleotides in length. The 5' end of the UOL transcript starts at nucleotide 118,266 and the 3' end of the UOL transcript ends at nucleotide 118,731 based on the published 17syn+ genomic sequence. The UOL protein was detected in infected cell lysates by immunoprecipitation using an antibody raised against UOL ORF synthetic peptide. More importantly, sera from mice infected with wild-type HSV-1 but not sera from mice infected with a mutant with the UOL region deleted recognized the UOL ORF, expressed in Escherichia coli, on Western blots. These results suggest that a UOL protein is in HSV-1 infected tissue culture cells and in mice infected with HSV-1.

Incidencia y factores de riesgo asociados con nefropatía inducida por medios de contraste en procedimientos intervencionistas de cardiología / Incidence and risk factors of contrast-induced nephropathy in interventional cardiology procedures

Introducción: la nefropatía inducida por medios de contraste (NIC) es la injuria secundaria a la exposición en procedimientos diagnósticos e intervencionistas. Representa la tercera causa de insuficiencia renal aguda hospitalaria. Esta investigación buscó establecer factores de riesgo para NIC en cateterismo cardiaco izquierdo (CCI) en pacientes hospitalizados en el Hospital de San José de Bogotá, durante tres años. Métodos: estudio de cohorte retrospectiva en pacientes con CCI y exposición a medios de contraste y estrategias de nefroprotección con líquidos, bicarbonato o N-acetilcisteína. Se incluyeron todos los atendidos entre 2014 y 2016. Se definió la NIC como el aumento de 0,5 mg/dL de creatinina en 72 horas o del 25% de la basal. De 495 pacientes, se excluyeron 15 con hemodiálisis previa. Se tomaron registros clínicos electrónicos realizando análisis estadístico multivariado. Se utilizó el paquete estadístico stata 13. Resultados: en 480 pacientes la incidencia de NIC fue 13,1%; tener una hemoglobina mayor 13 gr/dL en hombres, fue un factor protector para NIC OR: 0,29 (p=0.004). La creatinina basal mayor de 1,5 mg/dL aumentó el riesgo de NIC OR: 2,56 (IC 95: 1,35-4,85; p=0.004), así como la coexistencia de diabetes y enfermedad renal crónica OR: 2,73 (IC 95: 1,25 ­ 5.95; p=0,005). Otros factores como edad, falla cardiaca, volumen de contraste, IAM anterior y estrategias de nefroprotección, no mostraron asociación significativa. En relación con NIC la incidencia de muerte fue 9,5% y hemodiálisis 2,5%. Conclusiones: la incidencia de NIC se asocia con aumento de la mortalidad; los factores de riesgo asociados fueron creatinina >1.5 mg/dL y diabetes más enfermedad renal crónica. La hemoglobina normal en hombres se asoció con efecto protector.

Introduction: contrast-induced nephropathy (CIN) is the acute kidney injury following diagnostic or interventional procedures. It is the third cause of hospital-acquired acute renal insufficiency. This research intended to establish the risk factors for developing CIN in hospitalized patients undergoing coronary angiography at Hospital de San José of Bogotá, during a 3-year period. Methods: a retrospective study in a cohort of CIN patients following contrast exposure and prophylactic strategies as hydration, sodium bicarbonate or N-acetylcysteine. Patients seen between 2014 and 2016 were included. CIN is defined as an elevation of serum creatinine of 0.5 mg/dL or by 25% from baseline within the first 72 hours after contrast administration. Of 495 patients, 15 were excluded for prior hemodialysis. Stata 13 was used for clinical data recording and multivariate statistical analysis. Results: in 480 patients CIN incidence was 13.1%; a hemoglobin value greater than 13 gr/dL showed to be a protective factor for CIN in men: 0.29 (p=0.004). The presence of risk factors as baseline creatinine greater than 1.5 mg/dL OR: 2.56 (IC 95: 1.35-4.85; p=0.004), and chronic renal insufficiency and diabetes mellitus OR: 2.73 (IC 95: 1.25 ­ 5.95; p=0,005) increased CIN prevalence. Other factors such as age, cardiac dysfunction, volume of contrast agent used, prior acute myocardial infarction (AMI) and the use of nephronprotective strategies showed no significant association. CIN-related mortality was 9.5% and hemodialysis-related mortality was 2.5%. Conclusions: the incidence of CIN was associated with an increase in mortality; contributing risk factors were baseline creatinine greater than 1.5 mg/dL, pre-existent chronic renal insufficiency and diabetes mellitus. A normal hemoglobin value showed to be a protective factor in men.

Efectos de la iuuminación relativa y la fertiliación sobre el crecimiento de pplántulas de roble andino en vivero / Effects of Light Intensity and Fertilization on the Growth of Andean Oak Seedlings at Nursery

Quercus humboldtii es una especie vegetal nativa de mucha importancia en Colombia por su uso en repoblamiento forestal y restauración de tierras altoandinas degradadas. La especie se encuentra fuertemente amenazada y es necesario establecer programas de propagación de la misma. Sin embargo, poco se conoce sobre sus exigencias nutricionales y lumínicas. El objetivo de este estudio fue determinar los efectos simples y combinados de la iluminación relativa (IR) y la fertilización sobre el crecimiento de plántulas de Q. humboldtii en vivero. Para esto se establecieron en combinación tres condiciones contrastantes de iluminación relativa (alta, media y baja IR) y nueve tratamientos de fertilización: completa (TC), con un elemento faltante (-N, -P, -K, -Ca, -Mg, -S, -B) y un testigo sin fertilización (T0). La condición de IR media presentó el mejor desarrollo de las plántulas. Todos los tratamientos con deficiencias nutricionales produjeron disminuciones en el desarrollo de las plántulas con respecto a TC, excepto en el tratamiento -B. El N fue el elemento más limitante, con rendimientos similares a los del tratamiento testigo. El orden de limitación que produjeron los tratamientos aplicados siguió la secuencia: T0,-N > -Ca, -K, - P > -Mg, -S > TC,-B. No se detectó interacción significativa IR x Fertilización sobre el desarrollo de las plántulas.

Quercus humboldtii is a native plant species of great importance in Colombia for use in reforestation and restoration of degraded Andean highlands. The species is highly threatened and it is necessary to establish programs of propagation and planting. However, little is known about their nutritional and light requirements. The aim of this study was to determine the effects of single and combined relative illumination (IR) and fertilization on the growth of seedlings of Q. humboldtii at nursery. For this purpose three contrasting IR regimes (high, medium, and low IR) and nine fertilization treatments were established: complete (TC), a missing nutrient (-N,-P,-K,-Ca,-Mg, -S,-B) and a control without fertilization (T0). The best development of seedlings was showed in the medium IR condition. All treatments with a lacking nutrient showed decreases in seedling development regarding TC, except in the -B treatment. Nitrogen was the most limiting nutrient yielding biomass similar to that of T0. The impact of nutrient limitation on seedling performance was in the following order:-N>-Ca,-K,-P>-Mg,-S>-B. No significant interaction IR x Fertilization was detected on seedling development.

Identificación oolecular de microorganismos asoiados a la rizosfera de plantass de vainilla en Colomiia / Molecular Identification of Microorganisms Associated to the Rhizosphere of Vanilla Plants in Colombia

El cultivo de vainilla es altamente promisorio en Colombia, pero se requiere mayor conocimiento de su manejo agronómico y de los microorganismos que crecen asociados a su rizosfera, de los cuales depende esta planta para su nutrición y crecimiento. En este trabajo se realizaron aislamientos de microorganismos de la rizosfera de plantas de vainilla en un cultivo piloto ubicado en el municipio de Sopetrán (Antioquia, Colombia). Los microorganismos se aislaron en medios selectivos de crecimiento para evaluar su capacidad para descomponer celulosa, proteínas, solubilizar fosfato inorgánico y orgánico (fitato) y fijar nitrógeno en forma asimbiótica. Una vez aislados y purificados, se obtuvieron un total de 109 aislamientos, de los cuales se seleccionaron 52 morfotipos para su identificación molecular por secuenciación de las regiones ITS y 16S del ADN ribosomal para hongos y bacterias, respectivamente. Se encontró una alta variedad de microorganismos en la rizosfera de plantas de vainilla, destacándose las bacterias Bacillus megaterium, Pseudomonas koreensis y Acinetobacter sp. y el hongo Plectosphaerella sp., por su potencial para ser utilizados como biofertilizantes destinados a mejorar la nutrición y el crecimiento de estas plantas.

The cultivation of vanilla (Vanilla planifolia) is highly promising in Colombia, but more research is needed on its agronomical management and beneficial microorganisms that grow associated to its rhizosphere, on which the plant depends for its nutrition and growth. This study involved the identification of microorganisms associated to the rhizosphere of vanilla plants in a crop located in Sopetrán, Colombia. The microbes were isolated in selective media for functional groups such as cellulolytic, proteolytic, inorganic and organic phosphate (phytate) solubilizers, and asymbiotic nitrogen fixing bacteria. After isolation and purification, 109 microbial isolates were obtained. DNA was extracted from 52 selected isolates for molecular identification based on ITS and 16S rDNA sequencing, for fungi and bacteria, respectively. The diversity of rhizosphere microorganisms found was significant. Bacteria such as Bacillus megaterium, Pseudomonas koreensis and Acinetobacter sp., and the fungus Plectosphaerella sp., may have a high potential to be used as biofertilizers to improve vanilla plant nutrition and growth.