In vitro comparison of acyclovir, ganciclovir and cidofovir against equid alphaherpesvirus 3 and evaluation of their efficacy against six field isolates / Comparación in vitro de aciclovir, ganciclovir y cidofovir contra alfa-herpesvirus equino 3 y evaluación de la eficacia de los mismos frente a 6 aislamientos de campo del virus

Equid alphaherpesvirus 3 (EHV3) is the etiological agent of equine coital exanthema (ECE), which is a venereal, highly contagious disease, characterized by the formation of papules, vesicles, pustules and ulcers on the external genitalia of mares and stallions. EHV3 remains in a latent state after a successful infection and there are latently infected animals in which the virus is reactivated and generally re-excreted subclinically. There are no available vaccines for this condition and prevention is based on the clinical examination of mares prior to mating, which allows to segregate those showing clinical signs. As this approach does not eliminate the risk of contagion in stallions from subclinically infected mares, there is a need for a specific EHV3 treatment. Nowadays, there exist various antiviral compounds of proven effectiveness for other alphaherpesviruses affecting humans and animals. The aim of the present study was to compare the efficacy of three antiviral compounds, acyclovir, ganciclovir and cidofovir against EHV3 in vitro, and to assess their efficacy against six EHV3 Argentinian field isolates. To determine the efficacy of these compounds in vitro, three parameters were analyzed: reduction of plaque number, reduction of plaque size and reduction of viral production. Additionally, the effectiveness of the three compounds at an optimum concentration previously determined in this study was investigated for the EHV3 field isolates. Based on our results, ganciclovir was the most potent antiviral compound to reduce EHV3 replication in vitro and may thus be a valuable candidate for treatment and prevention of ECE in mares and stallions.

El alfa-herpesvirus equino 3 (EHV3) es el agente etiológico del exantema coital equino (ECE), enfermedad venérea, altamente contagiosa y caracterizada por la aparición de pápulas, vesículas, pústulas y úlceras en los genitales externos de yeguas y padrillos. Luego de la primo-infección, el EHV3 se mantiene en el animal en un estado de latencia a partir del cual puede reactivar y excretarse, generalmente de manera subclínica. No existen vacunas, por lo que la prevención se basa en la detección de las lesiones clínicas previo al servicio, y la segregación de estos animales. Sin embargo, este abordaje no previene la infección del padrillo por parte de yeguas que excretan el virus de manera subclínica, y por lo tanto existe la necesidad de un tratamiento específico contra el EHV3. En la actualidad, existen varios compuestos antivirales de probada eficacia contra herpesvirus humanos y veterinarios. El objetivo de este trabajo es comparar la eficacia de 3 compuestos antivirales, aciclovir, ganciclovir y cidofovir, contra EHV3 in vitro, y evaluar la eficacia de los mismos contra 6 cepas de campo argentinas de EHV3. Para determinar la eficacia de los compuestos in vitro se evaluaron 3 parámetros: reducción del número de placas de lisis, reducción del tamaño de placas de lisis y reducción de la producción de virus. Adicionalmente, la efectividad de los compuestos en una concentración óptima, previamente determinada en este estudio, fue determinada para 6 cepas de campo argentinas de EHV3. De acuerdo con los resultados obtenidos, ganciclovir fue el compuesto más potente en reducir la replicación del EHV3 in vitro, y por lo tanto podría considerarse un potencial candidato para el tratamiento y la prevención del ECE en yeguas y padrillos.

Cultural adaptation, content validity and inter-rater reliability of the "STAR Skin Tear Classification System" / Adaptação cultural, validade de conteúdo e confiabilidade interobservadores do "STAR Skin Tear Classification System" / Adaptación cultural, validez de contenido y confiabilidad interobservadores del "STAR Skin Tear Classification System"

AIMS: to perform the cultural adaptation of the STAR Skin Tear Classification System into the Portuguese language and to test the content validity and inter-rater reliability of the adapted version. METHODS: methodological study with a quantitative approach. The cultural adaptation was developed in three phases: translation, evaluation by a committee of judges and back-translation. The instrument was tested regarding content validity and inter-rater reliability. RESULTS: the adapted version obtained a regular level of concordance when it was applied by nurses using photographs of friction injuries. Regarding its application in clinical practice, the adapted version obtained a moderate and statistically significant level of concordance. CONCLUSION: the study tested the content validity and inter-rater reliability of the version adapted into the Portuguese language. Its inclusion in clinical practice will enable the correct identification of this type of injury, as well as the implementation of protocols for the prevention and treatment of friction injuries. .

OBJETIVOS: realizar a adaptação cultural do STAR Skin Tear Classification System, para a língua portuguesa e testar a validade de conteúdo e a confiabilidade interobservadores da versão adaptada. MÉTODOS: estudo metodológico com abordagem quantitativa. A adaptação cultural foi desenvolvida em três fases: tradução, avaliação por comitê de juízes e retrotradução. O instrumento foi testado quanto à validade de conteúdo e confiabilidade interobservadores. RESULTADOS: a versão adaptada obteve um nível regular de concordância quando aplicada por enfermeiros em fotografias de lesões por fricção. Quando aplicada na prática clínica, a versão adaptada obteve nível moderado e estatisticamente significativo de concordância. CONCLUSÃO: o estudo atestou a validade de conteúdo e a confiabilidade interobservadores da versão adaptada para a língua portuguesa. Sua inclusão na prática clínica possibilitará a correta identificação desse tipo de lesão, além da implementação de protocolos para a prevenção e tratamento das lesões por fricção. .

OBJETIVOS: realizar la adaptación cultural del STAR Skin Tear Classification System, para el idioma portugués y comprobar la validez de contenido y la confiabilidad interobservadores de la versión adaptada. MÉTODOS: estudio metodológico con abordaje cuantitativo. La adaptación cultural fue desarrollada en tres fases: traducción, evaluación por comité de jueces y retrotraducción. El instrumento fue comprobado en lo que se refiere a su validez de contenido y confiabilidad interobservadores. RESULTADOS: la versión adaptada obtuvo un nivel regular de concordancia cuando fue aplicada por enfermeros utilizando fotografías de lesiones por fricción. Cuando fue aplicado en la práctica clínica, la versión adaptada obtuvo un nivel moderado y estadísticamente significativo de concordancia. CONCLUSIÓN: el estudio comprobó la validez de contenido y la confiabilidad interobservadores de la versión adaptada para el idioma portugués. Su inclusión en la práctica clínica posibilitará la correcta identificación de ese tipo de lesión, además de la implementación de protocolos para la prevención y tratamiento de las lesiones por fricción. .

Búsqueda de mutaciones en el gen UL 97 asociadas a resistencia a ganciclovir en citomegalovirus obtenidos desde muestras biológicas de pacientes chilenos / Presence of mutations associated with ganciclovir resistance in cytomegalovirus UL 97 gene

Background: Long term use of ganciclovir (GCV) is associated with acquired resistance to it. Ninety percent of the responsible mutations occur in cytomegalovirus (CMV) UL 97 gene. Aim: To search for these mutations, comparing nucleotide sequences of CMV-positive samples from post transplant and immunocompromised patients receiving GCV, with sequences of CMV isolates obtained from subjects not exposed to the drug. Patients and Methods: Codons 440 to 465 of gene UL 97, in-cluding the most common mutations causing resistance to GCV, were amplifed in 33 plasma samples from patients exposed to GCV and in 15 urine samples of newborns. Both populations and their nucleotide sequences were compared with the prototype strain CMV AD169. Results: Samples of exposed patients had multiple mutations but only one had a mutation associated with clinical resistance (M460I). Eight subjects had the D605E mutation, whose role in resistance is controversial. The remaining 150 mutations were silent mutations. Conclusions: A low frequency of mutations associated with CMV resistance to GCV was found in these exposed and unexposed samples. These mutations may refect coexistence of multiple genetic variants of CMV. The absence of clinical expression of resistance, even with these mutations, can be explained by the use of GCV for a shorter lapse than that associated with the appearance of resistance.

Terapia con valganciclovir en coriorretinitis congénita por citomegalovirus: reporte de un caso / Valganciclovir therapy Chorioretinitis in congenital cytomegalovirus: a purpose of a case

La infección por citomegalovirus solamente es sintomática en un 2 por ciento de los recién nacidos vivos. Sin embargo, la expresión clínica es generalmente desbastante en el neonato, ocasionando procesos de Síndrome de Respuesta Inflamatoria Sistémica hasta generar daños irreversibles como ceguera por coriorretinitis y retardo psicomotor. El tratamiento de los casos sintomáticos se realiza a través de un inhibidor de la replicación viral como lo es el ganciclovir; cuya vía de administración es exclusivamente endovenosa, teniendo que hospitalizar al paciente por espacio de un intervalo mínimo de 21 días; ocasionando costos de hospitalización, riesgos de sobreinfección por agentes nosocomiales y separación temporal de la madre. Se presenta el siguiente caso de una lactante con coriorretinitis congénita, ocasionado por citomegalovirus, por comprobación de Reacción de Cadena de Polimerasa. Se inicia tratamiento ambulatorio con valganciclovir a una dosis de 30mg/kg/día. A los 03 meses del tratamiento, se realiza control de la actividad del citomegalovirus por Reacción de la Cadena de Polimerasa, la cual reporta negativa. Entre los efectos adversos se apreció un incremento leve de las transaminasas, las cuales se mantuvieron en dichos niveles a lo largo del tratamiento. No se observaron citopenias con el tratamiento ni otros efectos de importancia. El valganciclovir, una prodroga del ganciclovir, puede ofrecer una alternativa viable para el manejo de este tipo de pacientes, restando gastos de hospitalización y otras complicaciones derivadas a la vía endovenosa, pero con igual efecto terapéutico.

Imaging of Viral Thymidine Kinase Gene Expression by Replicating Oncolytic Adenovirus and Prediction of Therapeutic Efficacy

PURPOSE: We have used a genetically attenuated adenoviral vector which expresses HSVtk to assess the possible additive role of suicidal gene therapy for enhanced oncolytic effect of the virus. Expression of TK was measured using a radiotracer-based molecular counting and imaging system. MATERIALS AND METHODS: Replication-competent recombinant adenoviral vector (Ad-deltaE1B19/55) was used in this study, whereas replication-incompetent adenovirus (Ad-deltaE1A) was generated as a control. Both Ad-deltaE1B19/55-TK and Ad-deltaE1A-TK comprise the HSVtk gene inserted into the E3 region of the viruses. YCC-2 cells were infected with the viruses and incubated with 2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl-5-iodouracil (I-131 FIAU) to measure amount of radioactivity. The cytotoxicity of the viruses was determined, and gamma ray imaging of HSVtk gene was performed. MTT assay was also performed after GCV treatment. RESULTS: On gamma counter-analyses, counts/minute (cpm)/microgram of protein showed MOIs dependency with deltaE1B19/55-TK infection. On MTT assay, Ad-deltaE1B19/55-TK led to more efficient cell killing than Ad-deltaE1A-TK. On plate imaging by gamma camera, both Ad-deltaE1B19/55-TK and Ad-deltaE1A-TK infected cells showed increased I-131 FIAU uptake in a MOI dependent pattern, and with GCV treatment, cell viability of deltaE1B19/55-TK infection was remarkably reduced compared to that of Ad-deltaE1A-TK infection. CONCLUSION: Replicating Ad-deltaE1B19/55-TK showed more efficient TK expression even in the presence of higher-cancer cell killing effects compared to non-replicating Ad-deltaE1A-TK. Therefore, GCV treatment still possessed an additive role to oncolytic effect of Ad-deltaE1B19/55-TK. The expression of TK by oncolytic viruses could rapidly be screened using a radiotracer-based counting and imaging technique.

Atrophy of brown adipocytes in the adult mouse causes transformation into white adipocyte-like cells

Adipose tissue is an important endocrine regulator of glucose metabolism and energy homeostasis. Researches have focused on this tissue not only as a target for pharmacotherapy of obesity and insulin resistance but also as an endocrine tissue with leptin secretion and high insulin sensitivity. Brown adipose tissue (BAT) additionally plays a unique role in thermoregulation through the mitochondrial uncoupling protein 1 (UCP1), which uncouples oxidative phosphorylation. As a genetic tissue ablation model of BAT, we made transgenic mice expressing herpes simplex virus thymidine kinase (HSV-TK) driven by the brown adipocyte- specific UCP1 minimal regulatory element. The HSV-TK transgene was expressed specifically in BAT and more than 35% increase of apoptosis was induced by ganciclovir (GCV) treatment. Nevertheless, the expression level was not high enough to induce BAT ablation in GCV-treated adult mice. Importantly, however, we found that brown adipocytes in the periphery of interscapular BAT were transformed into white adipocyte-like unilocular cells. These cells express white adipocyte-specific leptin protein but are different in the ultrastructure of mitochondria from classical white adipocytes. Our data indicates that atrophy of BAT causes transformation into white adipocyte-like cells in the adult mouse and also suggests that further molecular understanding of adipocyte plasticity using our transgenic mouse model might be beneficial for the development of anti-obesity/anti-diabetic therapies.

Antiviral susceptibility of cytomegakovirus isolated

Two wild-types clinical cytomegalovirus [CMV] isolates were plaque purified and the individual plaque variants were then randomly picked and passed 3-5 times in human foreskin fibroblast [MRHF] to increase viral titer and test for susceptibility to the antiviral ganciclovir [DHPG]. Different susceptibility pattern to DHPG has been identified among the individual plaques tested with an inhibitory concentration 50 [ID50] ranged from 0.08-14.2 muM. Also, a fully sensitive [DHPG ID50 <5 muM], intermediate resistant [DHPG ID50 between 6-10 muM] and resistant [DHPG ID50 >10 muM] plaque purified viral isolates were further tested for susceptibility to other antiviral agents [foscarnet and HPMPC]. All the tested individual strains [DHPG sensitive and resistant] remained susceptible to foscarnet and HPMPC. It may be concluded that within any resistant population of CMV to DHPG, there was a marked heterogenicity with some subsets of the population remaining susceptible to the drug and others exhibiting considerable level of resistance. DHPC resistant CMV isolates remained susceptible to the other antiviral drugs [foscarnet and HPMPC meaning that therapy with these two antiviral agents would still be available

Terapia genica antitumoral mediante el uso de genes suicidas / Cancer gene therapy using suicide genes

Células tumorales transducidas con vectores retrovirales portanto el gen de la timidina kinasa del virus herpes simplex-1 (HSV-tk), son capaces de transformar la droga antiherpética ganciclovir (GCV) en un metabolito tóxico para células en división. Esta terapia suicida aumenta su eficiencia debido a un efecto "bystander" que induce la muerte de células no transducidas, vecinas a células modificadas. El mecanismo del mencionado efecto no se conoce totalmente, pero existe evidencia que asigna un rol preponderante al sistema inmune, para lograr una completa erradicación tumoral. En este trabajo estudiamos la efectividad del sistema en tres líneas celulares: un melanoma humano y uno murino, y un glioma de rata. Los tumores fueron estabelecidos por inyección de células tumorales s.c. en ratones nude y C57BI/6, e intracerebralmente por esterotaxis en ratas Sprague Dawley, respectivamente. Animales tratados fueron co-inyectados con células productoras de retrovirus expresando HSV-tk y posterior administración i.p. de GCV. En experimentos in vivo a corto plazo, se observó inhibición total o parcial del crecimiento tumoral en todos los modelos. En experimentos de supervivencia a largo plazo con células C6, el 50 por ciento de los animales sobrevivió más de 75 dias (p < 0 0001) y fue capaz de rechazar un desafio con células parentales C6 inyectadas en el hemisferio contralateral. El análisis histológico e inmunohistoquímico mostró la presencia de un infiltrado inflamatorio compuesto por linfocitos T, macrófagos y polimorfonucleares. Estos resultados demuestran que el uso de genes suicidas puede ser una herramienta de enorme importancia en el tratamiento de tumores de cerebro y de metástasis cerebrales.