Capsid-deficient alphaviruses generate propagative infectious microvesicles at the plasma membrane.

Alphavirus budding is driven by interactions between nucleocapsids assembled in the cytoplasm and envelope proteins present at the plasma membrane. So far, the expression of capsid and envelope proteins in infected cells has been considered an absolute requirement for alphavirus budding and propagation. In the present study, we show that Semliki Forest virus and Sindbis virus lacking the capsid gene can propagate in mammalian and insect cells. This propagation is mediated by the release of infectious microvesicles (iMVs), which are pleomorphic and have a larger size and density than wild-type virus. iMVs, which contain viral RNA inside and viral envelope proteins on their surface, are released at the plasma membrane and infect cells using the endocytic pathway in a similar way to wild-type virus. iMVs are not pathogenic in immunocompetent mice when injected intravenously, but can infect different organs like lungs and heart. Finally, we also show that alphavirus genomes without capsid can mediate the propagation of heterologous genes, making these vectors potentially interesting for gene therapy or vaccination studies. The minimalist infectious system described in this study shows that a self-replicating RNA able to express membrane proteins with binding and fusion properties is able to propagate, providing some insights into virus evolution.

A quick and efficient method to generate mammalian stable cell lines based on a novel inducible alphavirus DNA/RNA layered system.

We report a new method to generate high-expressing mammalian cell lines in a quick and efficient way. For that purpose, we developed a master cell line (MCL) containing an inducible alphavirus vector expressing GFP integrated into the genome. In the MCL, recombinant RNA levels increased >4,600-fold after induction, due to a doxycycline-dependent RNA amplification loop. The MCL maintained inducibility and expression during 50 passages, being more efficient for protein expression than a conventional cell line. To generate new cell lines, mutant LoxP sites were inserted into the MCL, allowing transgene and selection gene exchange by Cre-directed recombination, leading to quick generation of inducible cell lines expressing proteins of therapeutic interest, like human cardiotrophin-1 and oncostatin-M at several mg/l/24 h. These proteins contained posttranslational modifications, showed bioactivity, and were efficiently purified. Remarkably, this system allowed production of toxic proteins, like oncostatin-M, since cells able to express it could be grown to the desired amount before induction. These cell lines were easily adapted to growth in suspension, making this methodology very attractive for therapeutic protein production.

[Herpes simplex virus type 1 latency and reactivation: an update]. / Latence et réactivation du virus de l'herpès simplex de type 1 (HSV-1) - Une mise à jour.

Following primary infections HSV-1 replicates productively in epithelial cells and enters sensory neurons via nerve termini. After retrograde transport the virus genome is delivered into the cell nucleus, where it establishes lifelong latent infections. During latency, the virus genome remains as a chromatinized episome expressing only a set of latency-associated transcripts (LAT) and a group of microRNAs that inhibit expression of key lytic viral functions. Periodically the virus can reactivate to reinitiate lytic, secondary infections at peripheral tissues. The ability to establish both lytic and latent infections relies on the coexistence in the virus genome of two alternative gene expression programs, under the control of epigenetic mechanisms. Latency is an adaptive phenotype that allows the virus to escape immune host responses and to reactivate and disseminate to other hosts upon recognizing danger signals such as stress, neurologic trauma or growth factor deprivation.

In Vivo Efficacy of an Adhesive Bioresorbable Patch to Treat Aortic Dissections.

Endovascular repair of aortic dissection still presents significant limitations. Preserving the mechanical and biological properties set by the aortic microstructure is critical to the success of implantable grafts. In this paper, we present the performance of an adhesive bioresorbable patch designed to cover the entry tear of aortic dissections. We demonstrate the power of using a biomimetic scaffold in a vascular environment.

Multiresistant Candida Endophthalmitis Treated with Intravitreal Caspofungin: A Case Report.

BACKGROUND: Candida endophthalmitis is a severe complication of candidemia. Currently, the recommended treatment of fungal endophthalmitis is a combination of intravitreal and systemic antifungal drugs, and in some cases vitrectomy is also required. Intravitreal therapies that are commonly used are amphotericin B and voriconazole, although recently the use of intravitreal caspofungin has been described in a few case reports. However, clinical experience with intravitreal caspofungin is still limited. CASE PRESENTATION: We report a case of bilateral candida tropicalis endophthalmitis, initially managed with repeated 100 µg/0.1 ml caspofungin intravitreal injections and posteriorly treated with pars plana vitrectomy in both eyes. CONCLUSIONS: Intravitreal caspofungin could be a safe intravitreal alternative to habitual antimycotic drugs in cases with resistant candida endophthalmitis.Abbreviations: Intensive Care Unit (ICU); Best-Corrected Visual Acuity (BCVA).

Comparative analysis of the pregnancy rate via in vitro fertilization vs. previous artificial insemination in patients with unexplained infertility.

OBJECTIVE: To compare the clinical pregnancy rate among patients undergoing direct in vitro fertilization vs. in vitro fertilization after two cycles of intrauterine insemination in couples with unexplained infertility. METHODS: Comparative cross-sectional, retrospective study from 2016 to 2019, from the Centro Mexicano de Fertilidad Doctor Alberto Kably. The patients with unexplained infertility were divided into two groups, direct in vitro fertilization and a group of in vitro fertilization after intrauterine insemination, and we compared the rate of pregnancy and live births in both cases. RESULTS: 89 couples with unexplained infertility were included, the in vitro fertilization after intrauterine insemination group (n=46) and direct in vitro fertilization group (n=43). The direct in vitro fertilization group resulted in a higher clinical pregnancy rate throughout the study compared to the other group (55.8% vs. 34.8%, OR 2.37; 95% CI 1.008 - 5.57, p=0.046). However, there was no difference in the rate of live newborns (p=0.12). When analyzing the data by cycle, we noticed a statistical difference in both, the clinical pregnancy rate in the direct in vitro fertilization group (38.7% vs. 16.7%, OR 3.2; 95% CI 1.50-6.62), as well as the rate of live newborns (32.3 % vs. 14.6%, OR 2.79; 95% CI 1.28-6.07, p=0.008). CONCLUSIONS: In the in vitro fertilization group, as first-line treatment for unexplained infertility, the patients had a higher pregnancy rate.

Idiotype vaccines produced with a non-cytopathic alphavirus self-amplifying RNA vector induce antitumor responses in a murine model of B-cell lymphoma.

A promising therapy for patients with B-cell lymphoma is based on vaccination with idiotype monoclonal antibodies (mAbs). Since idiotypes are different in each tumor, a personalized vaccine has to be produced for each patient. Expression of immunoglobulins with appropriate post-translational modifications for human use often requires the use of stable mammalian cells that can be scaled-up to reach the desired level of production. We have used a noncytopathic self-amplifying RNA vector derived from Semliki Forest virus (ncSFV) to generate BHK cell lines expressing murine follicular lymphoma-derived idiotype A20 mAb. ncSFV/BHK cell lines expressed approximately 2 mg/L/24 h of A20 mAb with proper quaternary structure and a glycosylation pattern similar to that of A20 mAb produced by hybridoma cells. A20 mAb purified from the supernatant of a ncSFV cell line, or from the hybridoma, was conjugated to keyhole limpet hemocyanin and used to immunize Balb/c mice by administration of four weekly doses of 25 µg of mAb. Both idiotype mAbs were able to induce a similar antitumor protection and longer survival compared to non-immunized mice. These results indicate that the ncSFV RNA vector could represent a quick and efficient system to produce patient-specific idiotypes with potential application as lymphoma vaccines.

The cytotoxic effect of unconjugated bilirubin in human neuroblastoma SH-SY5Y cells is modulated by the expression level of MRP1 but not MDR1.

In vitro and in vivo studies have demonstrated that UCB (unconjugated bilirubin) is neurotoxic. Although previous studies suggested that both MRP1 (multidrug resistance-associated protein 1) and MDR1 (multidrug resistance protein 1) may protect cells against accumulation of UCB, direct comparison of their role in UCB transport was never performed. To this end, we used an inducible siRNA (small interfering RNA) expression system to silence the expression of MRP1 and MDR1 in human neuroblastoma SH-SY5Y cells. The effects of in vitro exposure to clinically-relevant levels of unbound UCB were compared between unsilenced (control) cells and cells with similar reductions in the expression of MRP1 or MDR1, documented by RT-PCR (reverse transcription-PCR) (mRNA), immunoblotting (protein), and for MDR1, the enhanced net uptake of a specific fluorescent substrate. Cytotoxicity was assessed by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] test. MRP1-deficient cells accumulated significantly more UCB and suffered greater cytotoxicity than controls. By contrast, MDR1-deficient cells exhibited UCB uptake and cytotoxicity comparable with controls. At intermediate levels of silencing, the increased susceptibility to UCB toxicity closely correlated with the decrease in the expression of MRP1, but not of MDR1. These data support the concept that limitation of cellular UCB accumulation, due to UCB export mediated by MRP1, but not MDR1, plays an important role in preventing bilirubin encephalopathy in the newborn.

Percepción de cambios en el ciclo menstrual posteriores a la vacunación contra SARS-CoV-2 / Perception of menstrual cycle changes following SARS-CoV-2 vaccination

Resumen OBJETIVO: Identificar si la vacunación contra la COVID-19 se asocia con cambios en el patrón menstrual. MATERIALES Y MÉTODOS: Estudio observacional y transversal anónimo efectuado entre el 4 y 29 de octubre del 2021 en un grupo de mujeres que completaron un cuestionario elaborado para saber si hubo cambios en el ciclo menstrual posteriores a la vacunación. La encuesta se aplicó durante un periodo de cuatro semanas. Para el análisis estadístico se utilizó el programa SPSS. Los cambios antes y después de la aplicación de la primera y segunda dosis de la vacuna contra COVID-19 se evaluaron con una prueba t de muestras pareadas y χ2. RESULTADOS: Se reunieron 4501 pacientes de las que se excluyeron 1815 por no cumplir con los criterios de inclusión; al final quedaron para el análisis 2686. De éstas el 37.9% (n = 1018) tuvieron cambios en el ciclo menstrual posteriores a la vacunación contra COVID-19; en 61.8% (n = 630) fueron en la cantidad del sangrado. De las mujeres que reportaron cambios en el ciclo menstrual, el 64.34% (n = 655) fueron posteriores a la aplicación de vacunas tipo ARN mensajero (35.06%; p = 0.19, IC95%: -0.35-0.19). En relación con la cantidad de dosis aplicadas 72.10% (n = 734) reportaron modificaciones en el ciclo menstrual después de la segunda vacuna (p = 0.01, IC95%: 0.58-0.98). CONCLUSIONES: La vacunación contra COVID-19 se asocia con pequeños cambios en el ciclo menstrual, sin significación estadística. Las mujeres que reciben dos dosis de la vacuna tuvieron cambios en la cantidad del sangrado.

Abstract OBJECTIVE: Identify whether vaccination against COVID-19 is associated with changes in menstrual pattern. MATERIALS AND METHODS: An anonymous observational, cross-sectional to be held from October 4 to 29, 2021 was conducted in a group of women who completed a questionnaire designed to inquire about changes in the menstrual cycle following vaccination. The survey was administered over a four-week period. SPSS software was used for statistical analysis. Changes before and after the application of the first and second doses of COVID-19 vaccine were evaluated with a paired samples t-test and 2. RESULTS: 4501 patients were collected of whom 1815 were excluded because they did not meet the inclusion criteria; in the end 2686 remained for analysis. Of these, 37.9% (n = 1018) had changes in the menstrual cycle following COVID-19 vaccination; in 61.8% (n = 630) it was in the amount of bleeding. Of the women who reported menstrual cycle changes, 64.34% (n = 655) were after application of messenger RNA vaccines (35.06%; p = 0.19, 95%CI: -0.35-0.19). In relation to the number of doses applied 72.10% (n = 734) reported modifications in the menstrual cycle after l second vaccine (p = 0.01, CI95%: 0.58-0.98). CONCLUSIONS: Vaccination against COVID-19 is associated with small changes in the menstrual cycle, without statistical significance. Women receiving two doses of vaccine had changes in the amount of bleeding.

Functional characterization and subcellular localization of the three malate dehydrogenase isozymes in Leishmania spp.

As part of a study on the malate dehydrogenase isozymes (MDHs) from Trypanosomatids, three different fractions with MDH activity were obtained from crude extracts of Leishmania mexicana promastigotes combining two different chromatographic steps. Gel filtration chromatography in native conditions showed that most of the MDH activity present in the crude extracts eluted in a single peak, which corresponded to a lower apparent molecular mass ( congruent with 57kDa) than the value expected for typical MDHs. To further characterize the leishmanial isozymes, three putative MDH genes, presumably corresponding to the mitochondrial, glycosomal and cytosolic isoforms were amplified by PCR, cloned into bacterial expression vectors, and the recombinant enzymes purified. Digitonin extraction of intact L. mexicana promastigotes and immunofluorescence microscopy of L. major promastigotes confirmed the subcellular compartmentation of each of the three isozymes. Western blot analysis showed that the three MDHs are developmentally regulated. At the protein level, these isozymes are remarkably more abundant in amastigotes than in promastigotes of L. mexicana. Altogether our results demonstrate the presence of three MDH isoforms with slightly distinct biochemical properties and different subcellular localization in Leishmania spp. Presumably the functional and biochemical features of these isozymes reflect the metabolic adaptation to the different nutrient sources these parasites have to face along their life cycle. These results also emphasize the differences among Trypanosomatids in this area of metabolism, since in the case of Trypanosoma brucei the cMDH is the only isoform expressed in bloodstream trypomastigotes, whereas in Trypanosoma cruzi cMDH is absent.

The malate dehydrogenase isoforms from Trypanosoma brucei: subcellular localization and differential expression in bloodstream and procyclic forms.

Trypanosoma brucei procyclic forms possess three different malate dehydrogenase isozymes that could be separated by hydrophobic interaction chromatography and were recognized as the mitochondrial, glycosomal and cytosolic malate dehydrogenase isozymes. The latter is the only malate dehydrogenase expressed in the bloodstream forms, thus confirming that the expression of malate dehydrogenase isozymes is regulated during the T. brucei life cycle. To achieve further biochemical characterization, the genes encoding mitochondrial and glycosomal malate dehydrogenase were cloned on the basis of previously reported nucleotide sequences and the recombinant enzymes were functionally expressed in Escherichia coli cultures. Mitochondrial malate dehydrogenase showed to be more active than glycosomal malate dehydrogenase in the reduction of oxaloacetate; nearly 80% of the total activity in procyclic crude extracts corresponds to the former isozyme which also catalyzes, although less efficiently, the reduction of p-hydroxyphenyl-pyruvate. The rabbit antisera raised against each of the recombinant isozymes showed that the three malate dehydrogenases do not cross-react immunologically. Immunofluorescence experiments using these antisera confirmed the glycosomal and mitochondrial localization of glycosomal and mitochondrial malate dehydrogenase, as well as a cytosolic localization for the third malate dehydrogenase isozyme. These results clearly distinguish Trypanosoma brucei from Trypanosoma cruzi, since in the latter parasite a cytosolic malate dehydrogenase is not present and mitochondrial malate dehydrogenase specifically reduces oxaloacetate.

Técnica de doble disparo (doubble trigger) en pacientes normorrespondedoras con bajo porcentaje de ovocitos maduros / Doubble Trigger in patients with a normal response with a low percentage of mature oocytes

Resumen OBJETIVO: Evaluar el efecto del doble disparo en pacientes con un ciclo previo con menos de 65% de ovocitos maduros respecto de los ovocitos capturados, en una población con respuesta normal a la inducción de la ovulación con hCG recombinante o urinaria. MATERIALES Y MÉTODOS: Estudio de cohorte, prospectivo, efectuado en pacientes con diagnóstico de infertilidad, en tratamiento con fertilización in vitro, evaluadas en el Centro Mexicano de Fertilidad (Hospital Ángeles Lomas) entre 2017 y 2019. El tratamiento se llevó a cabo en la misma paciente, en cuyo ciclo previo convencional con esquema de antagonista e inducción de la ovulación con hCG tuvo respuesta ovárica subóptima y captura ovocitaria con menos de 65% en fase M2 (Grupo 1). Posteriormente se les indicó un segundo ciclo con el mismo esquema de gonadotropinas e inducción de la ovulación con doble disparo: acetato de triptorelina 1 mg + 5000 UI de hCG urinaria 40 y 34 horas previas a la captura (Grupo 2). Se evaluaron el porcentaje y la cantidad de ovocitos capturados en fase M2. RESULTADOS: Se registraron 34 pacientes en quienes se llevaron a cabo 68 ciclos. La cantidad de ovocitos capturados fue mayor en el grupo 2 (agonista de GnRH + hCG urinaria; p = 0.03). El doble disparo aumentó el porcentaje de ovocitos maduros (65.4 ± 21.3 vs 74.6 ± 20.2; p = 0.07). CONCLUSIONES: La técnica de doble disparo es valiosa para el tratamiento de pacientes con captura de ovocitos deficiente, aun con desarrollo folicular normal y concentraciones de estradiol adecuadas y óptimas de hCG el día de la captura. Se requieren estudios prospectivos de gran tamaño para dilucidar la recomendación mencionada de la técnica de "doble disparo".

Abstract OBJECTIVE: To evaluate the effect of double trigger in patients with a previous cycle with less than 65% of mature oocytes compared to the captured oocytes, in a normorresponding population with induction of ovulation with recombinant or urinary hCG. MATERIALS AND METHODS: A prospective cohort study, conducted in patients diagnosed with infertility, treated with in vitro fertilization, evaluated at the Mexican Fertility Center (Hospital Angeles Lomas) between 2017 and 2019. The treatment was carried out in the same patient, in whose previous conventional cycle with antagonist scheme and induction of ovulation with hCG had suboptimal ovarian response and oocyte capture with less than 65% in M2 phase (Group 1). Subsequently, a second cycle was performed with the same scheme of gonadotropins and induction of ovulation with double shot: 1 mg triptorelin acetate + 5000 IU of urinary hCG 40 and 34 hours prior to capture (Group 2 or double trigger). Percentage and quantity of oocytes captured in M2 phase were evaluated. RESULTS: 34 patients were registered, in whom 68 cycles were performed. The number of oocytes captured was greater in group 2 (agonist of GnRH + urinary hCG; p = 0.03). The double shot increased the percentage of mature oocytes 65.4 ± 21.3 vs 74.6 ± 20.2 (p = 0.07). CONCLUSIONS: The double trigger technique is valuable for the treatment of patients with poor oocyte capture, even with normal follicular development and adequate and optimal hCG estradiol concentrations on the day of capture. Large prospective studies are required to elucidate the aforementioned recommendation of the "double shot" technique.

A simple and efficient method for the production of human glycosylated glial cell line-derived neurotrophic factor using a Semliki Forest virus expression system.

Human glial cell line-derived neurotrophic factor (hGDNF) is a very promising protein for the treatment of Parkinson's disease and other neurodegenerative disorders. The present work describes a quick and simple method to obtain a high amount of purified hGDNF using a mammalian cell-derived system. The method is based on the high expression level provided by a Semliki Forest virus vector and its ability to induce a strong shut-off of host-cell protein synthesis in mammalian cells. As a result, hGDNF is the only protein present in the supernatant and can be efficiently purified by a single chromatographic step. Using this system it was possible to eliminate other secreted proteins from the culture medium, like insulin-like growth factor-5, which are hard to remove using other hGDNF production methods. Purified hGDNF presents a complex glycosylation pattern typical of mammalian expression systems and is biologically active. This protocol could be extended to other secreted proteins and could be easily scaled up for industrial purposes.

Valor de la cuenta folicular antral como predictor de éxito en ciclos de fertilización in vitro / Value of the antral follicular count as a predictor of success in in vitro fertilization cycles

Resumen ANTECEDENTES: La asociación entre pruebas de reserva ovárica y respuesta a la estimulación está debidamente establecida aunque su capacidad para predecir embarazo clínico y recién nacido vivo es limitada. OBJETIVO: Evaluar la utilidad clínica de la cuenta folicular antral para predecir embarazo clínico y recién nacido vivo. MATERIALES Y MÉTODOS: Estudio de cohorte, retrospectivo, efectuado en el Instituto Nacional de Perinatología, entre 2011 y 2016 en ciclos de fertilización in vitro en fresco. Se incluyeron pacientes con diagnóstico de infertilidad a quienes se efectuó, in vitro, transferencia de embriones en fresco. Variables de estudio: edad, cuenta folicular antral, concentración basal de FSH y cantidad de ovocitos capturados. Se elaboró un modelo de regresión logística. Para el análisis estadístico se utilizó el programa Statistic Package for Social Sciences (SPSS). Se consideró significativa la probabilidad de error alfa menor de 5%. RESULTADOS: Se analizaron 923 ciclos de fertilización in vitro. La cuenta folicular antral tiene predicción para detectar embarazo clínico con un área bajo la curva ROC de 0.59 y para recién nacido vivo de 0.57. El número óptimo con mayor porcentaje de embarazo clínico (9%) y recién nacido vivo (10.4%) tuvo cuenta folicular antral ≥ 8. CONCLUSIONES: Cuando la cuenta folicular antral es más o menos mayor de 8 folículos se espera mayor cantidad de embarazos clínicos y de recién nacidos.

Abstract BACKGROUND: The association between ovarian reserve test and ovarian response is well established, however, its ability to predict clinical pregnancy and the live birth is limited. OBJETIVE: Evaluate the clinical usefulness of the antral follicle count (AFC) to predict clinical pregnancy and live newborn. MATERIALS AND METHODS: Retrospective cohort study was made. In fresh IVF cycles, performed at INPer between 2011-2016. Including patients diagnosed with infertility, who underwent in vitro fertilization with fresh embryo transfer. The study variables were age, antral follicle count, basal FSH concentration and number of oocytes captured. A binary logistic regression model was performed. Statistical Package for Social Sciences (SPSS) was used for the statistical analysis. The probability of error alpha <5% was considered significant. RESULTS: A total of 923 in vitro fertilization cycles were included. The antral follicle count has a prediction for clinical pregnancy (ABC 0.59) and live birth (ABC 0.57). The optimal cut-off value with the highest percentage of clinical pregnancy (9%) and live birth (10.4%) was presented with a CFA ≥ 8. A higher pregnancy rate is reported when there is a follicular count above ≥8 follicles. CONCLUSIONS: It is expected the highest number of clinical pregnancy and live birth when the antral follicle count is for ≥8 follicles.

Recent patents on alphavirus protein expression and vector production.

Alphaviruses contain a single-strand RNA genome that can be modified to express heterologous genes at high levels. Alphavirus vectors can be packaged within viral particles (VPs) or used as DNA/RNA layered systems. The broad tropism and high expression levels of alphavirus vectors have made them very attractive for applications like recombinant protein expression, vaccination or gene therapy. Expression mediated by alphavirus vectors is generally transient due to induction of apoptosis. However, during the last years several non-cytopathic mutations have been identified within the replicase sequence of different alphaviruses, allowing prolonged protein expression in culture cells. Some of these mutants, which have been patented, have allowed the generation of stable cell lines able to express recombinant proteins for extended periods of time in a constitutive or inducible manner. Production of alphavirus VPs usually requires cotransfection of cells with vector and helper RNAs providing viral structural proteins in trans. During this process full-length wild type (wt) genomes can be generated through recombination between different RNAs. Several new strategies to reduce wt virus generation during packaging, optimize VP production, increase packaging capacity, and provide VPs with specific targeting have been recently patented. Finally, hybrid vectors between alphavirus and other types of viruses have led to a number of patents with applications in vaccination, cancer therapy or retrovirus production.

Alphavirus vectors for cancer therapy.

Alphaviruses contain a single strand RNA genome that can be easily modified to express heterologous genes at very high levels in a broad variety of cells, including tumor cells. Alphavirus vectors can be used as viral particles containing a packaged vector RNA, or directly as nucleic acids in the form of RNA or DNA. In the latter case alphavirus RNA is cloned within a DNA vector downstream of a eukaryotic promoter. Expression mediated by these vectors is generally transient due to the induction of apoptosis. The high expression levels, induction of apoptosis, and activation of type I IFN response are the key features that have made alphavirus vectors very attractive for cancer treatment and vaccination. Alphavirus vectors have been successfully used as vaccines to induce protective and therapeutic immune responses against many tumor-associated antigens in animal models of mastocytoma, melanoma, mammary, prostate, and virally induced tumors. Alphavirus vectors have also shown a high antitumoral efficacy by expressing antitumoral molecules in tumor cells, which include cytokines, antiangiogenic factors or toxic proteins. In these studies induction of apoptosis in tumor cells contributed to the antitumoral efficacy by the release of tumor antigens that can be uptaken by antigen presenting cells, enhancing immune responses against tumors. The potential use of alphaviruses as oncolytic agents has also been evaluated for avirulent strains of Semliki Forest virus and Sindbis virus. The fact that this latter virus has a natural tropism for tumor cells has led to many studies in which this vector was able to reach metastatic tumors when administered systemically. Other "artificial" strategies to increase the tropism of alphavirus for tumors have also been evaluated and will be discussed.

A novel system for the production of high levels of functional human therapeutic proteins in stable cells with a Semliki Forest virus noncytopathic vector.

Semliki Forest virus (SFV) vectors lead to high protein expression in mammalian cells, but expression is transient due to vector cytopathic effects, inhibition of host cell proteins and RNA-based expression. We have used a noncytopathic SFV mutant (ncSFV) RNA vector to generate stable cell lines expressing two human therapeutic proteins: insulin-like growth factor I (IGF-I) and cardiotrophin-1 (CT-1). Therapeutic genes were fused at the carboxy-terminal end of Puromycin N-acetyl-transferase gene by using as a linker the sequence coding for foot-and-mouth disease virus (FMDV) 2A autoprotease. These cassettes were cloned into the ncSFV vector. Recombinant ncSFV vectors allowed rapid and efficient selection of stable BHK cell lines with puromycin. These cells expressed IGF-I and CT-1 in supernatants at levels reaching 1.4 and 8.6 microg/10(6)cells/24 hours, respectively. Two cell lines generated with each vector were passaged ten times during 30 days, showing constant levels of protein expression. Recombinant proteins expressed at different passages were functional by in vitro signaling assays. Stability at RNA level was unexpectedly high, showing a very low mutation rate in the CT-1 sequence, which did not increase at high passages. CT-1 was efficiently purified from supernatants of ncSFV cell lines, obtaining a yield of approximately 2mg/L/24 hours. These results indicate that the ncSFV vector has a great potential for the production of recombinant proteins in mammalian cells.

Resolucion artroscopica del quiste popliteo. Evaluacion preliminar. Tecnica quirurgico, año 2006 / Arthroscopic treatment of Baker cyst. Preliminar report

Introduccion: El quiste popliteo o de Baker se caracteriza por la coleccion liquida en la bursa del semimembranoso y gemelo interno. El objetivo principal del trabajo es evaluar la efectividad del tratamiento artroscopico del quiste popliteo recurrente, secundariamente detallar la tecnica quirurgica detallada. Materiales y metodos: Presentamos una serie de 6 pacientes operados entre junio del 2004 y enero del 2005 con tecnica artroscopica, con un promedio de edad de 54,8 años. Los mismos fueron estudiados por ecografia y RNM. Se incluyeron aquellos pacientes con quistes popliteos mayores a 4 cm, quienes habiendo agotado medidas conservadoras de tratamiento continuaban con sintomas. Se utilizo el store de Rauschning y Lindgren para la evaluacion clinica de los casos. El primer registro se realizo previo a la cirugia, luego a los 15 dias del postoperatorio, al mes y a los 6 meses. En este ultimo control se solicito una RNM. Resultados: Dividimos a los pacientes en 2 grupos. Aquellos a los que se les realizo la reseccion de las paredes del quiste y a los que no. A los 15 didas del postoperatorio 4 de 6 pacientes con dolor, tumefaccion y disminución del rango de movilidad presentaron movilidad completa, indolora y sin tumefacción ni edema. Dos pacientes llegaron al grado 0 al segundo control postoperatorio. Estos 2 pacientes presentaron complicaciones menores. A los 6 meses el resultado fue satisfactorio. Ningun caso presento recidivas en la RNM. Conclusiones: La resolucion artroscopica del quiste popliteo, combinado siempre con el tratamiento de las lesiones intraarticulares y la eliminación del mecanismo de válvula unidireccional mostro ser efectivo en el tratamiento del quiste popliteo. Creemos que la reseccion de las paredes del quiste aumenta la morbilidad del procedimiento.