BrdU does not induce hepatocellular damage in experimental Wistar rats.

Bromodeoxyuridine (BrdU) is used in studies related to cell proliferation and neurogenesis. The multiple intraperitoneal injections of this molecule could favor liver function profile changes. In this study, we evaluate the systemic and hepatocellular impact of BrdU in male adult Wistar rats in 30 %-partial hepatectomy (PHx) model. The rats received BrdU 50 mg/Kg by intraperitoneal injection at 0.5, 1, 2, 3, 6, 9 and 16 days after 30 %-PH. The rats were distributed into four groups as follows, control, sham, PHx/BrdU(-) and PHx/BrdU(+). On day 16, we evaluated hepatocellular nuclei and analyzed histopathological features by haematoxylin-eosin stain and apoptotic profile was qualified by caspase-3 presence. The systemic effect was evaluated by liver markers such as alanine transferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), alkaline phosphatase (AP), bilirubin, total proteins and serum albumin content. The statistical analysis consisted of a student t-test and one-way ANOVA. BrdU did not induce apoptosis or hepatocellular damage in male rats. Multiple administrations of BrdU in male rats did not induce significant decrease body weight, but increased serum ALT and LDH levels were found. Our results show that the BrdU does not produce hepatocellular damage.

In Vivo Models for the Study of Hyaluronic Acid Fillers: A Review.

BACKGROUND: Hyaluronic acid (HA) filler application is one of the most frequent minimally invasive aesthetic procedures used worldwide. Its properties and characterization, performance, effects in other tissues, and response to complication treatments have been studied in several animal models. This review aims to categorize animal models considering the advantages and disadvantages regarding the purpose of the study. METHODS: Literature research was made using MEDLINE via PubMed by two reviewers using keywords "hyaluronic acid" "filler" and "animal model". Full-text articles published in English and with an in vivo animal model were included for data extraction. RESULTS: The rat model was the most common animal used to evaluate properties or characteristics and degradation of HA fillers. Rabbits were preferred for evaluating HA embolism treatments; however, anatomical names of the arteries differ in some studies. Mice and rats used as vascular occlusion model are challenging due to the size of the vessels and viscosity of the filler. CONCLUSION: There is a wide variability of options of in vivo animal models to evaluate HA fillers. The animal characteristics, laboratory resources, and HA properties should be considered in accordance with the objective of the study, when choosing the ideal model. LEVEL OF EVIDENCE III: This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Enhanced antitumor activity induced by a DNA vaccine encoding E7 antigen fused to an ERAD-targeting sequence.

The endoplasmic reticulum (ER) is a key organelle in cell homeostasis and cell health through antigen presentation to immune cells. Thus, the ER has become a therapeutic target to induce cellular immune responses. We previously reported the antitumor effect of a DNA vaccine that expresses the E7 antigen fused to the cyclooxygenase-2 (COX-2) protein. This inflammation-related enzyme contains a degradation cassette associated with the endoplasmic reticulum-associated degradation (ERAD) pathway. To avoid the use of full-length COX-2 and any risk of adverse effects due to the activity of its catalytic site, we designed new versions of the fusion protein. These new constructs encode the E7 antigen fused to the signal peptide and the ERAD sequence of COX-2 with or without the membrane-binding domain (MBD) as well as deletion of the catalytic site. We evaluated the antigen-specific antitumor effect of these DNA constructs in murine prophylactic and therapeutic cancer models. These assays showed that the ERAD cassette is the minimum sequence in the COX-2 protein that induces an antitumor effect when fused to the E7 antigen with the advantage of eliminating any potential adverse effects from the use of full-length COX-2.

Chronic diseases: Origin and cell mechanisms involved.

Chronic diseases are a worldwide health problem directly related to society, lifestyle, and the development of unhealthy habits over time. Cardiovascular disease, cancer, chronic respiratory disease, and diabetes are the main causes of death. Environmental factors, such as air pollutants, poor diet, genetic predisposition, or a combination of these, are related to the development of these diseases. These factors activate cell mechanisms, such as DNA damage, oxidative stress, endoplasmic reticulum stress, autophagy, inflammation, and cell death. Depending on the dose and duration of exposure to causative agents, this cell damage can be acute or chronic. Activating these cell mechanisms can rescue normal cell function and cause permanent damage, unleashing the degeneration of tissues and organs over time. A wide variety of treatments help control chronic diseases; however, they cannot be cured completely. This fact leads to complications, dysfunctions, and disabilities. Herein, we discuss some of the principal mechanisms involved and how cellular stress can lead to these diseases when they persist for a long time.

Plasmid DNA for Therapeutic Applications in Cancer.

Recently, the interest in using nucleic acids for therapeutic applications has been increasing. DNA molecules can be manipulated to express a gene of interest for gene therapy applications or vaccine development. Plasmid DNA can be developed to treat different diseases, such as infections and cancer. In most cancers, the immune system is limited or suppressed, allowing cancer cells to grow. DNA vaccination has demonstrated its capacity to stimulate the immune system to fight against cancer cells. Furthermore, plasmids for cancer gene therapy can direct the expression of proteins with different functions, such as enzymes, toxins, and cytotoxic or proapoptotic proteins, to directly kill cancer cells. The progress and promising results reported in animal models in recent years have led to interesting clinical results. These DNA strategies are expected to be approved for cancer treatment in the near future. This review discusses the main strategies, challenges, and future perspectives of using plasmid DNA for cancer treatment.

Targeting E7 antigen to the endoplasmic reticulum degradation pathway promotes a potent therapeutic antitumor effect.

It has been previously reported that targeting and retaining antigens in the endoplasmic reticulum (ER) can induce an ER stress response. In this study, we evaluated the antitumor effect of E7 antigen fused to an ERresident protein, cyclooxygenase-2, which possesses a 19-aminoacid cassette that directs it to the endoplasmic reticulum-associated protein degradation (ERAD) pathway. The featured DNA constructs, COX2-E7 and COX2-E7ΔERAD, with a deletion in the 19-aminoacid cassette, were used to evaluate the importance of this sequence. In vitro analysis of protein expression and ER localisation were verified. We observed that both constructs induced an ER stress response. This finding correlated with the antitumor effect in mice injected with TC-1 cells and treated with different DNA constructs by biolistic vaccination. Immunisation with COX2-E7 and COX2-E7ΔERAD DNA constructs induced a significant antitumor effect in mice, without a significant difference between them, although the COX2-E7 construct induced a significant E7-specific immune response. These results demonstrate that targeting the E7 antigen to the ERAD pathway promotes a potent therapeutic antitumor effect. This strategy could be useful for the design of other antigen-specific therapies.

Caspase-3-related apoptosis prevents pathological regeneration in a living liver donor rat model.

PURPOSE: The main goal of this study was to determine the relationship of cleaved-caspase-3 (C3)-related apoptosis and hepatic proliferation, during the liver repopulation in a living liver donor rat model. MATERIAL/METHODS: Thirty-three animals were randomized into eleven groups and evaluated on postoperative from 3 â€‹h until 384 â€‹h after 30%-partial hepatectomy (30%-PHx). Liver sections (5 â€‹µm) were processed by hematoxylin-eosin, and immunostaining for C3, accompanied by hepatic function test. C3 content and the hepatic lobule enlargement were analyzed by optical density, followed by cell counting. RESULTS: Transient variations of alanine transferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) were found. Significant increase in the C3 levels, and cell nuclei number, were detected at 12 â€‹h and 48 â€‹h after 30%-PHx, evidencing a correlation of p â€‹= â€‹-0.3679. CONCLUSION: In the 30%-PHx rat model, C3-related apoptosis prevents proliferative pathological conditions during the hepatic lobule re-modeling.

Advances in gene therapy for hemophilia.

Hemophilia is a hereditary disorder that can be life-threatening in individuals who have severe spontaneous bleeding resulting from minor trauma or surgery. Although replacement therapy of the missing exogenous factor has improved patients' quality of life, it has not been possible to establish a long-term treatment. Due to the severity of the disease and the need for repetitive doses throughout the patient's life, replacement therapy has become a high-cost treatment option; therefore, the development of self-sustainable long-term therapies is critical. Hemophilia is a good candidate for gene therapy because it is a monogenic disease that can be counteracted by expression of the missing factor. In this article, we review some of the most relevant advances in gene therapy for this illness.

Antitumor effect of adenoviruses expressing mutant non-oncogenic E7 versions from HPV-16 fused to calreticulin.

PURPOSE: To compare the antitumor effect of adenoviruses that express mutant variants of the protein E7 from HPV-16 fused to calreticulin. METHODS: Recombinant adenoviruses were generated to express calreticulin fused to mutant versions of E7 (CRT/E7m and CRT/E7dm). Western blot and immunofluorescence assays were made to demonstrate protein expression. Antitumor assays were performed in C57BL6 mice injected with TC-1 cell line. RESULTS: When HEK293 cells were infected with these adenoviruses, we detected that all the recombinant proteins were expressed at endoplasmic reticulum, as expected. Next, the antitumor effect was tested on a murine tumor model established by inoculation of TC-1 cell line. We detected that both Ad CRT/E7m and Ad CRT/E7dm were capable of reducing the antitumor volume when compared to Ad LacZ, which was used as negative control. No significant difference was observed when compared to Ad CRT/E7, a positive control. CONCLUSIONS: Here we demonstrated that the mutant versions of E7 HPV-16 fused to calreticulin generate similar antitumor effect than the wild type version.

Four main therapeutic keys for Parkinson's disease: A mini review.

OBJECTIVES: Parkinson's disease (PD) is characterized by motor and cognitive dysfunctions. The progressive degeneration of dopamine-producing neurons that are present in the substantia nigra pars compacta (SNpc) has been the main focus of study and PD therapies since ages. MATERIALS AND METHODS: In this manuscript, a systematic revision of experimental and clinical evidence of PD-associated cell process was conducted. RESULTS: Classically, the damage in the dopaminergic neuronal circuits of SNpc is favored by reactive oxidative/nitrosative stress, leading to cell death. Interestingly, the therapy for PD has only focused on avoiding the symptom progression but not in finding a complete reversion of the disease. Recent evidence suggests that the renin-angiotensin system imbalance and neuroinflammation are the main keys in the progression of experimental PD. CONCLUSION: The progression of neurodegeneration in SNpc is due to the complex interaction of multiple processes. In this review, we analyzed the main contribution of four cellular processes and discussed in the perspective of novel experimental approaches.

Sex and age morphometric variations in bony nasolacrimal duct and fossa for lacrimal gland in Mexican population / Variaciones morfométricas de sexo y edad en el conducto nasolagrimal óseo y la fosa de la glándula lagrimal en la población mexicana

Primary acquired nasolacrimal duct obstruction is greater in women over 40 years and has been associated with morphometric variations in the osseous nasolacrimal duct, which varies according to age and sex. The objective is to determine variations regarding sex and age of the nasolacrimal duct and osseous fossa for lacrimal gland. One hundred sixteen dry orbits from Mexican population were analyzed; subdivided into four groups based on age and sex. The length, transverse and anteroposterior diameters of the bone entrance of the nasolacrimal duct, and the length and width of the fossa for lacrimal gland were determined. Statistical tests were applied to determine the significance of the differences found between groups. The nasolacrimal duct in women had shorter length than men in both age groups. The entrance had a wider transverse diameter in women than men independently of age and its anteroposterior diameter was shorter in men under 40 years than over 40 years. The fossa for lacrimal gland was larger in women under 40 years than in men of same age group and women over 40 years old. The lower third of the was wider in women under 40 years than in women over 40 years. Our study confirms significant differences between sex and age groups in some of the morphometric measurements of bony nasolacrimal duct and fossa for lacrimal gland in Mexican population. Comparative studies with and without clinical illness are needed to clarify if the bony characteristics of those structures participate in the etiopathogenesis and distribution differences observed in sex, age and ethnicity of thisillness.

La obstrucción de la vía lagrimal inferior es mayor en mujeres mayores de 50 años y se ha asociado a variaciones morfométricas en la entrada ósea del conducto nasolagrimal (CNL), las cuales varían según edad y sexo. El objetivo del presente estudio es determinar las variaciones respecto a sexo y edad de los diámetros de la entrada ósea del CNL, longitud del CNL y fosa de la glángula lagrimal (FL) ósea. Se analizaron 116 órbitas secas de población mexicana, los cuales a su vez fueron subdivididos en dos subgrupos en base a la edad y sexo. Se determinaron los diámetros transverso y anteroposterior de la entrada ósea del CNL, la longitud del CNL y la longitud y anchura de la FL. Se aplicaron pruebas estadísticas para determinar la significancia de las diferencias entre los grupos de estudio. La longitud del CNL fue menor en mujeres que en hombres en ambos grupos de edad. En cuanto a la entrada ósea del CNL, el diámetro transverso de fue significativamente menor en hombres que en mujeres independientemente de la edad y el diámetro anteroposterior fue menor en hombres < 40 que en > 40. La longitud de la FL fue mayor en mujeres < 40 años que en hombres del mismo rango de edad y que en mujeres > 40 años. La anchura de la FL en el tercio inferior fue menor en mujeres > 40 años que < 40 años. El resto de las mediciones de la FL no presentó diferencias significativas por sexo ni grupo de edad. Nuestro estudio confirmó que existen diferencias entre sexo y grupo de edad en algunas mediciones morfométricas del CNL y FL óseas en población mexicana. Se necesitan estudios comparativos entre personas con y sin obstrucción clínica de vía lagrimal inferior para esclarecer si las características óseas de estas estructuras participan o no en la etiopatogenia y las diferencias de distribución en género, edad y etnia de esta enfermedad.

Antitumor Response by Endoplasmic Reticulum-Targeting DNA Vaccine Is Improved by Adding a KDEL Retention Signal.

Directing an antigen to the endoplasmic reticulum (ER) improves the antigen-specific immune response, revealing a potentially useful strategy in cancer immunotherapy using tumor-associated antigens (TAAs). This can be achieved by fusing the antigen to an ER chaperone protein, such as calreticulin (CRT). We previously reported the antitumor response by fusing the CRT signal peptide (SP) and its ER retention sequence (KDEL) to full-length human papillomavirus type 16 (HPV-16) E6 and E7 antigens, obtaining a potent antitumoral effect. In this article, we compare the antitumor response due to the use of each signal (SP and/or KDEL) fused to HPV16 E6 and E7 antigens in a DNA vaccination model. Using both SP and KDEL signals promotes higher interferon (IFN)-γ production and a faster antitumor response than using only the SP, resulting in better tumor growth restraint and higher survival, indicating that the KDEL addition to an ER-directed antigen helps by shortening the time to response. Meanwhile, antigens without signals or only the KDEL signal showed no induction of antigen-specific IFN-γ or antitumor response. Our results indicate that directing the E6E7m antigen to the ER by the SP signal is sufficient to promote an efficient antitumor response. Importantly, this effect is stronger and faster when the antigen also has an ER retention sequence, such as the KDEL signal.

Invasion of TC-1 cells to skeletal muscle fibers protect them from peroxisomicine A1 (T-514) treatment in a murine model of cancer.

T-514 or Peroxisomicine A1 (PA1) is a toxin isolated from plants of genus Karwinskia. In vitro studies described selective toxicity of PA1 on malignant cell lines. A toxic effect of PA1 on TC-1 cells in vivo was reported. The objective was to evaluate the effect of PA1 over invasion of TC-1 cells to muscle fibers in vivo. TC-1 cells were implanted in 36 mice divided in two groups (n: 18): treated with PA1 or with vehicle, a control group was included. At 10 days, nine mice of each group were euthanized. TC-1 implant site was analyzed by light and electron microscopy, a morphometric study was also performed. Remaining mice were used to evaluate tumor growth and survival time. Results show tumor cells between muscle fibers, with diminution in diameter, change in the staining pattern, loss of continuity of external lamina, and sarcoplasm with tumor cells. Statistically difference was observed between treated group vs control group. PA1 decreased tumor growth and increased the survival time in treated mice. The degree of resistant activity, aggressiveness, and invasiveness of TC-1 cells described in present work; should be taken into account in studies that evaluate effectiveness of therapies using this cancer model.

Morphological Analysis of Major Segments of Coronary Artery Occlusion: Importance in Myocardial Revascularization Surgery / Análisis Morfológico de los Principales Segmentos de Oclusión de las Arterias Coronarias: Importancia en la Cirugía de Revascularización Miocárdiaca

Revascularization surgery should ensure morphological similarity between the coronary artery and the graft. This is an important factor for its duration and permeability. The aim of this study was to analyze the morphological characteristics and morphometrics of the coronary artery segments with greater occlusion. This was an observational, cross-sectional descriptive study that consisted of two phases. A macroscopic phase in which 11 cadaveric hearts were extracted and coronary dominance and length of the anterior interventricular artery (AIA), the right coronary artery (RCA) and the circumflex artery (CXA) were determined. In the microscopic phase a total of 77 segments of these arteries were obtained and the luminal diameter, wall thickness, and amount of elastic fibers and the presence and size of the atheroma were determined. Right coronary dominance was the most frequent. Total vessel length was 15.65±1.17 cm for the AIA, 12.67±2.02 cm for the RCA and 8.79±2.5 cm for the CXA. Diameters ranged from 2.3 mm in the proximal segments and between 1.1 mm to 1.8 mm in the distal segments. Wall thickness in the proximal segments was between 354 µm and 396 µm and in the distal segments it ranged from 120 µm to 305 µm. The amount of elastic fibers showed that they were muscular arteries. Atheromas were present in 35% in the CXA, and in 32.5% in the AIA and the RCA. The largest ones were found in the proximal segments. This study examined the morphology and morphometry of the segments of the coronary arteries that are more frequently occluded. It provides information on the most significant parameters to be considered for election of the vascular graft in myocardial revascularization surgery.

En la cirugía de revascularización miocárdica se debe asegurar la similitud morfológica entre la arteria coronaria y el injerto. Esto es un factor importante en su duración y permeabilidad. El objetivo fue analizar las características morfológicas y morfométricas de los segmentos de mayor oclusión de las arterias coronarias. Estudio observacional, transversal y descriptivo que constó de dos fases. Una macroscópica en la que se extrajeron 11 corazones de cadáveres y se determinó la dominancia coronaria y longitud de la arteria interventricular anterior (AIA), la arteria coronaria derecha (ACD) y la arteria circunfleja (ACX). En la fase microscópica se obtuvo un total de 77 segmentos de estas arterias y se determinó el diámetro luminal, grosor de pared, cantidad de fibras elásticas y presencia y tamaño de ateroma. La dominancia coronaria derecha fue la más frecuente. La longitud total de los vasos fue de 15,65±1,17 cm para la AIA, de 12,67±2,02 cm para la ACD y 8,79±2,5 cm para la ACX. Los diámetros oscilaron entre los 2,3 mm en los segmentos proximales y entre 1,1 mm a 1,8 mm en los segmentos distales. Los grosores de pared en los segmentos proximales se presentaron entre 354 µm y 396 µm y en los segmentos distales oscilaron entre 120 µm a 305 µm. La cantidad de fibras elásticas demostró que son arterias musculares. El 35% de los ateromas se presentó en la ACX y el 32,5% en la AIA al igual que en la ACD. Los de mayor tamaño se encontraron en los segmentos proximales. Este estudio analizó la morfología y la morfometría de los segmentos de las arterias coronarias que se ocluyen con mayor frecuencia. Los resultados encontrados aporta información sobre los parámetros más significativos que se deben considerar para la elección del injerto vascular en la cirugía de revascularización miocárdica.

Production of biologically active human lymphotactin (XCL1) by Lactococcus lactis.

Lymphotactin-XCL1 is a chemokine produced mainly by activated CD8+ T-cells and directs migration of CD4+ and CD8+ lymphocytes and natural killer (NK) cells. We expressed human lymphotactin (LTN) by the lactic-acid bacterium Lactococcus lactis. Biological activity of LTN was confirmed by chemo-attraction of human T-cells by chemotaxis demonstrating, for the first time, how this chemokine secreted by a food-grade prokaryote retains biological activity and chemoattracts T lymphocytes. This strain thus represents a feasible well-tolerated vector to deliver active LTN at a mucosal level.