Development of a validated molecular analytical method to determine the viral safety of F(AB´)2 products: A novel application for a well-known technique.

The immunotherapy agents derived from horses are biological products that allow the neutralization of clinically relevant immunogens, such as the SARS-CoV-2 virus that causes COVID-19, or the neutralization of toxins present in the venoms of snakes, spiders, and other poisonous animals. Due to their importance, detecting adventitious viruses in equine hyperimmune serum (raw material in industrial processes) is a critical step to support the safety of products for human use, and, in consequence, it is a requirement for commercialization and distribution. The safety of the finished product is based on three complementary approaches: (i) testing of the source material (horse serum) donations, (ii) release of the starting material (i.e., pool of horse serum) based on non-reactivity for a range of human infectious or pathogenic viruses, and (iii) validate (selected) steps of the manufacturing process for their capacity to inactivate and/or remove a wide range of viruses potentially present in the starting material. Orthogonal approaches to reduce viral contamination risk include implementing a reliable and validated system for detecting adventitious viruses. Thus, it is necessary to establish trustworthy and sufficiently sensitive analytical methods to evidence the lack of viruses to assure the safety of the therapeutic product. Therefore, in this research, an analytical method based on end-point Reverse Transcription Polymerase Chain Reaction (RT-PCR) was developed, implemented, and validated in hyperimmune equine serum samples to detect Venezuelan equine encephalitis virus, West Nile virus, and Rabies virus.

Methotrexate Induces an Antioxidant Hormetic Response in Primary Rat Astrocytes.

Neurodegenerative diseases have increased worldwide in recent years. Their relationship with oxidative stress has motivated the research to find therapies and medications capable of suppressing oxidative damage and therefore slowing the progression of these diseases. Glutathione (GSH) is the most important cellular antioxidant in living beings and is responsible for regulating the cellular redox state. However, GSH cannot be administered by any route of administration, so molecules that increase its levels by activating Nrf2-ARE signaling pathway are explored; since Nrf2 regulates the main genes involved in GSH de novo synthesis and recycling. Astrocytes are the most important cell-type in the antioxidant cell response and are responsible for providing GSH and other substrates for neurons to have an efficient antioxidant response. Methotrexate (MTX) is an anti-inflammatory agent that has different cellular effects when administered at low or high concentrations. So in this study, we used MTX different concentrations and exposure times to induce a hormetic antioxidant response in rat primary astrocytes. Our results showed that 20 nM MTX pre-conditioning for 12 h augmented the GSH/GSSG ratio and protected cellular viability against a toxic MTX and H2O2 insult, which was abrogated when Nrf2 was inhibited by brusatol. Hence, MTX subsequent studies as a drug to counteract the progression of some stress-associated neurodegenerative diseases are suggested.

Long-term sulforaphane-treatment restores redox homeostasis and prevents cognitive decline in middleaged female and male rats, but cannot revert previous damage in old animals.

Aging is a complex and detrimental process, which disrupts most organs and systems within the organisms. The nervous system is morphologically and functionally affected during normal aging, and oxidative stress has been involved in age-related damage, leading to cognitive decline and neurodegenerative processes. Sulforaphane (SFN) is a hormetin that activates the antioxidant and anti-inflammatory responses. So, we aimed to evaluate if SFN long-term treatment was able to prevent age-associated cognitive decline in adult and old female and male rats. Memory was evaluated in adult (15-month-old), and old (21-month-old) female and male Wistar rats after three months of SFN treatment. Young rats (4-month-old) were used as age controls. The antioxidant response induction, the redox state (GSH/GSSG), and oxidative damage were determined in the brain cortex (Cx) and hippocampus (Hc). Our results showed that SFN restored redox homeostasis in the Cx and Hc of adult rats, thus preventing cognitive decline in both sexes; however, the redox responses were not the same in males and females. Old rats were not able to recover their redox state as adults did, but they had a mild improvement. These results suggest that SFN mainly prevents rather than reverts neural damage; though, there might also be a range of opportunities to use hormetins like SFN, to improve redox modulation in old animals.

Senescence in Primary Rat Astrocytes Induces Loss of the Mitochondrial Membrane Potential and Alters Mitochondrial Dynamics in Cortical Neurons.

The decline in brain function during aging is one of the most critical health problems nowadays. Although senescent astrocytes have been found in old-age brains and neurodegenerative diseases, their impact on the function of other cerebral cell types is unknown. The aim of this study was to evaluate the effect of senescent astrocytes on the mitochondrial function of a neuron. In order to evaluate neuronal susceptibility to a long and constant senescence-associated secretory phenotype (SASP) exposure, we developed a model by using cellular cocultures in transwell plates. Rat primary cortical astrocytes were seeded in transwell inserts and induced to premature senescence with hydrogen peroxide [stress-induced premature senescence (SIPS)]. Independently, primary rat cortical neurons were seeded at the bottom of transwells. After neuronal 6 days in vitro (DIV), the inserts with SIPS-astrocytes were placed in the chamber and cocultured with neurons for 6 more days. The neuronal viability, the redox state [reduced glutathione/oxidized glutathione (GSH/GSSG)], the mitochondrial morphology, and the proteins and membrane potential were determined. Our results showed that the neuronal mitochondria functionality was altered after being cocultured with senescent astrocytes. In vivo, we found that old animals had diminished mitochondrial oxidative phosphorylation (OXPHOS) proteins, redox state, and senescence markers as compared to young rats, suggesting effects of the senescent astrocytes similar to the ones we observed in vitro. Overall, these results indicate that the microenvironment generated by senescent astrocytes can affect neuronal mitochondria and physiology.

Low-Intensity Exercise Routine for a Long Period of Time Prevents Osteosarcopenic Obesity in Sedentary Old Female Rats, by Decreasing Inflammation and Oxidative Stress and Increasing GDF-11.

The loss of skeletal muscle mass and strength is known as sarcopenia; it is characterized as a progressive and generalized muscle disorder associated with aging. This deterioration can seriously compromise the elderly's health and reduce their quality of life. In addition to age, there are other factors that induce muscle mass loss, among which are sedentary lifestyle, chronic diseases, inflammation, and obesity. In recent years, a new clinical condition has been observed in older adults that affects their physical capacities and quality of life, which is known as osteosarcopenic obesity (OSO). Osteoporosis, sarcopenia, and obesity coexist in this condition. Physical exercise and nutritional management are the most widely used interventions for the treatment and prevention of sarcopenia. However, in older adults, physical exercise and protein intake do not have the same outcomes observed in younger people. Here, we used a low-intensity exercise routine for a long period of time (LIERLT) in order to delay the OSO appearance related to sedentarism and aging in female Wistar rats. The LIERLT routine consisted of walking at 15 m/min for 30 min, five days a week for 20 months. To evaluate the effects of the LIERLT routine, body composition was determined using DXA-scan, additionally, biochemical parameters, inflammatory profile, oxidative protein damage, redox state, and serum concentration of GDF-11 at different ages were evaluated (4, 8, 12, 18, 22, and 24 months). Our results show that the LIERLT routine delays OSO phenotype in old 24-month-old rats, in a mechanism involving the decrease in the inflammatory state and oxidative stress. GDF-11 was evaluated as a protein related to muscle repair and regeneration; interestingly, rats that perform the LIERLT increased their GDF-11 levels.

Estrogen Receptors Alpha and Beta in POA-AHA Region Regulate Asymmetrically Ovulation.

We examined the role of the estrogen receptors alpha (ERα) and beta (ERß) in of the preoptic-anterior hypothalamic area (POA-AHA) in the regulation of ovulation in rats. The number of ERα- and ERß-immunoreactive (-ir) cells was determined at 09:00, 13:00, and 17:00 h of each stage of the estrous cycle in intact rats. Additionally, the effects of blocking ERα and ERß on ovulation rate at 09:00 h on diestrus-2 or proestrus day through the microinjection of methyl-piperidino-pyrazole (MPP) or cyclofenil in either side of POA-AHA were evaluated. The number of ERα-ir and ERß-ir cells in POA-AHA varied in each phase of estrous cycle. Either MPP or cyclofenil in the right side of POA-AHA on diestrus-2 day reduced the ovulation rate, while at proestrus day it was decreased in rats treated in either side with MPP, and in those treated with cyclofenil in the left side. MPP or cyclofenil produced a decrease in the surge of luteinizing hormone levels (LH) and an increase in progesterone and follicle stimulating hormone (FSH). Replacement with synthetic luteinizing hormone-releasing hormone in non-ovulating rats treated with MPP or cyclofenil restored ovulation. These results suggest that activation of estrogen receptors on the morning of diestrus-2 and proestrus day asymmetrically regulates ovulation and appropriately regulates the secretion of FSH and progesterone in the morning and afternoon of proestrus day. This ensures that both, the preovulatory secretion of LH and ovulation, occur at the right time.

Muscarinic Receptors Types 1 and 2 in the Preoptic-Anterior Hypothalamic Areas Regulate Ovulation Unequally in the Rat Oestrous Cycle.

Muscarinic receptors types 1 (m1AChR) and 2 (m2AChR) in the preoptic and anterior hypothalamus areas (POA-AHA) were counted, and the effects of blocking these receptors on spontaneous ovulation were analysed throughout the rat oestrous cycle. Rats in each phase of the oestrous cycle were assigned to the following experiments: (1) an immunohistochemical study of the number of cells expressing m1AChR or m2AChR in the POA-AHA and (2) analysis of the effects of the unilateral blockade of the m1AChR (pirenzepine, PZP) or m2AChR (methoctramine, MTC) on either side of the POA-AHA on the ovulation rate. The number of m2AChR-immunoreactive cells was significantly higher at 09:00 h on each day of the oestrous cycle in the POA-AHA region, while no changes in the expression profile of m1AChR protein were observed. The ovulation rate in rats treated with PZP on the oestrous day was lower than that in the vehicle group. Animals treated on dioestrous-1 with PZP or MTC had a higher ovulation rate than those in the vehicle group. In contrast, on dioestrous-2, the MTC treatment decreased the ovulation rate. These results suggest that m1AChR or m2AChR in the POA-AHA could participate in the regulation of spontaneous ovulation in rats.

Gran adenocarcinoma de cabeza pancreática sin ictericia / Big Pancreatic Head Adenocarcinoma without Jaundice

Se presentó una paciente femenina de 28 años de edad, raza negra y procedencia rural con antecedentes de litiasis vesicular hace siete años, la cual acudió a consulta de Cirugía General por presentar vómitos ocasionales al ingerir alimentos, de color amarillo, escasos en cantidad y de contenido alimentario, al ser examinada se encuentra tumor palpable en hipocondrio derecho de ocho centímetros, por lo que se ingresó para estudio, luego de este fue sometida a tratamiento quirúrgico, encontrándose gran tumor de la cabeza pancreática, con compresión del duodeno, sin ictericia, ni dilatación de vías biliares extrahepáticas, realizándose pancreatoduodenectomía con reconstrucción del tránsito gastrointestinal y biliodigestivo.

A 28 - year-old female patient, black and rural origin with a history of lithiasis during seven years ago was presented in this paper. She assisted to General Surgery consultation because when eating food occasionally presented vomiting of yellow color, few in number and food content. When examination was performed a palpable tumor in the right upper quadrant of eight centimeters was found, so she was admitted for evaluation, after the surgical treatment was performed a big pancreatic head adenocarcinoma was found, with compression of the duodenum, neither jaundice nor extrahepatic bile duct dilatation were observed. Pancreatoduodenectomy with reconstruction of gastrointestinal and biliodigestive traffic was performed.

Gran adenocarcinoma de cabeza pancreática sin ictericia / Big Pancreatic Head Adenocarcinoma without Jaundice

Se presentó una paciente femenina de 28 años de edad, raza negra y procedencia rural con antecedentes de litiasis vesicular hace siete años, la cual acudió a consulta de Cirugía General por presentar vómitos ocasionales al ingerir alimentos, de color amarillo, escasos en cantidad y de contenido alimentario, al ser examinada se encuentra tumor palpable en hipocondrio derecho de ocho centímetros, por lo que se ingresó para estudio, luego de este fue sometida a tratamiento quirúrgico, encontrándose gran tumor de la cabeza pancreática, con compresión del duodeno, sin ictericia, ni dilatación de vías biliares extrahepáticas, realizándose pancreatoduodenectomía con reconstrucción del tránsito gastrointestinal y biliodigestivo(AU)

A 28 year-old female patient, black and rural origin with a history of lithiasis during seven years ago was presented in this paper. She assisted to General Surgery consultation because when eating food occasionally presented vomiting of yellow color, few in number and food content. When examination was performed a palpable tumor in the right upper quadrant of eight centimeters was found, so she was admitted for evaluation, after the surgical treatment was performed a big pancreatic head adenocarcinoma was found, with compression of the duodenum, neither jaundice nor extrahepatic bile duct dilatation were observed. Pancreatoduodenectomy with reconstruction of gastrointestinal and biliodigestive traffic was performed(AU)

Hepatic dysfunction in typhoid fever during pregnancy.

We described the hepatic dysfunction found in 10 cases out of 32 women with typhoid fever during pregnancy. This was associated with late diagnosis and maternal and perinatal complications.

Comparative study of the venoms of three subspecies of Lachesis muta (bushmaster) from Brazil, Colombia and Costa Rica

A comparative study was performed on the pharmacology and biochemistry of venoms from three subspecies of Lachesis muta (L. m. stenophrys, L. m. muta and L. m. rhombeata) from Brazil, Colombia and Costa Rica. All venoms induced lethal, hemorrhagic, edema-forming, myotoxic, coagulant and defibrinating effects, showing also proteolytic and indirect hemolytic activities. The venoms of L. m. stenophrys from Costa Rica and L. m. muta from Cascalheira, Brazil, had the highest lethal and hemorrhagic activities and the venom of L. m. rhombeata showed the highest coagulant activity, whereas no significant differences were observed in myotoxic and edema-forming activities at most of the time intervals studied. In addition, venoms showed similar electrophoretic patterns on SDS–polyacrylamide gel electrophoresis. In conclusion, despite quantitative differences in toxic and enzymatic activities, together with subtle variations in electrophoretic patterns, our results indicate that experimental envenomation by these venoms induce a qualitatively similar pathophysiological profile.

Accidente ofídico en Antioquia y Chocó. Aspectos clínicos y epidemiológicos (marzo de 1989 - febrero de 1990)

Se presentan las caracteristicas clínicas y epidemiológicas de 244 mordeduras de serpientes, estudiadas durante un año, 190 Antioquia y 54 en Chocó, Colombia. La incidencia por 10.00 habitentes fue dos veces más altas en el Chocó y las mordeduras fuerón más frecuentes en agricultores jóvenes de sexo masculino (45.9 por ciento), 71 por ciento afectaban las extremidades inferiores, especialmente los pies (52.5 por ciento). El accidente bothropico fue el más común (89.4 por ciento); luego las morderuras por serpientes no venenosas (7 por ciento), por Lachesis muta (2 por ciento), por especies de Micrurus (corales) (0.8 por ciento) y por Crotatus durissis terificus (0.8 por ciento) en manipuladores de esta serpiente. La Bothrops (mapana) fue la especie prevalente (44.5 por ciento). El edema (97.2 por ciento) y la desfibrinacion (61.8 por ciento) fueron las caraterísticas clasicas del envenenamiento bothropico; la necrossis solo ocurrio en 9,2 por ciento de los pacientes y 35,8 por ciento no tienen envenenamiento sistémico. En las mordeduras por la L. muta los cinco pacientes tenían envenenamiento local y sistémico tres fueron séveros, pero las hemorragias en otras fueran raras, en contrarse con el envenenamiento bothropico en el cual fue frecuente la gingivorrigia (23 por ciento). La infeccion secundaria y la insificiencia renal aguda fueron fueron complicaciones en ambos accidentes (10.6 por ciento). En las mordeduras por C.d. terrificus, los signos locales fueron moderados y un paciente tuvo envenenamiento sistémico severo. En las mordeduras por Micrurus, los síntomas neurológicos fueron leves o moderados. Las mordeduras por Colubridae (cazadoras) no produjeron envenenamiento. Doce fallecieron (5.3 por ciento), 11 por Bothrops y uno por C.d. terrificus; 16 tuvieron secuelas (7 por ciento), 13 por Bothrops y tres por L. muta, principalmente por la necrosis. Hubo asociación estadistica (p<0,05) entre las prácticas de medicina tradicional y retardo de 6 o más horas en la consulta con envenenamiento bothrópico severo; y entre mortalidad y secuelas con demora de >= 12 horas en la consulta. Los primeros auxilios no modificaron el curso del envenenamiento.