Inmunoterapia autóloga con células dendríticas en modelos caninos con tumor venéreo transmisible

La inmunoterapia autóloga utiliza células del mismo cuerpo para estimular y restaurar las defensas naturales del sistema inmunológico. Algunas de las células que han sido utilizado en años recientes son: linfocitos infiltrantes de tumor, linfocitos T citotóxicos, células asesinas activadas por linfocinas y células dendríticas (CD). Las CD son células especializadas y su función es el capturar, procesar y presentar antígenos a los linfocitos para iniciar una respuesta inmune. El tumor venéreo transmisible (TVT), también conocido como sarcoma infeccioso o tumor de Sticker, es un cáncer transmisible en perros que afecta mayormente los genitales externos y se transmite durante el coito. En este trabajo se implementó por primera vez un modelo de TVT en el órgano genital (vagina) de los pacientes y se les administró la inmunoterapia autóloga con CD específicas contra el tumor. Para estudiar esta terapia se utilizaron tres grupos experimentales: el tumor sin tratamiento, el tumor tratado con sangre completa autóloga, y el tumor tratado con CD autólogas específicas para el TVT. Por último se evaluó la capacidad inmunológica del extracto tumoral total (ETT) del tumor como método de prevención in vivo. Para el tratamiento autólogo con las CD, se esperó que el tumor midiese 3cm, se realizó un cultivo primario de las células de TVT y se les extrajo el 4% de su peso corporal de sangre a los pacientes para realizar una diferenciación de los monocitos a CD. Para evaluar el efecto de la inmunoterapia autóloga con CD se observaron los efectos secundarios, el tamaño tumoral, las poblaciones de linfocitos, los niveles de IFN-γ en sangre y los linfocitos infiltrantes de tumor por histopatología. Los monocitos se diferenciaron a CD y se les realizó un análisis fenotípico mediante citometría de flujo. Los monocitos mostraron una expresión de CD14+ de 80.1%, CD80+ de 15.6%, CD83+ de 0.4% y un DLA II de1.8%. En las CD se obtuvo una expresión de 8.7% para CD14+, 80.3% para CD80+, 76.4% para CD83+ y 86.5% para DLA II y 62% en la prueba de fagocitosis. La terapia no mostró ningún efecto secundario en nuestro grupo experimental y hubo una regresión tumoral del 100% para la semana doce. Se encontró un aumento de expresión celular en sangre de CD4+ de 29%, de CD8+ de 34% y de IFN-γ de 120 pg/mL. Nuestros resultados demuestran que la inmunoterapia autóloga con CD específicas inducen una regresión del TVT en caninos

Terapia alternativa de células dendríticas cargadas con exosomas derivados de tumor venéreo transmisible.

La inmunoterapia celular estimula y restaura las defensas naturales del sistema inmune para combatir el cáncer. Las células dendríticas (CD) son células especializadas del sistema inmune y su función es capturar, procesar y presentar antígenos a los linfocitos para iniciar una respuesta inmune. Una nueva fuente de antígenos para las CD son los exosomas, pequeñas vesículas (30-120 nm) que contienen ARN, ADN y proteína. Se han identificado proteínas tales como tetraspaninas (CD9, CD63, CD81), proteínas de choque térmico (Hsp), moléculas del complejo mayor de histocompatibilidad (CMH) de clase I y II y antígenos asociados a tumores (AAT). Un excelente modelo in vivo para estudios de inmunoterapia es el tumor venéreo transmisible canino (TVTC), uno de los cuatro tumores transmisibles en el mundo, es un cáncer transmisible en perros que afecta principalmente los genitales externos y se transmite durante el coito. En este trabajo utilizamos CD pulsadas con exosomas de TVTC como tratamiento en caninos con TVT. Para estudiar la inmunoterapia, se usaron tres grupos experimentales: grupo no vacunado; vacunados con exosomas de TVTC y vacunados con CD pulsadas con exosomas de TVTC. Para la inmunoterapia, se realizó un cultivo primario de células de TVTC, se aislaron los exosomas de TVTC y se caracterizaron por un ensayo de microscopía de transmisión electrónica, dot blot y cuantificación de proteínas. Luego se extrajo el 4% de sangrea los pacientes para diferenciar monocitos en CD. Para evaluar el efecto de la inmunoterapia con CD se observaron efectos secundarios, tamaño tumoral, poblaciones de linfocitos y los niveles de IFN-γ en suero sanguíneo. Los exosomas aislados de TVTC mostraron una muestra homogénea con una forma circular de aproximadamente 90 nm. En el dot blot, todos los marcadores de proteína (CD81, CD63, CD9 y Hsp70) fueron positivos, excepto el control (albúmina). La cuantificación de proteínas de cinco muestras representativas de los exosomas fue de 9.15 mg/mL, 13.65 mg/mL, 10.21 mg/mL, 11.43 mg/mL y 10.79 mg/mL. Los monocitos se diferenciaron en CD y se sometieron a un análisis fenotípico mediante citometría de flujo. Los monocitos mostraron una expresión de 85.71% para CD14+, 12.3% para CD80+, 0.1% para CD83+ y 0.8% para DLA II. Las CD mostraron5.1% para CD14+, 86.7% para CD80+, 90.1% para CD83+ y 92.6% para DLA II y un porcentaje de fagocitosis de 63% que se obtuvo mediante la prueba de FITC-Dextran. No se observó ningún efecto secundario en nuestro grupo experimental para nuestra terapia y la regresión tumoral fue del 100% durante la séptima semana. Un aumento de expresión en la población de CD4+de28% y de 34% en la población de CD8+ y los niveles de IFN-γ aumentaron a 142 pg/mL. Estos resultados muestran que lasCD pulsadas con exosomas tumorales inducen la regresión del TVT en perros

Insoluble chitosan complex as a potential adsorbent for aflatoxin B1 in poultry feed

As a class of secondary metabolites or toxins produced by fungi, aflatoxins can poison humans and animals; among them, aflatoxin B1 (AFB1) is the most dangerous one owing to its carcinogenic and mutagenic properties that increase risks for hepatocellular carcinoma in humans; hence, adsorbents such as smectites are commonly included in poultry feed to mitigate their effects. In this study, chitosan was crosslinked with sodium dodecyl sulfate (SDS) to form an insoluble polymer complex that is stable at the relevant physiological pH levels. The characterization via Fourier transforms infrared spectroscopy revealed the interaction between the sulfate groups of the SDS and the amine group of chitosan (1,016 and 819 cm−1); this result was further confirmed by the X-ray diffraction patterns with a change in the crystalline structure of the chitosan-insoluble complex (2θ = 4.76°, 7°, and 22°). The morphology of the chitosan-insoluble complex obtained using a field emission scanning electron microscope (FE-SEM) revealed that particles were slightly porous. After characterization, the performance of the chemically modified polymer complex was evaluated as an adsorbent for AFB1 and compared with those of the unmodified chitosan, soluble chitosan complex, and commercial montmorillonite clay binder. In addition, the polymer complex was investigated as an adsorbent in an in vitro model for the poultry gastrointestinal system. Sequestration of AFB1 by a chemically modified polymer complex was 93.4%, equivalent to that of commercial montmorillonite clay (99.5%). However, these treatments also sequestered microminerals, particularly selenium and iron. This pH-stable, high-capacity adsorbent could be used in poultry feed to reduce the uptake of AFB1

Clinical trial evaluating the effectiveness of biocompound IMMUNEPOTENT CRP in the third-molar extraction

ABSTRACT A controlled, parallel, randomized and comparative trial was carried out to evaluate the antiinflammatory efficacy of IMMUNEPOTENT CRP versus ibuprofen in patients after third-molar surgery over seven days. The anti-inflammatory efficacy of IMMUNEPOTENT CRP was evaluated using the method of Amin and Laskin, and the analysis of cytokine production (IL-2, IL-4, IL-6, IL-10, TNF-a, INF-g) in saliva was done by flow cytometry. The swelling process after surgery was significant (p < 0.05) and the treatments with IMMUNEPOTENT CRP or ibuprofen controlled this process properly; no difference between the groups was found (p < 0.05). Both treatments were shown to modulate the cytokine production. These results demonstrate the anti-inflammatory activity of the natural compound IMMUNEPOTENT CRP and suggest it could be used in clinical dental practice

Evaluation of a canine transmissible venereal tumour cell line with tumour immunity capacity but without tumorigenic property

Introduction: Canine transmissible venereal tumour (CTVT) is a sexually transmitted tumour affecting dogs worldwide, imposing a financial burden on dog owners. A stable culture cell line in continuous passages for >18 months has only been achieved once. The present study investigated a stable CTVT cell line isolated from a bitch and its potential as a vaccine. Material and Methods: A biopsy from a 2-year-old mongrel bitch with CTVT was obtained for histopathological confirmation and isolation of tumour cells. The isolated cells were cultured to passage 55 and characterised by flow cytometry, with karyotyping by GTG-banding and by PCR detection of myc S-2 and LINE AS1. The isolated CTVT cell line was also used as a preventive vaccine in a canine model. Results: Histopathological analysis of the isolated tumour cells revealed typical CTVT characteristics. Constant proliferation and stable morphological characteristics were observed during culture. Phenotypic analysis determined the expression of HLA-DR+, CD5.1+, CD14+, CD45+, CD83+, CD163+, and Ly-6G-Ly-6C+. GTG-banding revealed a mean of 57 chromosomes in the karyotype with several complex chromosomal rearrangements. LINE-c-myc insertion in the isolated CTVT cell line at 550 bp was not detected. However, a 340-bp band was amplified. Isolated CTVT cell line inoculation at a concentration of 1×108 did not induce tumour growth in bitches, nor did a challenge with primary CTVT cells. Conclusion: The present study successfully identified and isolated a stable CTVT cell line that may be useful in CTVT prevention

A Novel Oral Endoscopic Biopsy Procedure to Obtain Rumen Epithelial Samples

Most in vivo studies related to ruminal development in calves use invasive techniques involving rumen-fistulated or euthanized animals. In consideration of animal welfare, we developed an oral endoscopic biopsy procedure to allow the obtaining of rumen epithelial samples, thus serving as an alternative for measuring the height and width of rumen papillae in calves in a safe, quick, and efficient manner that allows the slaughtering of calves to be avoided. This procedure was tested on 12 Brangus crossbred calves randomly distributed in two groups, with one fed a meal starter and the other an extruded starter feed. Calves underwent a 12-h fasting period, were restrained in a squeeze chute, administered a dose of atropine, and sedated with xylazine before the oral endoscopic biopsy procedure. A 120 cm long Olympus® oral flexible video endoscope and forceps were used to collect cranial–dorsal sac rumen epithelial tissue samples of approximately 0.5 mm. Endoscopy was successful in all 12 calves and the collected tissue samples were placed in formalin (10%) for further processing for obtaining rumen papillae measurements. Consumption of the extruded starter feed resulted in the increased (p = 0.035) width of rumen papillae. The oral endoscopic biopsy procedure implemented in this study was demonstrated to be successful and is thus an alternative technique for studying rumen epithelial development and morphometric alterations in calf rumen tissue

Aluminosilicates as a Double-Edged Sword: Adsorption of Aflatoxin B₁ and Sequestration of Essential Trace Minerals in an In Vitro Gastrointestinal Poultry Model

Aflatoxins can cause intoxication and poisoning in animals and humans. Among these molecules, aflatoxin B1 (AFB1) is the most dangerous because of its carcinogenic and mutagenic properties. To mitigate these effects, clay adsorbents are commonly included in the diet of animals to adsorb the carcinogens and prevent their absorption in the gastrointestinal tract. In this study, four clays, three smectites (C-1, C-2, and C-3), and one zeolite (C-4), were compared as adsorbents of AFB1 and trace inorganic nutrients using an in vitro gastrointestinal model for poultry. Characterization of the clays using Fourier transform infrared spectroscopy revealed characteristic bands of smectites in C-1, C-2, and C-3 (stretching vibrations of Si-O, Al-O-Si, and Si-O-Si). The C-4 presented bands related to the bending vibration of structural units (Si-O-Si and Al-O-Si). X-ray diffraction analysis showed that C-1 is a montmorillonite, C-2 is a beidellite, C-3 is a beidellite-Ca-montmorillonite, and C-4 is a clinoptilolite. The elemental compositions of the clays showed alumina, silica, iron, calcium, and sodium contents. The cation exchange capacity was higher in C-3 clay (60.2 cmol(+)/kg) in contrast with the other clays. The AFB1 adsorption of C-1 was the highest (98%; p ˂ 0.001), followed by C-2 (94%). However, all the clays also sequestered trace inorganic nutrients (Fe, Mn, Zn, and Se). Both smectites, montmorillonite and beidellite, were the most suitable for use as adsorbents of AFB1

Clinical trial evaluating the effectiveness of biocompound IMMUNEPOTENT CRP in the third-molar extraction

A controlled, parallel, randomized and comparative trial was carried out to evaluate the anti-inflammatory efficacy of IMMUNEPOTENT CRP versus ibuprofen in patients after third-molar surgery over seven days. The anti-inflammatory efficacy of IMMUNEPOTENT CRP was evaluated using the method of Amin and Laskin, and the analysis of cytokine production (IL-2, IL-4, IL-6, IL-10, TNF-α, INF-γ) in saliva was done by flow cytometry. The swelling process after surgery was significant (p < 0.05) and the treatments with IMMUNEPOTENT CRP or ibuprofen controlled this process properly; no difference between the groups was found (p < 0.05). Both treatments were shown to modulate the cytokine production. These results demonstrate the anti-inflammatory activity of the natural compound IMMUNEPOTENT CRP and suggest it could be used in clinical dental practice

Table1_Insoluble chitosan complex as a potential adsorbent for aflatoxin B1 in poultry feed.docx

As a class of secondary metabolites or toxins produced by fungi, aflatoxins can poison humans and animals; among them, aflatoxin B1 (AFB1) is the most dangerous one owing to its carcinogenic and mutagenic properties that increase risks for hepatocellular carcinoma in humans; hence, adsorbents such as smectites are commonly included in poultry feed to mitigate their effects. In this study, chitosan was crosslinked with sodium dodecyl sulfate (SDS) to form an insoluble polymer complex that is stable at the relevant physiological pH levels. The characterization via Fourier transforms infrared spectroscopy revealed the interaction between the sulfate groups of the SDS and the amine group of chitosan (1,016 and 819 cm−1); this result was further confirmed by the X-ray diffraction patterns with a change in the crystalline structure of the chitosan-insoluble complex (2θ = 4.76°, 7°, and 22°). The morphology of the chitosan-insoluble complex obtained using a field emission scanning electron microscope (FE-SEM) revealed that particles were slightly porous. After characterization, the performance of the chemically modified polymer complex was evaluated as an adsorbent for AFB1 and compared with those of the unmodified chitosan, soluble chitosan complex, and commercial montmorillonite clay binder. In addition, the polymer complex was investigated as an adsorbent in an in vitro model for the poultry gastrointestinal system. Sequestration of AFB1 by a chemically modified polymer complex was 93.4%, equivalent to that of commercial montmorillonite clay (99.5%). However, these treatments also sequestered microminerals, particularly selenium and iron. This pH-stable, high-capacity adsorbent could be used in poultry feed to reduce the uptake of AFB1.</p