RESUMEN
Few vaccine adjuvants have been approved for human use although several are currently being studied in preclinical and clinical trial. MPL is a toll-like receptor agonist able to trigger a high and persistent antibody response via-TLR-4 while QS-21 activates the NLRP3 inflammasome. Data suggest that there is a cross-talk between Notch and TLR signaling pathways modulating the polarization of the immune response in a MyD88-dependent manner. However, the role of Notch on the mechanism action of immunogenic adjuvants has not been addressed yet. This study aims to evaluate the in vitro toxicity and inflammatory response triggered by MPL and QS-21 using an in vitro human cell co-culture model and to determine whether NFκB or Notch signaling pathways are involved in their mechanism of immunotoxicity. In order to do this, we evaluated the effect of QS- 21/MPL alone or in combination using a co-culture of PBMC and HUVEC using cytotoxicity, surface expression of ECAMs, cell adhesion and cytokine release, NF-κB activation and NOTCH1 expression as observation endpoints. We found that both MPL and QS-21 were cytotoxic at concentrations over 5 µg/mL. Both adjuvants were able to trigger the expression of ECAMs and induce firm adhesion of PBMC to the endothelium. QS-21 and MPL combination demonstrated a synergistic effect on cellular recruitment and cytokine release generating a switch from Th2 to Th1 cytokine profile. Both MPL and QS-21 by themselves were able to generate significant NF-κB activation. However, this effect was not observed when both adjuvants were combined. On the contrary, the adjuvants alone and combined induced an overexpression of NOTCH-1. This is an important finding, as it provides new evidence that these adjuvants could modulate reactogenicity of vaccines through Notch signaling.
Asunto(s)
Adyuvantes Inmunológicos/toxicidad , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Leucocitos Mononucleares/efectos de los fármacos , Lípido A/análogos & derivados , Receptor Notch1/genética , Saponinas/toxicidad , Adhesión Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Técnicas de Cocultivo , Citocinas/metabolismo , Interacciones Farmacológicas , Células Endoteliales de la Vena Umbilical Humana/fisiología , Humanos , Leucocitos Mononucleares/fisiología , Lípido A/toxicidad , FN-kappa B/metabolismoRESUMEN
Context: Influenza is a severe, life-threatening viral disease that can be prevented by vaccination. However, the anti-influenza human vaccine failed to show the required efficacy both in infants under 5 years old and in the elder population, who are among those with the highest risk of developing severe complications after influenza infection. Therefore, it is of high importance to improve the vaccine efficacy and ensure its safety in these susceptible populations. GK-1, a novel 18-aa peptide adjuvant, has been proved to increase the immunogenicity of the human influenza vaccine in both young and aged mice. Objective: A preclinical study of the toxicity profile of GK-1 following the World Health Organization guidelines to support its use was herein conducted. Material and methods: GK-1 was synthetically produced following Good Manufacturing Practices. The toxicological evaluation of GK-1 peptide was performed in rats after repeated dose-ranging trials by the subcutaneous route. The mutagenic potential of GK-1 was assessed by the micronucleus, chromosomal aberration, and Ames tests, in accordance with OECD Guidelines. Results: GK-1 did not show toxic effects at doses up to 12.5mg/kg, corresponding to 25 times the dose intended for human use. No indications of mutagenic potential were observed. GK-1 after dermal administration was well tolerated locally. Conclusion: The efficacy of GK-1 to improve influenza vaccine protection, along with the absence of toxicity and mutagenicity, as reported herein, support the evaluation of this peptide in a clinical trial as a novel adjuvant for human use.
Asunto(s)
Adyuvantes Inmunológicos/toxicidad , Aberraciones Cromosómicas/efectos de los fármacos , Daño del ADN , Vacunas contra la Influenza/inmunología , Péptidos Cíclicos/toxicidad , Animales , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Femenino , Humanos , Gripe Humana/prevención & control , Inyecciones Subcutáneas , Masculino , Pruebas de Mutagenicidad , Péptidos Cíclicos/inmunología , Ratas Wistar , Salmonella typhimurium/efectos de los fármacos , Salmonella typhimurium/genética , Pruebas de Toxicidad CrónicaRESUMEN
Catabolic conditions like acquired immunodeficiency syndrome, cancer, and burn can cause immunosuppression. Amino acids such as alanine and glutamine are essential for the activity of the immune system. Propolis is immunostimulant and the waste of propolis extraction has been reused with technological and therapeutic purposes. Therefore, this study describes the association of propolis byproduct extract (BPE) with pectin to prepare spray-dried microparticles containing the dipeptide l-alanyl-l-glutamine as stimulant systems of neutrophils. The use of a factorial design allowed selecting the best formulation, which was characterized by morphology, size, and entrapment efficiency analyses. In addition, the systems were characterized by thermal and X-ray diffraction analysis, Fourier-transform infrared spectroscopy, in vitro drug release, and in vitro cytotoxicity and stimulation test of neutrophils. Small well-structured microparticles with good entrapment efficiency values were achieved. Thermal stability of formulation was observed, and it was proved that pectin, BPE and l-alanyl-l-glutamine were dispersed throughout the matrix. The drug was released from the microparticles during 24 h governed by swelling and diffusion. The drug-loaded formulations showed a significant stimulating effect on neutrophils. These structures could increase the activity of immune cells, and other in vitro and in vivo studies should be performed in the future.
Asunto(s)
Dipéptidos/administración & dosificación , Neutrófilos/efectos de los fármacos , Pectinas/química , Própolis/química , Adyuvantes Inmunológicos/administración & dosificación , Adyuvantes Inmunológicos/farmacología , Adyuvantes Inmunológicos/toxicidad , Química Farmacéutica/métodos , Dipéptidos/farmacología , Dipéptidos/toxicidad , Portadores de Fármacos/química , Liberación de Fármacos , Humanos , Técnicas In Vitro , Microesferas , Neutrófilos/metabolismo , Espectroscopía Infrarroja por Transformada de Fourier , Factores de Tiempo , Difracción de Rayos XRESUMEN
Sporotrichosis is an important zoonosis in Brazil and the most frequent subcutaneous mycosis in Latin America, caused by different Sporothrix species. Currently, there is no effective vaccine available to prevent this disease. In this study, the efficacy and toxicity of the adjuvant Montanide™ Pet Gel A (PGA) formulated with S. schenckii cell wall proteins (ssCWP) was evaluated and compared with that of aluminum hydroxide (AH). Balb/c mice received two subcutaneous doses (1st and 14th days) of either the unadjuvanted or adjuvanted vaccine candidates. On the 21st day, anti-ssCWP antibody levels (ELISA), the phagocytic index, as well as the ex vivo release of IFN-γ, IL-4, and IL-17 by splenocytes and IL-12 by peritoneal macrophages were assessed. Cytotoxicity of the vaccine formulations was evaluated in vitro and by histopathological analysis of the inoculation site. Both adjuvanted vaccine formulations increased anti-ssCWP IgG, IgG1, IgG2a, and IgG3 levels, although IgG2a levels were higher in response to PGA+CWP100, probably contributing to the increase in S. schenckii yeast phagocytosis by macrophages in the opsonophagocytosis assay when using serum from PGA+CWP100-immunized mice. Immunization with AH+CWP100 led to a mixed Th1/Th2/Th17 ex vivo cytokine release profile, while PGA+CWP100 stimulated a preferential Th1/Th2 profile. Moreover, PGA+CWP100 was less cytotoxic in vitro, caused less local toxicity and led to a similar reduction in fungal load in the liver and spleen of S. schenckii- or S. brasiliensis-challenged mice as compared with AH+CWP100. These results suggest that PGA may be an effective and safe adjuvant for a future sporotrichosis vaccine.
Asunto(s)
Adyuvantes Inmunológicos , Hidróxido de Aluminio/inmunología , Vacunas Fúngicas/efectos adversos , Vacunas Fúngicas/inmunología , Sporothrix/inmunología , Esporotricosis/prevención & control , Adyuvantes Inmunológicos/toxicidad , Hidróxido de Aluminio/toxicidad , Animales , Anticuerpos Antifúngicos/biosíntesis , Anticuerpos Antifúngicos/sangre , Anticuerpos Antifúngicos/inmunología , Brasil , Vacunas Fúngicas/administración & dosificación , Vacunas Fúngicas/química , Inmunidad Celular , Inmunogenicidad Vacunal , Interleucina-17/inmunología , Ratones , Ratones Endogámicos BALB C , Fagocitosis , Esporotricosis/inmunología , Balance Th1 - Th2 , VacunaciónRESUMEN
Local reactions are the most frequent adverse event associated with vaccines. Adjuvants are major constituents of many vaccines and they are frequently involved in these reactions, associated with their irritating effect and the stimulation of local inflammation. The hen's egg test on chorioallantoic membrane (HET-CAM) is an alternative toxicological method widely used to determine ocular irritation potential, but very few studies have demonstrated the utility of this method for assessing the irritant properties of vaccine adjuvants. In this work, known/experimental adjuvants were evaluated by both HET-CAM and an in vivo local toxicity study in mice to compare irritation scores to determine whether there was a correlation (Pearson test). Based on these data (r = 0.9034; P < 0.0001), the HET-CAM assay can be used as an alternate method for the prediction of the local toxicity potential of adjuvant candidates to be used in vaccines.
Asunto(s)
Adyuvantes Inmunológicos/toxicidad , Membrana Corioalantoides/efectos de los fármacos , Irritantes/toxicidad , Alternativas a las Pruebas en Animales , Animales , Bioensayo , Pollos , Femenino , Ratones Endogámicos BALB C , Piel/efectos de los fármacos , VacunasRESUMEN
In this study, a preparation of saponins (QB-90U) extracted from leaves of Quillaja brasiliensis collected in Uruguay was evaluated as a vaccine adjuvant by comparison with alum and the well known saponin-based adjuvant, Quil A. The haemolytic activity and cellular toxicity of the saponin preparations were also evaluated. QB-90U was only slightly haemolytic and showed a low cytotoxicity when compared to Quil A. The adjuvant properties of QB-90U were assayed by sub-cutaneous immunization of mice with a preparation of inactivated bovine herpesvirus 5 (BoHV-5) either with no adjuvant or adjuvanted with QB-90U, Quil A or alum. Serum levels of anti-BoHV-5 IgG, IgG1, IgG2a, IgG2b and also IgG3 were significantly increased by QB-90U and were of the same order as those elicited by Quil A. Furthermore, high titres of neutralizing antibodies were found to be present in the serum of immunized animals from both groups. The cellular response induced by QB-90U did also reproduce the one elicited by Quil A. In fact, a robust DTH response was observed in mice immunized with both saponin preparations; as well as increased splenocytes levels of Th1-type cytokines, namely IFN-γ and IL-2. Taken together, the above results confirm and extend our previous observation regarding the similarity of the responses elicited by Quil A and the saponin preparation from Q. brasiliensis (Fleck et al., 2006) and indicate that QB-90U is worth of further studies as a safe and potent vaccine adjuvant.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Infecciones por Herpesviridae/prevención & control , Herpesvirus Bovino 5/patogenicidad , Quillaja/química , Saponinas/inmunología , Adyuvantes Inmunológicos/toxicidad , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Antígenos Virales/inmunología , Linfocitos T CD4-Positivos/inmunología , Chlorocebus aethiops , Citocinas/inmunología , Femenino , Hemólisis , Infecciones por Herpesviridae/inmunología , Inmunidad Humoral , Ratones , Saponinas de Quillaja , Saponinas/farmacología , Saponinas/toxicidad , Bazo/inmunología , Células VeroRESUMEN
The type I and type II heat-labile enterotoxins (LT-I and LT-II) are strong mucosal adjuvants when they are coadministered with soluble antigens. Nonetheless, data on the parenteral adjuvant activities of LT-II are still limited. Particularly, no previous study has evaluated the adjuvant effects and induced inflammatory reactions of LT-II holotoxins or their B pentameric subunits after delivery via the intradermal (i.d.) route to mice. In the present report, the adjuvant and local skin inflammatory effects of LT-IIa and its B subunit pentamer (LT-IIaB(5)) were determined. When coadministered with ovalbumin (OVA), LT-IIa and, to a lesser extent, LT-IIaB(5) exhibited serum IgG adjuvant effects. In addition, LT-IIa but not LT-IIaB(5) induced T cell-specific anti-OVA responses, particularly in respect to induction of antigen-specific cytotoxic CD8(+) T cell responses. LT-IIa and LT-IIaB(5) induced differential tissue permeability and local inflammatory reactions after i.d. injection. Of particular interest was the reduced or complete lack of local reactions, such as edema and tissue induration, in mice i.d. inoculated with LT-IIa and LT-IIaB(5,) respectively, compared with mice immunized with LT-I. In conclusion, the present results show that LT-IIa and, to a lesser extent, LT-IIaB(5) exert adjuvant effects when they are delivered via the i.d. route. In addition, the low inflammatory effects of LT-IIa and LT-IIaB(5) in comparison to those of LT-I support the usefulness of LT-IIa and LT-IIaB(5) as parenterally delivered vaccine adjuvants.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Toxinas Bacterianas/administración & dosificación , Toxinas Bacterianas/inmunología , Enterotoxinas/administración & dosificación , Enterotoxinas/inmunología , Proteínas de Escherichia coli/administración & dosificación , Proteínas de Escherichia coli/inmunología , Piel/efectos de los fármacos , Piel/inmunología , Adyuvantes Inmunológicos/toxicidad , Animales , Toxinas Bacterianas/toxicidad , Linfocitos T CD8-positivos/inmunología , Enterotoxinas/toxicidad , Proteínas de Escherichia coli/toxicidad , Femenino , Inmunoglobulina G/sangre , Inflamación/patología , Inyecciones Intradérmicas , Ratones , Ratones Endogámicos C57BL , Ovalbúmina/administración & dosificación , Ovalbúmina/inmunología , Linfocitos T Citotóxicos/inmunologíaRESUMEN
Over the last twenty years research has provided an important insight into the mechanisms responsible for the immunotoxicity of both local and systemic adverse reactions following the use of immunostimulating drugs and adjuvants. In this article we provide an update of the present knowledge relating to the various parameters and reactants of the immune system at the cellular as well as molecular level that are believed to play a key role in reactogenicity. We discuss evidence obtained from observations in vitro, in vivo in animal models and from clinical applications, including adjuvants used in large scale vaccination today. The data discussed are mainly taken from animal models following hyperstimulation of the immune system; either by the use of very powerful adjuvants, like Freund's that are too toxic for use in practical vaccination, by deliberate high dose application of adjuvants or by the in vivo application of cytokines. Although such hyperstimulating regimens are unlikely to find their way into practical vaccination of humans, this information is of great value as it may facilitate the understanding of the toxicity mechanisms, aid the design of standardised models for the assessment of adjuvant safety and the possible application of new adjuvants in vaccines for humans.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Adyuvantes Inmunológicos/toxicidad , Animales , Humanos , Hipersensibilidad/inmunología , Sistema Inmunológico/efectos de los fármacos , Vacunación/métodosRESUMEN
UNLABELLED: We used multiple pain models to investigate the effects of (-)-linalool, a monoterpene alcohol present in the essential oil of plants, on chronic inflammatory and neuropathic hypersensitivity in adult Swiss mice. Inflammatory or neuropathic hypersensitivity was induced by intraplantar (i.pl.) injection of complete Freund's adjuvant (CFA) or partial sciatic nerve ligation (PSNL), respectively. Twenty-four hours after CFA injection, we used Von Frey filaments and acetone-evoked cooling to evaluate tactile and thermal hypersensitivity, respectively. A single i.p. injection of (-)-linalool (50 or 200 mg/kg) administered 30 minutes before testing reduced CFA-induced mechanical hypersensitivity. Similarly, (-)-linalool reduced acetone-evoked hypersensitivity up to 4 hours after treatment. Compared with vehicle, (-)-linalool produced a marked reduction in CFA-induced paw edema. (-)-Linalool also reduced mechanical hypersensitivity induced by PSNL 7 days after injury. Multiple (-)-linalool treatments given chronically (twice a day for 10 days; 50 mg/kg, i.p.) significantly reduced mechanical hypersensitivity induced by CFA and PSNL. This multidose strategy did not cause tolerance. We also reasoned that (-)-linalool might reduce nociceptive behavior in response to direct administration of inflammatory mediators. Therefore, we injected the cytokines IL-1ß (.1 pg/site) and TNF-α (1 pg/site) intrathecally. (-)-Linalool inhibited the biting response induced by IL-1ß and TNF-α. PERSPECTIVE: The article adds information about antinociceptive properties of (-)-linalool in chronic inflammatory and neuropathic hypersensitivity. It also indicates that (-)-linalool might be potentially interesting in the development of new clinically relevant drugs for the management of persistent pain.
Asunto(s)
Mediadores de Inflamación/farmacología , Monoterpenos/farmacología , Neuralgia/prevención & control , Monoterpenos Acíclicos , Adyuvantes Inmunológicos/toxicidad , Animales , Enfermedad Crónica , Modelos Animales de Enfermedad , Femenino , Adyuvante de Freund/toxicidad , Hiperalgesia/tratamiento farmacológico , Hiperalgesia/patología , Hiperestesia/tratamiento farmacológico , Hiperestesia/patología , Mediadores de Inflamación/uso terapéutico , Ratones , Monoterpenos/uso terapéutico , Neuralgia/tratamiento farmacológico , Neuralgia/patología , Neuropatía Ciática/tratamiento farmacológico , Neuropatía Ciática/patologíaRESUMEN
La Solución CM-95 tratada magnéticamente es un producto en desarrollo que mostró propiedades inmunoestimulantes en ensayos preclínicos, característica que la hacen adecuada como candidata a inmunopotenciador. En este trabajo se evaluaron los posibles efectos tóxicos preclínicos de la Solución CM-95 tratada magnéticamente, por el método de las Clases de Toxicidad Aguda y el de irritación de la mucosa oral, adaptando las normas OECD 423 y la ISO 10993-10, respectivamente. En el método de las Clases de Toxicidad Aguda se utilizó el ensayo límite, en ratas Sprague Dawley hembras, en el cual la dosis estuvo relacionada con el nivel de inducción magnética, en este caso 0,16 T, aplicado a la Solución CM-95; y el volumen a administrar de la misma, calculado sobre la base de 2 ml de la solución por 100 g de peso corporal. La determinación de la irritación de la mucosa oral se llevó a cabo en hámster Sirios Dorados hembras mediante un ensayo a dosis repetidas durante7 días de tratamiento en la bolsa gular derecha, con pellet de algodón impregnado con 0,5 ml de la solución tratada magnéticamente con la misma inducción. No se encontró mortalidad ni evidencias de signos tóxicos para el ensayo de toxicidad aguda, y se obtuvo un índice de irritación sobre mucosa oral de 0, por lo que la sustancia estudiada se enmarcó como No clasificaday No irritante según la metodología empleada. Estos resultados complementarán otros estudios toxicológicos para avalar la seguridad de esta Solución para su uso futuro como fármaco por vía oral.
CM-95 solution magnetically treated is a product which showed immunologic properties in preliminary tests, characteristic that makes it adequate as inmunopotentiator candidate. In this study the possible preclinical toxic effects of CM-95 Solution magnetically treated were evaluated, by the Acute Toxicity Class method and oral mucosa irritation test, adapting guideline OECD423 and ISO 10993-10. In Acute Toxicity Class method was used the Limit Test, in Sprague Dawley females rats, where the dose was related to the magnetic induction level, in this case 0.16 T, applied to CM-95 Solution; and the administration volume of aqueous solution was calculated on base 2 ml per 100 g body weight. Determination of oral mucosa irritation was carried out in female golden Syrian hamsters by means of a repeated doses test during 7 days of treatment in right gular bag, with a cotton pellet impregnated with 0.5 ml of CM-95 Solution magnetically treated with the same induction. Neither mortality nor evidences of toxic signs were observed for the test of acute toxicity, and an irritation index of 0 was obtained on oral mucosa irritation test, reason why the studied substance was framed as not classified and non irritating according to the applied methodology. These results will complement other toxicological studies to guarantee the safety of this Solution for its future use as a drug by oral route.
Asunto(s)
Animales , Femenino , Ratas , Pruebas de Toxicidad Aguda , Adyuvantes Inmunológicos/efectos adversos , Adyuvantes Inmunológicos/toxicidad , Mucosa Bucal , Cricetinae , Sistema Inmunológico , Mesocricetus , Ratas Sprague-DawleyRESUMEN
The aim of the present work was to study some of the adverse effects produced by hyperandrogenism on the uterine function. Daily injection of dehydroepiandrosterone (DHEA: 6 mg/ 100 g body weight, sc) for 20 consecutive days induced polycystic ovaries in BALB/c mice. In this model, we found that DHEA produced alterations on uterine histology closely related to the development of tumour structures. In addition, hyperandrogenism induced a pro-inflammatory and a pro-oxidant condition represented by increased levels of prostaglandin F2 alpha production and uterine nitric oxide synthase (NOS) activity and by a decrease in both superoxide dismutase (SOD) and catalase (CAT) activities together with a decrease in the levels of the antioxidant metabolite glutathione (GSH). DHEA also induced an increase in CD4+ together with a decrease in the CD8+ T lymphocytes that infiltrate the uterine tissue. We conclude that this intricate network of regulators could be responsible for the low rate of implantation observed in women with polycystic ovary syndrome.
Asunto(s)
Adyuvantes Inmunológicos/toxicidad , Deshidroepiandrosterona/toxicidad , Hiperandrogenismo/fisiopatología , Síndrome del Ovario Poliquístico/inducido químicamente , Útero/fisiopatología , Animales , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Catalasa/antagonistas & inhibidores , Catalasa/metabolismo , Ciclooxigenasa 1/efectos de los fármacos , Ciclooxigenasa 2/efectos de los fármacos , Dinoprost/biosíntesis , Femenino , Glutatión/antagonistas & inhibidores , Glutatión/metabolismo , Hiperandrogenismo/patología , Proteínas de la Membrana/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Óxido Nítrico Sintasa/efectos de los fármacos , Óxido Nítrico Sintasa/metabolismo , Ovario/patología , Estrés Oxidativo/efectos de los fármacos , Síndrome del Ovario Poliquístico/inmunología , Síndrome del Ovario Poliquístico/patología , Prostaglandinas E/biosíntesis , Superóxido Dismutasa/antagonistas & inhibidores , Superóxido Dismutasa/metabolismo , Útero/inmunología , Útero/patologíaRESUMEN
En este trabajo se realizó la evaluación toxicológica a dosis repetidas por el método de test límite del candidato ainmunopotenciador, la solución CM-95 tratada magnéticamente, acorde con las regulaciones de la Organización para la Colaboración Económica y el Desarrollo, incluida en la Guía 407. El objetivo fue establecer las posibles lesiones orgánicas y funcionales ocasionadas por la solución CM-95, tratada magnéticamente con la máxima inducción magnética permisible (0,16 T), para la obtención del candidato a inmunopotenciador. Se emplearon tres grupos: Experimental, Control y Satélite. Como Biomodelo experimental se utilizaron ratas Sprague Dawley machos y hembras con pesos de 150 a 200 g. Durante el estudio no se registraron signos clínicos de toxicidad ni muertes en ninguno de los animales de los grupos tratados, ni en los controles. No hubo afectación del peso corporal durante elensayo. Aunque hubo variaciones en los valores de algunos parámetros hematológicos y bioquímicos, estos no tuvieron significación biológica. No se encontraron lesiones macroscópicas, ni microscópicas; solo se observaron efectos proliferativos en el tejido linfoide de timo y bazo, relacionados con la respuesta del sistema inmune. Lasolución CM-95 tratada magnéticamente, no mostró toxicidad en el modelo animal y nivel de dosis utilizado, y bajo las condiciones experimentales ensayadas(AU)
In this study the toxicological evaluation at repeated dose by the method of limit test of the candidate to magnetically treated immunopotentiator CM-95 Solution was carried out; according with regulations of the Organization for Economic Cooperation and Development included in guide 407. The objective was to establish the possible organic and functional lesions caused bythe magnetically treated CM-95 Solution with maximum permissible magnetic induction (0,16 T), for obtaining the candidate to immunopotentiator. Three groups were used: Experimental, Control and Satellite. As experimental Biomodels, male and female Sprague Dawley rats were used, with 150 to 200 g weights. During the study neither clinical signs of toxicity nor deathsin any of the animals of the treated groups were registered. There was not affectation of the corporal weight during the test. There was not change of the corporal weight during the trial. Although there were variations of some haematology and biochemical parameters, they had no biological significance. Macroscopic or microscopic lesions were not found; justproliferation effects were observed, in the lymphoid tissues of the thymus and spleen, related to the response of immune system. Magnetically treated CM-95 Solution, showed no toxicity in the animal model and dose level used, and under the observed experimental conditions(AU)
Asunto(s)
Animales , Ratas , Adyuvantes Inmunológicos/toxicidad , Dosis Repetida/efectos adversos , Ratas Sprague-DawleyRESUMEN
Chronic pain is associated with N-methyl-D-aspartate (NMDA) receptor activation and downstream production of nitric oxide, which has a pivotal role in multisynaptic local circuit nociceptive processing in the spinal cord. The formation of nitric oxide is catalyzed by three major nitric oxide synthase (NOS) isoforms (neuronal, nNOS; inducible, iNOS; endothelial, eNOS), which are increased in the spinal cord of rodents subjected to some tonic and chronic forms of experimental pain. Despite the important role of NOS in spinal cord nociceptive transmission, there have been no studies exploring the effect of NMDA receptor blockade on NOS expression in the dorsal horn during chronic pain. Furthermore, NOS isoforms have not been fully characterized in the dorsal horn of animals subjected to arthritic pain. The aim of this work was therefore to study the expression of nNOS, iNOS and eNOS in the dorsal horns of monoarthritic rats, and the modifications in NOS expression induced by pharmacological blockade of spinal cord NMDA receptors. Monoarthritis was produced by intra-articular injection of complete Freund's adjuvant into the right tibio-tarsal joint. At week 4, monoarthritic rats were given either the competitive NMDA antagonist (+/-)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) or the uncompetitive NMDA antagonist ketamine. After 6 and 24 hours, animals were killed and posterior quadrants of the lumbar spinal cord were dissected. Sample tissues were homogenized and subjected to immunoblotting with anti-nNOS, anti-iNOS or anti-eNOS monoclonal antibodies. The nNOS isoform, but not the iNOS and eNOS isoforms, were detected in the dorsal horns of control rats. Monoarthritis increased the expression of nNOS, iNOS and eNOS in the dorsal horns ipsilateral and contralateral to the inflamed hindpaw. Intrathecal administration of CPP and ketamine reduced nNOS expression in monoarthritic rats but increased the expression of iNOS and eNOS. Results suggest that blockade of spinal cord NMDA receptors produces complex regulatory changes in the expression of NOS isoforms in monoarthritic rats that may be relevant for nitridergic neuronal/glial mechanisms involved in the pathophysiology of monoarthritis and in the pharmacological response to drugs interacting with NMDA receptors.
Asunto(s)
Artritis Experimental/enzimología , Artritis Experimental/fisiopatología , Óxido Nítrico Sintasa/biosíntesis , Dolor/fisiopatología , Células del Asta Posterior/metabolismo , Adyuvantes Inmunológicos/toxicidad , Animales , Artritis Experimental/tratamiento farmacológico , Western Blotting , Antagonistas de Aminoácidos Excitadores/administración & dosificación , Adyuvante de Freund/toxicidad , Lateralidad Funcional , Inyecciones Espinales , Isoenzimas/biosíntesis , Isoenzimas/efectos de los fármacos , Masculino , Óxido Nítrico Sintasa/efectos de los fármacos , Dolor/etiología , Células del Asta Posterior/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidoresRESUMEN
The chemical characterization of aqueous extracts (AE) of barks, leaves and branches and the saponin fraction denominated QB-90 obtained from Quillaja brasiliensis, a native species from Southern Brazil, show remarkable similarities to Quillaja saponaria saponins which are known as adjuvants in vaccine formulations. In vivo toxicity assays of AE and QB-90 showed not to be lethal for mice in doses ranging from 50 to 1600 microg and 50-400 microg, respectively. Experimental vaccines prepared with bovine herpesvirus type 1 (BHV-1) antigen and either AE (barks 100 microg, leaves 400 microg, branches 400 microg) or QB-90 (100 microg) were able to enhance the immune responses of mice in a comparable manner to saponins from Q. saponaria (QuilA, 100 microg). BHV-1 specific IgG, IgG1 and IgG2a antibody levels in serum were also significantly enhanced by AE, QB-90 and QuilA compared to control group (p<0.05). These results showed that AE and QB-90 from Q. brasiliensis are potential candidates as adjuvants in vaccines.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Herpesvirus Bovino 1/inmunología , Quillaja/química , Saponinas/farmacología , Adyuvantes Inmunológicos/toxicidad , Animales , Anticuerpos Antivirales/biosíntesis , Ensayo de Inmunoadsorción Enzimática , Hidrólisis , Esquemas de Inmunización , Inmunoensayo , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/inmunología , Inyecciones Subcutáneas , Espectroscopía de Resonancia Magnética , Ratones , Corteza de la Planta/química , Extractos Vegetales/química , Extractos Vegetales/toxicidad , Hojas de la Planta/química , Saponinas/toxicidad , Estimulación QuímicaRESUMEN
We evaluated the adjuvant properties and toxicity of purified Neisseria meningitidis serogroup B lipopolysaccharide (LPS) conjugated with tetanus toxoid (TT) using a new method of conjugation to obtain amine groups in the polysaccharide structure. The endotoxic activity of treated LPS was reduced 2400 times as determined by Limulus amoebocyte assay and no mortality was observed in Balb/c mice inoculated with detoxified LPS versus 100% mortality in native LPS inoculated mice. The conjugated LPS-TT elicited in mice higher anti-TT IgG2a and IgG1 than unconjugated TT. In addition, high levels of anti-LPS IgG and IgG subclasses were detected in sera. These results evidence the adjuvant activity of detoxified LPS and may suggest that the conjugation to TT changes the LPS immune response from thymus-independent to thymus-dependent.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Lipopolisacáridos/farmacología , Neisseria meningitidis Serogrupo B , Toxoide Tetánico/inmunología , Adyuvantes Inmunológicos/química , Adyuvantes Inmunológicos/toxicidad , Animales , Lipopolisacáridos/química , Lipopolisacáridos/toxicidad , Ratones , Ratones Endogámicos BALB C , Vacunas Conjugadas/inmunologíaRESUMEN
The aim of this study was to investigate the effects of chronically administered aluminum on erythropoiesis in rats. After treatment (i.p. injections of Al(2)(SO(4))(3), 50 micromol/kg body weight, five times a week) for 3 months, the treated (Al) group showed significantly decreased hemoglobin concentration (32%) and hematocrit (24%) compared with the control group. Serum iron decreased significantly in the Al group, whereas total iron binding capacity did not change. Treatment did not alter the activity of hepatic, renal or cerebral delta-ALA-D. Biochemical measurements related to 2-thiobarbituric acid-reactive substance (TBARS) levels from serum and hepatic, renal and cerebral homogenates also did not change after treatment. Hepatic concentrations of aluminum were higher in the Al group than in the control group. Renal and cerebral aluminum concentrations did not vary between groups. The present results indicate that exposure to aluminum sulfate promotes signs of anemia in rats as a consequence of alterations in iron status.
Asunto(s)
Adyuvantes Inmunológicos/toxicidad , Compuestos de Alumbre/toxicidad , Eritropoyesis/efectos de los fármacos , Adyuvantes Inmunológicos/farmacocinética , Compuestos de Alumbre/farmacocinética , Animales , Inyecciones Intraperitoneales , Hierro/sangre , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Ratas , Ratas Wistar , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismo , Distribución TisularRESUMEN
In the search for more potent and less toxic immunomodulators, adamantylamide dipeptide (AdDP) was synthesized by the covalent union of amantadine with the L-alanyl-D-isoglutamine residue of muramyldipeptide (MDP). The present experiments demonstrate the ability of AdDP, co-administered with a protein immunogen, to raise or enhance a humoral response in immunized animals. BALB/c mice were immunized either by the intraperitoneal (ip) or oral route with ovalbumin (Ova) alone or combined with either AdDP or CpG oligonucleotide (ODN-CpG), a proved adjuvant. A clear adjuvant dose-response relationship was observed on the increment of Ova-specific serum antibody titers when AdDP was used as adjuvant, irrespectively of the administration route. The IgG isotype analysis showed that AdDP promotes a consistent increment in IgG1 antibodies associated with a dominant Th2 response pattern. When administered by the oral route, AdDP was at least as efficient as ODN-CpG as adjuvant. Similar results were obtained in rabbits immunized by the oral route, suggesting that the adjuvanticity of AdDP is not restricted to the murine system. In conclusion, AdDP was shown to be a powerful and non-toxic adjuvant at both systemic and mucosal levels, which makes it a promising tool for vaccine development.
Asunto(s)
Adyuvantes Inmunológicos , Amantadina/análogos & derivados , Amantadina/inmunología , Dipéptidos/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Adyuvantes Inmunológicos/toxicidad , Administración Oral , Amantadina/administración & dosificación , Amantadina/toxicidad , Animales , Islas de CpG/inmunología , Dipéptidos/administración & dosificación , Dipéptidos/toxicidad , Relación Dosis-Respuesta Inmunológica , Heces/química , Inmunoglobulina A/biosíntesis , Inmunoglobulina A/inmunología , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/inmunología , Isotipos de Inmunoglobulinas/biosíntesis , Isotipos de Inmunoglobulinas/inmunología , Inyecciones Intraperitoneales , Linfocinas/metabolismo , Ratones , Ratones Endogámicos BALB C , Mucosa Bucal/inmunología , Ovalbúmina/inmunología , Conejos , Especificidad de la Especie , Células Th2/inmunología , Células Th2/metabolismoRESUMEN
Crotoxin isolated from the venom of Crotalus durissus terrificus (South American rattlesnake) was incorporated into liposomes by the dehydration-rehydration vesicle method using different membrane compositions and the co-encapsulation of immunostimulants. Crotoxin was also encapsulated into liposomes formed from a non-phospholipid amphiphile, a mixture of polyoxyethylene 2-cetyl ether, dicetyl phosphate and cholesterol. The preparations were characterized in relation to stability, toxicity and the protection of mice against whole venom after immunization. All liposome preparations were quite stable, retaining more than 75% of the originally encapsulated crotoxin after 1 week of incubation at physiological temperature. Co-encapsulation with lipopolysaccharide increased the leakage of crotoxin. In contrast, co-encapsulation of the lipid moiety of lipopolysaccharide did not influence the stability of liposomes. Toxicity of liposomes was dependent on membrane composition. Liposomes made with phospholipids that were resistant to phospholipase A(2) activity were less toxic. Mice immunized with three doses of the 1 x LD50 of crotoxin encapsulated into liposomes, and with associated immunostimulants, were protected against challenge with 8 x subcutaneous LD50 of C. durissus terrificus venom. Using the same immunization schedule, liposomes made from a non-phospholipid mixture and without immunostimulants achieved 100% protection.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Adyuvantes Inmunológicos/química , Crotoxina/administración & dosificación , Crotoxina/inmunología , Membranas Artificiales , Adyuvantes Inmunológicos/toxicidad , Animales , Crotoxina/toxicidad , Femenino , Inyecciones Subcutáneas , Dosificación Letal Mediana , Liposomas , Ratones , Ratones Endogámicos BALB CRESUMEN
Neste trabalho, nos investigamos o efeito da 8-Bromoguanosina, um composto imunoestimulador, na citotoxicidade de macrofagos infectados com Leishmania amazonensis em um sistema in vitro. Os resultados mostraram que macrofagos tratados com 8-Bromoguanosina pre- ou pos-infeccao foram capazes de reduzir a carga parasitaria, monitorada pelo numero de amastigotas por macrofago e a percentagem de celulas infectadas (i.e. indice fagocitico). Sendo a 8-Bromoguanosina inocua para promastigotas, concluimos que o composto induz ativacao celular. Os macrofagos produziriam interferon alfa e beta e teriam seus mecanismos leishmanicidas estimulados. Esses resultados sugerem que compostos como a 8-Bromoguanosina (ribonucleosideos de guanina) podem auxiliar no tratamento contra patogenos intracelulares
Asunto(s)
Animales , Ratones , Células Asesinas Naturales , Nucleótidos de Guanina/toxicidad , Técnicas In Vitro , Leishmania/inmunología , Adyuvantes Inmunológicos/toxicidad , Técnicas de Cultivo de Célula , Enfermedades Parasitarias/terapia , Interferón-alfa/farmacología , Interferón beta/farmacología , Leishmania/clasificaciónRESUMEN
An 87.7% (P < 0.01) and 84% (P < 0.001) of protection against visceral leishmaniasis was achieved in CB hamsters and Balb/c mice, respectively, with saponin combined to the fucose-mannose ligand of Leishmania donovani (FML). However, an undesirable haemolytic effect was described for several saponins. Aiming to improve the formulation with FML/saponin, we comparatively analysed the haemolytic potential of recently characterized plant saponins and currently used adjuvants. The haemolytic activity of steroidic saponins from Agave sisalana; Smilax officinalis as well as commercial saponin (Riedel De Haën's), was higher than that of triterpenoid ones (Bredemeyera floribunda; Periandra mediterranea) and the Freund's complete adjuvant. The concentration resulting in 50% haemolysis was 500 micrograms ml-1 for aluminum hydroxide. The low haemolytic effect of P. mediterranea saponin was abolished by removal of its glycidic moiety and its sapogenin fraction as well as the Freund's Incomplete Adjuvant were non-haemolytic within this range. Furthermore, the adjuvant effect of three doses of P. mediterranea saponin injected with the FML antigen of L. donovani, was assayed in mice, either by the intraperitoneal (i.p.) or the subcutaneous (s.c.) route. The anti-FML IgG antibody levels increased and detectable levels were observed up to 3 months in the s.c. group. The response was expanded in both groups after an injection with a fourth vaccine dose. The IgG response showed increased levels of IgG2a only in the i.p. group, while IgG2b and IgG1 but not IgG3 antibodies were higher than controls in both groups. In conclusion, the results suggest that the recently described triterpenoid fractions of P. mediterranea can be safely used as adjuvant with low or non-haemolytic effect.