RESUMEN
In vitro fertilization (IVF) can be used to assess the fertilization capacity of sperm. Heterologous IVF may be useful when assessing that of wild animals as it is often difficult to obtain adequate numbers of naturally corresponding oocytes. The aim of the present study was to assess the fertilization capacity of frozen-thawed ibex epididymal spermatozoa via heterologous IVF involving the oocytes of prepubertal domestic goats. The effect on fertilization and embryo development of adding oestrous sheep serum (ESS) to the fertilization medium was also examined. Cumulus-oocyte complexes (COCs) were matured in TCM-199 for 24-27 h at 38.5°C in a 5% CO2 in air atmosphere. Frozen-thawed epididymal spermatozoa were selected by density gradient centrifugation. After maturation, the oocytes were co-incubated with spermatozoa in synthetic oviductal fluid (SOF) with different concentrations of ESS: SOF-C (0%), SOF-2 (2%) and SOF-20 (20%). At 17 h post-insemination (hpi), zygotes with one female and one male pronucleus (2PN) were categorised as normal; zygotes with 3PN were recorded as polyspermic, and oocytes with 1PN as asynchronous. Cleavage and blastocyst development were assessed at 48 and 168 hpi respectively. The percentage of zygotes with 2PN was higher in the SOF-2 than in the SOF-20 treatment group (27.7% versus 2.9% P < 0.05). The percentage of blastocysts formed with the SOF-C, SOF-2 and SOF-20 treatments were 1.1%, 7.5% and 0% respectively. These results show that the presence of 2% ESS achieves better results than the use of no serum or the standard 20% concentration. Heterologous IVF may be an effective method for predicting the fertilization capacity of ibex spermatozoa, and therefore perhaps that of other wild mountain ungulates.
Asunto(s)
Epidídimo/citología , Fertilización In Vitro/métodos , Cabras , Técnicas de Maduración In Vitro de los Oocitos/métodos , Preservación de Semen/métodos , Espermatozoides/fisiología , Animales , Blastocisto/fisiología , Criopreservación/métodos , Estro/sangre , Femenino , Fertilización , Masculino , Suero , Motilidad EspermáticaRESUMEN
The aim of this work was to evaluate the protective effect of catalase (CAT) on frozen/thawed ibex epididymal sperm recovered post mortem, and to detect any harmful effect this might have on sperm fertilisation capacity. Epididymal spermatozoa were diluted using a Tris-citric acid-glucose medium (TCG) composed of 3.8% Tris (w/v), 2.2% citric acid (w/v), 0.6% glucose (w/v), 5% glycerol (v/v), and 6% egg yolk (v/v). Sperm masses from the right epididymis were diluted with TCG medium, while those from the left were diluted with TCG medium supplemented with 200IU/mL CAT. Heterologous in vitro fertilisation (IVF) was used to assess the fertilisation capacity of this sperm. The addition of CAT to the extender did not improve frozen/thawed sperm variables. Moreover, a reduced fertilisation capacity was detected: sperm diluted with TCG provided 25.5% 2PN zygotes, while just 13.2% was recorded for that diluted with TCG-CAT (P<0.01). The percentage of cleaved embryos at 48hpi was higher (P<0.01) with the TCG sperm than with the TCG-CAT sperm (16.7% vs. 7.6%). The use of 200IU/mL CAT as an additive cannot, therefore, be recommended for the preservation of ibex epididymal sperm. Other antioxidants should, however, be tested in both this and related wild mountain ungulates.
Asunto(s)
Catalasa/metabolismo , Criopreservación/veterinaria , Fertilización In Vitro/veterinaria , Cabras/fisiología , Preservación de Semen/veterinaria , Espermatozoides/citología , Animales , Antioxidantes/metabolismo , Criopreservación/métodos , Crioprotectores/metabolismo , Epidídimo/citología , Femenino , Fertilización In Vitro/métodos , Masculino , Preservación de Semen/métodos , Espermatozoides/metabolismoRESUMEN
Possible effects of repeated hormonal treatments and laparoscopic ovum pick-up (LOPU) on the efficiency of oocyte recovery rate and quality were determined in sheep and goats. In six adult Merino sheep and five Criolla goats, ovarian status was synchronized by a prostaglandin F(2 alpha) analogue and the insertion of an intravaginal sponge 48 h later. Follicle development was stimulated by a single dose of FSH (60 mg NIH-FSH-P1) plus a single dose of equine chorionic gonadotrophin (eCG; 300 UI). The first FSH/eCG doses were administered 48 h after the sponge insertion, being repeated every 4 days to complete a total of four treatments in sheep and three in goats. Follicles in both ovaries were categorized according to their diameter and follicular fluid was aspirated under laparoscopic observation without a vacuum pump. In sheep, during a 12-day-period, a total of 347 follicles were aspirated with a recovery rate of 46.9%. In goats, during an 8-day-period, 219 follicles were aspirated with a recovery rate of 45.6%. In both species, there were no significant differences in the number of aspirated follicles, oocyte recovery rate and good quality oocyte recovery rate. However, in sheep the oocyte recovery rate was higher for large follicles, whereas in goats no such effect was detected. In summary, current results indicate that retrieval of oocytes can be maximized, without affecting oocyte quality, by repeating 'oneshot' FSH/eCG regimes and LOPUs at intervals as short as 4 days.
Asunto(s)
Cabras/fisiología , Donación de Oocito/veterinaria , Oocitos/fisiología , Ovinos/fisiología , Animales , Femenino , Fertilización In Vitro/veterinaria , Recolección de Tejidos y Órganos/veterinariaRESUMEN
BACKGROUND: The role that growth factors and their receptors play in human cancer growth and progression makes them interesting targets for novel treatment modalities. Our approach consisted of active immunotherapy with the epidermal growth factor (EGF). Two pilot clinical trials were conducted to examine the safety and immunogenicity of a five-dose immunization protocol and to compare different adjuvants and treatment designs. PATIENTS AND METHODS: Forty patients with advanced non-small-cell lung cancer were enrolled in both trials. They were randomized to be treated with aluminum hydroxide or montanide ISA 51 as adjuvants in the EGF vaccine preparation. The use of cyclophosphamide prevaccination treatment was evaluated in the second trial. RESULTS: Pooled data from both trials showed that the use of montanide as adjuvant increased the percentage of good antibody responders (GAR). Cyclophosphamide prevaccination treatment did not provoke improvements in antibody response. GAR had a significant increase in survival as compared with poor antibody responders. Response duration was also related to a significant improvement in survival rates. CONCLUSIONS: Vaccination with five doses of EGF vaccine is safe and immunogenic. Montanide ISA 51 increased the percentage of GAR. There is a direct relationship between anti-EGF antibody titers and immune response duration with survival time.
Asunto(s)
Vacunas contra el Cáncer/farmacología , Carcinoma de Pulmón de Células no Pequeñas/inmunología , Carcinoma de Pulmón de Células no Pequeñas/terapia , Factor de Crecimiento Epidérmico/inmunología , Factor de Crecimiento Epidérmico/farmacología , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/terapia , Manitol/análogos & derivados , Adyuvantes Inmunológicos/administración & dosificación , Adulto , Anciano , Anciano de 80 o más Años , Hidróxido de Aluminio/administración & dosificación , Formación de Anticuerpos , Vacunas contra el Cáncer/efectos adversos , Vacunas contra el Cáncer/inmunología , Femenino , Humanos , Masculino , Manitol/administración & dosificación , Persona de Mediana Edad , Ácidos Oléicos/administración & dosificación , Análisis de Supervivencia , VacunaciónRESUMEN
Ior egf/r3, a neutralizing monoclonal antibody (mAb) against Epidermal Growth Factor Receptor (EGFR) was generated at the Cuban Institute of Oncology. Immunoscintigraphic studies in 148 patients with this 99-m Technetium (99Tc) labeled mAb, showed a high sensitivity and specificity for in vivo detection of epithelial tumors. To study safety, pharmacokinetic and immunogenicity of ior egf/r3 at high doses, a phase I clinical trial was conducted. Nineteen patients with advanced epithelial tumors received 4 mAb intravenous infusions at 6 dose levels: from 50 to 500 mg. Previously, immunoscintigraphic images using the same mAb labeled with 99Tc were acquired. Blood samples were collected for pharmacokinetic analysis and HAMA response. After mAb therapy, objective response was classified according to WHO criteria. Ior egf/r3 was well tolerated in spite of the high-administered doses. Only a severe adverse reaction consisting of hypotension and lethargy was observed. In 13 patients, selective accumulation of 99Tc-labeled mAb was observed at the site of the primary tumor or the metastasis. Pharmacokinetic analysis revealed that elimination half-life and the area under the time-concentration curve increased linearly with dose. HAMA response was detected in 17 patients. After 6 months of mAb therapy, 4 patients had stable disease. One patient had a tumor partial remission after 3 cycles of ior egf/r3.
Asunto(s)
Anticuerpos Monoclonales , Anticuerpos Antineoplásicos , Carcinoma/diagnóstico por imagen , Receptores ErbB/inmunología , Proteínas de Neoplasias/inmunología , Neoplasias/diagnóstico por imagen , Radioinmunodetección , Radiofármacos , Tecnecio , Adulto , Anciano , Animales , Anticuerpos Antiidiotipos/biosíntesis , Anticuerpos Heterófilos/biosíntesis , Anticuerpos Monoclonales/efectos adversos , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacocinética , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Antineoplásicos/efectos adversos , Anticuerpos Antineoplásicos/inmunología , Anticuerpos Antineoplásicos/uso terapéutico , Especificidad de Anticuerpos , Carcinoma/radioterapia , Carcinoma/terapia , Estudios de Cohortes , Terapia Combinada , Ensayo de Inmunoadsorción Enzimática , Femenino , Fiebre/inducido químicamente , Estudios de Seguimiento , Semivida , Humanos , Hipotensión/inducido químicamente , Neoplasias Pulmonares/diagnóstico por imagen , Neoplasias Pulmonares/radioterapia , Neoplasias Pulmonares/terapia , Masculino , Ratones , Persona de Mediana Edad , Neoplasias/radioterapia , Neoplasias/terapia , Pruebas de Neutralización , Radioinmunoterapia , Radiofármacos/efectos adversos , Radiofármacos/farmacocinética , Radiofármacos/uso terapéutico , Seguridad , Especificidad de la Especie , Tecnecio/efectos adversos , Tecnecio/farmacocinética , Tecnecio/uso terapéutico , Distribución TisularRESUMEN
BACKGROUND: There is evidence of a relationship between epidermal growth factor (EGF) and tumor cell proliferation, such as the overexpression of EGF receptor (EGF-R) in different human tumors, which makes this system an interesting target for cancer treatment. Up to now, passive immunotherapy with monoclonal antibodies against the EGF-R has been assayed in clinics. Our approach consists of active immunotherapy with human EGF (hu-EGF). We conducted a pilot clinical trial to define the safety, toxicity and immunogenicity of vaccination with hu-EGF coupled to a carrier protein. PATIENTS AND METHODS: Ten patients with histologically-proven malignant carcinomas (colon, lung, stomach and prostate) in advanced clinical stages were enrolled. Patients were immunized twice (on days 0 and 15) with hu-EGF linked to either tetanic toxoid (TT, five patients) or P64K Neisseria Meningitidis recombinant protein (P64k, five patients), intradermically, using aluminium hydroxyde as adjuvant. RESULTS: In both groups 60% of patients developed anti-EGF antibody titers without evidence of toxicity. Secondary reactions were very mild, limited to erythema and itching at the site of injection, which disappeared without medication. CONCLUSIONS: We conclude that the proposed vaccination with hu-EGF was well tolerated and that antibody titers against self EGF were developed. The results of this trial may be useful in the design of new clinical trials with higher dose immunization protocols and using more effective adjuvants.