RESUMEN
Aim of this study was to test the reliability of Trypan blue/Giemsa staining to evaluate sperm membrane integrity, acrosomal intactness and morphology in stallion to verify whether it could be applied in vitro as useful tool for sperm fertilizing ability. Fertility data on inseminated mares were collected to evaluate the relationship of sperm quality to pregnancy rates. Forty-one ejaculates were collected from 3 stallions of Salernitano Horse Breed and evaluated for gross appearance, volume, visual motility and membrane integrity with Trypan blue/Giemsa staining and thirty-five mares were inseminated during the breeding season from April to July. Differences among stallions were found in volume, sperm concentration (p < 0.05) and visual motility (p < 0.01). A decrease in sperm motility, concentration (p < 0.05) and total sperm number was found in June-July (p < 0.01). Live sperm with intact acrosome (LSIA) and proximal droplets (PD) were lower (p < 0.01) in June-July, while acrosome reacted sperm (ARS) percentage increased (p < 0.05). No fertility differences were found among stallions with an average fertility per cycle of 44.6% and a pregnancy rate of 68.6%. Higher percentages of LSIA were found in the ejaculates used to inseminate mares that became pregnant vs those used in mares not pregnant (p < 0.05). The significance of LSIA as test variable to verify the reliability of Trypan blue/Giemsa staining was confirmed by Receiver operating characteristic ROC analysis and the sensitivity of the test was 85% at a cut-off value of 48% LSIA. Trypan blue-Giemsa showed to be an accurate method that can be applied on field to evaluate sperm membrane integrity and to identify poor-quality ejaculates.
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Colorantes Azulados , Membrana Celular/ultraestructura , Caballos , Espermatozoides/ultraestructura , Coloración y Etiquetado/veterinaria , Azul de Tripano , Acrosoma/ultraestructura , Reacción Acrosómica , Animales , Cruzamiento , Femenino , Fertilidad , Inseminación Artificial/veterinaria , Masculino , Embarazo , Índice de Embarazo , Curva ROC , Sensibilidad y Especificidad , Recuento de Espermatozoides/veterinaria , Motilidad Espermática , Espermatozoides/fisiología , Coloración y Etiquetado/métodosRESUMEN
Fetal genotyping has important applications in the horse, but currently necessitates embryo recovery and biopsy. We investigated whether fetal genotyping could be performed on yolk-sac fluid recovered from pregnant mares via transvaginal aspiration. Fluid was collected before Day 30 to provide results before establishment of the endometrial cups (Day 37). Genotyping and assessment of maternal DNA contamination was performed by analyzing histograms of PCR results for 19 loci. In Exp. 1, mares underwent yolk-sac aspiration on Days 22-28 of gestation. Fluid (0.56-1.02â¯mL) was recovered from five of seven mares. Four of the five mares maintained pregnancy. One pregnancy was electively terminated at Day 75; the other three mares delivered healthy foals. Extraction of DNA from the fluid sample followed by direct PCR allowed the highest rate of determination of fetal alleles. Fetal genotype was correctly determined in three samples, and for 14/19 alleles in one sample. In Exp. 2, we evaluated whether recovery of more fluid (up to 1.6â¯mL), and fractionation of the sample, would minimize maternal DNA contamination. One of four mares maintained pregnancy. Evaluation at informative loci showed no difference in maternal contamination among fractions. We determined that mares can maintain pregnancy after aspiration of yolk-sac fluid, and that fetal genotype can be accurately determined from the sample obtained. Further work is needed on factors affecting maintenance of pregnancy after the procedure. The ability to access the yolk sac in early pregnancy opens the door to novel potential clinical and research applications.
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Embrión de Mamíferos , Genotipo , Caballos/genética , Animales , Femenino , Embarazo , Saco VitelinoRESUMEN
The present study aimed to determine the influence of water pH on survival of curimbatá, Prochilodus lineatus, larvae. Forty-five 2-l glass aquaria with artificial aeration and no water exchange were stocked at a density of 10 larvae l(-1). The pH values used as treatments were 3.7 +/- 0.0, 4.0 +/- 0.0, 4.4 +/- 0.2, 4.6 +/- 0.2, 4.8 +/- 0.2, 5.1 +/- 0.2, 5.3 +/- 0.2, 5.6 +/- 0.2, 7.2 +/- 0.2, 8.7 +/- 0.4, 9.0 +/- 0.4, 9.2 +/- 0.4, 9.4 +/- 0.6, 9.7 +/- 0.5 and 10.0 +/- 0.5 in a completely random experimental design with three repetitions. Water pH was maintained by the addition of NaOH or H(2)SO(4) solutions. After 72 h of experiment, no survival was registered at pH below 4.6, 1.5% survival at pH 4.6, and about 50% survival at pH 9.4 and above. Survival rates between 70 and 80% were registered at pH 4.8-5.6 and at pH 7.2, whereas over 90% survival was registered at pH between 8.7 and 9.2.
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Peces/fisiología , Agua Dulce/química , Animales , Concentración de Iones de Hidrógeno , Larva/fisiología , Distribución Aleatoria , Análisis de Regresión , Análisis de Supervivencia , Factores de TiempoRESUMEN
Commercially available vaginal lubricants, typically labeled as non-spermicidal, are used to lubricate equine artificial vaginas prior to semen collection. Improper type or amount of lubricant might affect stallion sperm quality, either after short-time exposure or following cooled storage of extended semen previously exposed to lubricant. The aim of this study was to evaluate stallion sperm quality following exposure to lubricant-containing extender for 1â¯hâ¯(T1h) or 24â¯h (T24h). Three ejaculates were collected from each of four stallions using a small volume of petrolatum to lubricate artificial vaginas, and gel-free semen was diluted to 30â¯×â¯106 sperm/mL in extender containing: no lubricant (control), or 1 or 5% (v/v) HR® Lubricating Jelly (HR1 or HR5); K-Y® Jelly (KY1 or KY5); Therio-gel® (TG1 or TG5); Priority Care® Sterile Lubricating Jelly (PC1 or PC5); or Clarity® A.I. Lubricating Jelly (CL1 or CL5). Sperm were evaluated at T1h and T24h for percentages of: total and progressive sperm motility (TMOT and PMOT); curvilinear velocity (VCL; µm/s); and straightness (STR; %); viable acrosome intact sperm (VAI); sperm with abnormal DNA (COMP-αt); viable lipid peroxidation negative sperm (VLPN); and sperm with no detectable DNA oxidative injury [8OHdG(-)]. Following short-term exposure of sperm to lubricants, KY5 reduced TMOT, PMOT, VCL, VAI, VLPN, and COMP-αt in comparison with controls (i.e., Pâ¯<â¯0.05). PC5 reduced TMOT, PMOT, VCL, VAI, and 8OHdG(-), and KY1 reduced TMOT, VAI, VLPN in comparison to controls (Pâ¯<â¯0.05). Lubricant CL1, HR1 and HR5 yielded similar values to controls for all 8 endpoints, and CL5 yielded similar values to controls for all 8 endpoints (Pâ¯>â¯0.05), except for VCL. Following long-term exposure, KY5 decreased TMOT, PMOT, VCL, VAI, VLPN, and COMP-αt as compared to controls (i.e., Pâ¯<â¯0.05), PC5 decreased TMOT, VCL, VAI, and 8OHdG(-)as compared to controls in PC5, and KY1 decreased TMOT, VAI, VLPN, and COMP-αt (Pâ¯<â¯0.05). TG5 decreased TMOT, PMOT, and VCL as compared to controls (Pâ¯<â¯0.05). Lubricant CL5 decreased VCL (Pâ¯<â¯0.05), and CL1, HR5, HR1, PC1, and TG1 were similar to controls for all 8 endpoints (Pâ¯>â¯0.05). Overall, lubricant KY was the most detrimental to sperm quality, with most profound changes detected at a 5% concentration. Lubricants CL and HR were generally similar to controls and were less affected by lubricant concentration.
Asunto(s)
Caballos , Lubricantes/toxicidad , Espermatozoides/efectos de los fármacos , Animales , Masculino , Análisis de Semen/veterinaria , Motilidad Espermática/efectos de los fármacosRESUMEN
The tolerance of sperm DNA structure to seminal plasma and freezing conditions has both clinical and basic biologic relevance. In this study, fresh (FS) or flash-frozen (FZ) stallion epididymal sperm were exposed (SP+) or unexposed (SP-) to seminal plasma. Sperm were then evaluated to monitor the degree of change in DNA structure following challenge with chemical (dithiothreitol-DTT), oxidative (iron sulfate; FeSO4) or enzymatic (DNase I) potentiators of DNA damage. For sperm not treated with potentiators (controls), there was no effect of SP treatment (SP- vs. SP+) or freezing treatment (FS vs. FZ; non-significant) on measures of any DNA assays (i.e., 8-hydroxy, 2'deoxyguanosine [8OHdG], TUNEL, or sperm chromatin structure [SCSA] assays). Group FZ was more susceptible than Group FS to potentiators of DNA damage. Percent 8OHdG-positive sperm was higher in Group FZ/SP- treated with FeSO4 than all other groups (Pâ¯<â¯0.05). Percent TUNEL-positive sperm was similar among FZ/SP- groups treated with DTT, FeSO4, or DNase (non-significant) and was higher in these groups than all other treatments (Pâ¯<â¯0.05). Percent COMP-αt was higher following treatment with DNase or DTT, as compared to their respective controls, regardless of prior exposure to SP (Pâ¯<â¯0.05). Overall, sperm DNA structure was unaffected by seminal plasma or freezing treatment when samples were not exposed to potentiators of sperm DNA damage; however, marked differences were identified in DNA structure when sperm were challenged with chemical, oxidative or enzymatic treatments. These results highlight the importance of challenging DNA structure prior to analysis. The use of potentiators of DNA damage provided a model to evaluate sperm DNA structure following exposure of sperm to various experimental treatments.
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Criopreservación/veterinaria , Daño del ADN , ADN/ultraestructura , Caballos , Preservación de Semen/veterinaria , Semen , Animales , Criopreservación/métodos , Desoxirribonucleasa I/farmacología , Ditiotreitol/farmacología , Etiquetado Corte-Fin in Situ , Masculino , Estrés Oxidativo , Análisis de Semen/veterinaria , Preservación de Semen/métodos , Sulfatos/farmacologíaRESUMEN
Two experiments were conducted to evaluate the effects of antibiotic-containing extender of on sperm quality and control of bacterial growth. In Experiment 1, ejaculates were diluted in extender containing no antibiotics, potassium penicillin G-amikacin disulfate (PEN-AMIK), ticarcillin disodium-potassium clavulanate (TICAR-CLAV), piperacillin sodium/tazobactam sodium (PIP-TAZ), or meropenem (MERO). In freshly extended semen, only slight differences were detected among some antibiotic treatments for total sperm motility, curvilinear velocity, and viable acrosome-intact sperm (Pâ¯<â¯0.05). In cool-stored semen, slight differences were also detected among certain antibiotic treatments for curvilinear velocity and chromatin integrity (Pâ¯<â¯0.05). In Experiment 2, ejaculates were diluted in extender and subjected to no bacterial spiking, or inoculated with lower or higher doses of K. pneumoniae or P. aeruginosa. Following cooled storage of semen, colony forming units/ml (CFU/mL) were less in PEN-AMIK (706⯱â¯244) and MERO (1576⯱â¯1076) treatment groups than in TICAR-CLAV (4678⯱â¯1388) or PIP-TAZ (8108⯱â¯3198) treatment groups (Pâ¯<â¯0.05). The CFU/mL were lower in all antibiotic-containing treatment groups than the control group (18478⯱â¯4374; Pâ¯<â¯0.05). The percentage of culture plates containing no bacterial growth in unspiked semen was greater in PEN-AMIK (75%) than PIP-TAZ (15%) or TICAR-CLAV (20%; Pâ¯<â¯0.05). The percentages of culture plates containing no bacterial growth in semen spiked with a lower doses of K. pneumoniae or P. aeruginosa were higher in PEN-AMIK (70% and 50%, respectively) then in all other treatment groups (0-40% and 0-15% for K. pneumonia and P. aeruginosa, respectively; Pâ¯<â¯0.05); however, complete control of bacterial load was only modest even with PEN-AMIK. In both experiments, freezing and thawing extender prior to use did not have any appreciable detrimental effect on sperm quality or antibiotic efficacy. In summary, all antibiotics tested had minimal effects on measures of sperm quality in fresh or cool-stored semen extenders; however, PEN-AMIK, followed by MERO, yielded the best results in terms of antimicrobial efficacy. None of the antibiotic types controlled bacterial growth, in comparison with the antibiotic-free control group, when extended semen was spiked with a high concentration of Pseudomonas aeruginosa. Cooled storage of extended semen reduced bacterial growth in comparison with freshly extended semen.
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Caballos , Preservación de Semen/veterinaria , Espermatozoides/fisiología , Animales , Antibacterianos/farmacología , Masculino , Semen/microbiología , Análisis de Semen/veterinaria , Preservación de Semen/métodos , Motilidad Espermática , Espermatozoides/efectos de los fármacosRESUMEN
The effect of flash-freezing storage temperature on stallion sperm DNA has not been evaluated. Commonly, sperm are flash-frozen at various temperatures to preserve sperm DNA prior to analysis. It is unclear whether the temperature at which sperm are frozen and stored may affect the results of DNA assays. In this study, the neutral comet assay was used to evaluate the effect of flash-freezing storage temperature (freezer [-60 °C], dry ice [-78.5 °C], liquid nitrogen [-196 °C]) compared to fresh sperm DNA structure. In addition, intra- and inter-assay and intra- and inter-stallion variabilities were determined. All comet tail measures were higher following any flash-freezing method, as compared to fresh sperm DNA (P < 0.05), with no difference among flash-frozen treatments (P > 0.05). For most comet variables, intra- and inter-assay variabilities were <10%. Intra- and inter-stallion variabilities revealed that comet head length (HL) and width (CW) were less variable as compared to comet tail values, i.e., % comet tail DNA (T-DNA), tail length (TL), tail moment (OTM), and tail migration (TM). Certain comet tail values in fresh (% T-DNA, and OTM) and flash-frozen sperm (OTM, % T-DNA, TL, and TM) were correlated to the Sperm Chromatin Structure Assay (SCSA) variable, COMP-αt. The comet tail measures were negatively correlated to % morphologically normal sperm (P < 0.05) and positively correlated to % abnormal heads and premature germ cells (P < 0.05). Variables COMP-αt and % total sperm motility were not correlated to any morphologic sperm feature in this group of stallions (P > 0.05). While significant differences in the structure of the sperm DNA were identified in the flash-frozen as compared to the fresh sperm DNA with the neutral comet assay, it cannot be assumed that these changes are fertility limiting.
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Daño del ADN , Congelación , Caballos , Espermatozoides/citología , Animales , Ensayo Cometa/veterinaria , Criopreservación/veterinaria , Masculino , TemperaturaRESUMEN
Much of the current understanding of epilepsy mechanisms has been built on data recorded with one or a few electrodes from temporal lobe slices of normal young animals stimulated with convulsants. Mechanisms of adult, extratemporal, neocortical chronic epilepsy have not been characterized as much. A more advanced understanding of epilepsy mechanisms can be obtained by recording epileptiform discharges simultaneously from multiple points of an epileptic focus so as to define their sites of initiation and pathways of spreading. Brain slice recordings can characterize epileptic mechanisms in a simpler, more controlled preparation than in vivo. Yet, the intrinsic hyper-excitability of a chronic epileptic focus may not be entirely preserved in slices following the severing of connections in slice preparation. This study utilizes recordings of multiple electrode arrays to characterize which features of epileptic hyper-excitability present in in vivo chronic adult neocortical epileptic foci are preserved in brain slices. After tetanus toxin somatosensory cortex injections, adult rats manifest chronic spontaneous epileptic discharges both in the injection site (primary focus) and in the contralateral side (secondary focus). We prepared neocortical slices from these epileptic animals. When perfused with 4-Aminopyridine in a magnesium free medium, epileptic rat slices exhibit higher voltage discharges and broader spreading than control rat slices. Rates of discharges are similar in slices of epileptic and normal rats, however. Ictal and interictal discharges are distributed over most cortical layers, though with significant differences between primary and secondary foci. A chronic neocortical epileptic focus in slices does not show increased spontaneous pacemakers initiating epileptic discharges but shows discharges with higher voltages and broader spread, consistent with an enhanced synchrony of cellular and synaptic generators over wider surfaces.
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Epilepsia/fisiopatología , Neocórtex/fisiopatología , 4-Aminopiridina , Animales , Enfermedad Crónica , Modelos Animales de Enfermedad , Electrocorticografía , Técnicas In Vitro , Magnesio , Masculino , Ratas Sprague-Dawley , Convulsiones/fisiopatología , Toxina Tetánica , Técnicas de Cultivo de TejidosRESUMEN
Using flow cytometry in conjunction with a voltage-sensitive fluorescent indicator dye (oxonol), we have identified and separated embryonic hippocampal cells according to the sensitivity of their functionally expressed GABAA receptors to zolpidem. Immunocytochemical and RT-PCR analysis of sorted zolpidem-sensitive (ZS) and zolpidem-insensitive (ZI) subpopulations identified ZS cells as postmitotic, differentiating neurons expressing alpha2, alpha4, alpha5, beta1, beta2, beta3, gamma1, gamma2, and gamma3 GABAA receptor subunits, whereas the ZI cells were neuroepithelial cells or newly postmitotic neurons, expressing predominantly alpha4, alpha5, beta1, and gamma2 subunits. Fluctuation analyses of macroscopic Cl- currents evoked by GABA revealed three kinetic components of GABAA receptor/Cl- channel activity in both subpopulations. We focused our study on ZI cells, which exhibited a limited number of subunits and functional channels, to directly correlate subunit composition with channel properties. Biophysical analyses of GABA-activated Cl- currents in ZI cells revealed two types of receptor-coupled channel properties: one comprising short-lasting openings, high affinity for GABA, and low sensitivity to diazepam, and the other with long-lasting openings, low affinity for GABA, and high sensitivity to diazepam. Both types of channel activity were found in the same cell. Channel kinetics were well modeled by fitting dwell time distributions to biliganded activation and included two open and five closed states. We propose that short- and long-lasting openings correspond to GABAA receptor/Cl- channels containing alpha4beta1gamma2 and alpha5beta1gamma2 subunits, respectively.
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Canales de Cloruro/química , Hipocampo/citología , Hipnóticos y Sedantes/farmacología , Piridinas/farmacología , Receptores de GABA-A/química , Animales , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Canales de Cloruro/análisis , Canales de Cloruro/genética , Diazepam/farmacología , Femenino , Feto/química , Feto/fisiología , Citometría de Flujo , Moduladores del GABA/farmacología , Expresión Génica/fisiología , Hipocampo/efectos de los fármacos , Hipocampo/embriología , Cinética , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Neuronas/química , Neuronas/citología , Neuronas/efectos de los fármacos , Técnicas de Placa-Clamp , Embarazo , Estructura Terciaria de Proteína , Ratas , Ratas Sprague-Dawley , Receptores de GABA-A/análisis , Receptores de GABA-A/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Zolpidem , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
Unilateral orchiectomy (UO) may interfere with thermoregulation of the remaining testis caused by inflammation surrounding the incision site, thus altering normal spermatogenesis and consequently sperm quality. Two measures of sperm DNA quality (neutral comet assay and the sperm chromatin structure assay [SCSA]) were compared before UO (0 days) and at 14, 30, and 60 days after UO to determine whether sperm DNA changed after a mild testis stress (i.e., UO). The percent DNA in the comet tail was higher at 14 and 60 days compared to 0 days (P < 0.05) after UO. All other comet tail measures (i.e., length, moment, migration) were higher at all time periods after UO compared to 0 days (P < 0.05). Two SCSA measures (mean-αt, mode-αt) increased at 14 days after UO (P < 0.05), whereas two measures (SD-αt and COMP-αt) did not change. This study identified a decrease in sperm DNA quality using both the neutral comet assay and the SCSA, which was not identified using traditional measures of sperm quality.
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Caballos , Orquiectomía/veterinaria , Animales , Cromatina/ultraestructura , Ensayo Cometa/veterinaria , Daño del ADN , Masculino , Orquiectomía/efectos adversos , Análisis de Semen/métodos , Análisis de Semen/veterinariaRESUMEN
In this study we examined whether the potency of quinolinic acid (Quin) in inducing neurodegeneration in vivo was dependent on the exposure time of the tissue to the excitotoxin. The effect of chronic infusion of Quin into rat striatum and hippocampus was examined at the light microscopic level and by cell count on 40 microm Cresyl violet stained brain sections. Continuous infusion was at a constant speed (0.5 microl/h) for various times (15 h - 2 weeks) by osmotic minipumps (Alzet 2002). No build up of [3H]Quin occurred in the tissue during infusion; this was assessed by measuring the radioactivity 3 - 14 days after minipump placement. Intrastriatal infusion of 6 and 10 nmol/h Quin, but not of nicotinic acid, for 1 week induced a dose-dependent neurodegeneration (70 and 90% loss of neurons, respectively, compared to the contralateral striatum) extending 1.2 - 2 mm from the centre of the injection. The onset of the neurotoxicity caused by 10 nmol/h Quin was >24 h. One week's infusion of 4 nmol/h Quin did not induce neurotoxicity, but a 40% drop of neurons, compared to the contralateral side, occurred after 2 weeks. One week's intrahippocampal infusion of 2.4 and 6 nmol/h Quin, but not of nicotinic acid, caused a dose-dependent neurodegeneration with a radius of approximately 1 - 1.5 mm around the injection track. The onset of the neurotoxicity induced by 2.4 nmol/h Quin was < 15 h. The pattern of nerve cell loss induced by 1.2 nmol/h Quin after 1 week (CA4 cells lost in 50% of the rats) did not differ from that observed after 2 weeks of infusion. Nerve cell loss caused by Quin in the striatum and in the hippocampus was restricted to the injected area and antagonized by coinfusion with d(-)-2-amino-7-phosphonoheptanoic and kynurenic acids in molar ratios of 1:0.1 and 1:3, respectively. These data show that Quin's potency in inducing neurodegeneration in the striatum, but not in the hippocampus, depends on the exposure time of the tissue to the excitotoxin. In addition, neurodegeneration is induced faster by Quin in the hippocampus than in the striatum. The usefulness of this model to study the sequelae of the neurotoxic process in vivo will be discussed.
RESUMEN
We evaluated the immunological reconstitution of patients who underwent high-dose chemotherapy and autologous blood stem cell transplantation (ABSCT) for advanced ovarian cancer. Sixty days after transplantation a complete reconstitution of lymphocytes and of the CD3, CD4, CD8, CD19, and CD16/56 subsets was observed in this series. A significant increase in the count of interleukin-2 receptor expressing lymphocyte (CD25) was found on day +60 after transplantation compared to that obtained at diagnosis and before transplantation. A significantly higher lymphokine-activated killer (LAK) precursor activity was seen on day +60 compared to the values obtained at diagnosis and before transplantation while natural killer activity did not show any significant variation. We conclude that ABSCT gives prompt and complete immunohaematopoietic reconstitution after high-dose treatment. Moreover, our data support the feasibility of interleukin-2/LAK therapy as consolidative therapy after ABSCT.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Trasplante de Células Madre Hematopoyéticas , Neoplasias Ováricas/terapia , Cisplatino/administración & dosificación , Ciclofosfamida/administración & dosificación , Femenino , Humanos , Células Asesinas Activadas por Linfocinas , Células Asesinas Naturales , Recuento de Leucocitos , Subgrupos Linfocitarios , Masculino , Neoplasias Ováricas/inmunología , Receptores de Interleucina-2/inmunología , Factores de TiempoRESUMEN
20 patients with stage III-IV ovarian cancer were submitted to induction chemotherapy (ICT) (40 mg/m2 cisplatin, days 1-4; 1.5 g/m2 cyclophosphamide, day 4; every 4 weeks for 2 cycles) followed by intensified CT (100 mg/m2 cisplatin, day 1; 650 mg/m2 etoposide, day 2; 1.8 g/m2 carboplatin by 24 h infusion, day 3). Haematological support consisted of autologous peripheral stem cells (APSC) and bone marrow (ABM) transplant (T) in 16 and 4 patients, respectively. All patients were evaluable for toxicity and 19 for pathological response (PR), one patient dying of systemic mycosis after ABMT. Severe (grade 3-4) non-haematological toxic effects were gastrointestinal (100%), neurological (10%) and hepatic (10%). PR was observed in 84% of patients (complete response 37%, partial response with microscopic residual disease 26%, partial response with macroscopic residual disease 21%). Five year overall survival was 60% and progression-free survival was 51% with 9 patients still disease-free (DFS). APSCT significantly reduced the duration of aplasia compared with ABMT, and toxicity was acceptable in those patients undergoing APSCT. The prolonged DFS in patients showing PCR suggests that this new approach may have a therapeutic impact.
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Adenocarcinoma/tratamiento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Trasplante de Médula Ósea , Trasplante de Células Madre Hematopoyéticas , Neoplasias Ováricas/tratamiento farmacológico , Adenocarcinoma/terapia , Adulto , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Cisplatino/administración & dosificación , Cisplatino/efectos adversos , Terapia Combinada , Ciclofosfamida/administración & dosificación , Ciclofosfamida/efectos adversos , Supervivencia sin Enfermedad , Esquema de Medicación , Femenino , Enfermedades Hematológicas/inducido químicamente , Enfermedades Hematológicas/terapia , Humanos , Persona de Mediana Edad , Neoplasias Ováricas/terapia , Resultado del TratamientoRESUMEN
Electroencephalographic (EEG) seizures were measured in rats after intrahippocampal injection of 120 nmol quinolinic acid into the stratum radiatum CA1 or 0.19 nmol kainic acid in the dentate gyrus or in the stratum radiatum. Injection of 5 micrograms SMS 201-995, a peptidase-resistant cyclic octapeptide analogue of somatostatin, into the stratum radiatum, 15 min before quinolinic acid, did not significantly modify the number of seizures and the total time in seizures. Five micrograms SMS 201-995 injected into the stratum radiatum reduced the number of seizures induced by kainic acid in the same area and the total time spent in seizures by 58% and 75%, respectively (Student's t-test; P less than 0.01). In both instances the latency to the first ictal episode was not significantly modified. Lesions of the medial septum, which reduced the activity of choline-o-acetyl-transferase (CAT) in the dorsal hippocampus by greater than 90% after one week did not significantly affect seizures induced by quinolinic acid. In rats lesioned in the medial septum, 5 micrograms SMS 201-995 reduced the total time spent in seizures by 43%, without changing the number of ictal episodes and raised the latency to the first quinolinic acid-induced seizure by 53% (ANOVA 2 x 2, P less than 0.05) but had no effect on these measures in the corresponding sham-operated group.(ABSTRACT TRUNCATED AT 250 WORDS)
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Convulsivantes/antagonistas & inhibidores , Hipocampo/efectos de los fármacos , Ácido Kaínico/antagonistas & inhibidores , Octreótido/farmacología , Ácidos Quinolínicos/antagonistas & inhibidores , Convulsiones/prevención & control , Animales , Atropina/farmacología , Electroencefalografía/efectos de los fármacos , Hipocampo/fisiopatología , Masculino , Picrotoxina/farmacología , Ácido Quinolínico , Ratas , Ratas Endogámicas , Convulsiones/inducido químicamenteRESUMEN
Twenty bone marrow aspirates harvested for autotransplantation from 20 patients suffering from several oncohematological diseases were processed using the automated Du Pont SteriCell processor. In 15 bone marrow harvests, the interface buffy coat cells were collected using the SteriCell processor in manual mode with a semiautomated procedure. The procedure yielded an average red cell removal of 84% and an average mononuclear cell (MNC) recovery of 86%. Cloning efficiencies of hematopoietic progenitor cells (CFU-GM and BFU-e) did not differ between processed and recovered MNCs. Four cryopreserved bone marrow buffy coats were thawed and reinfused into four patients who had undergone high dose chemotherapy. Stable engraftment was observed in all cases. In five bone marrow harvests, the SteriCell automated density gradient MNC isolation procedure was performed after buffy coat collection. The whole two-step procedure allowed an average MNC recovery of 69%. CFU-GM and BFU-e assays did not show a significant difference in cloning efficiency between processed and recovered bone marrow MNCs. We conclude that the SteriCell processor offers rapid, safe and feasible procedures for the semiautomated processing of human bone marrow for transplantation. The clinical efficacy of density gradient separated bone marrow employing the automated step and the opportunity to use fully automated processing must be investigated.
Asunto(s)
Células de la Médula Ósea , Trasplante de Médula Ósea/métodos , Separación Celular/métodos , Adulto , Separación Celular/instrumentación , Humanos , Persona de Mediana Edad , Trasplante AutólogoRESUMEN
We tested in vitro the effect of recombinant human erythropoietin (rhEPO) plus recombinant human G-CSF (rhG-CSF) on purified human CD34+ haemopoietic progenitors (HP) and in vivo in patients who had undergone anti-cancer chemotherapy for advanced ovarian cancer. In this preliminary experience we found that, in vitro, rhEPO potentiates the effect of rhG-CSF on HP growth and differentiation toward the granulocyte-macrophage lineage. rhEPO plus rhG-CSF produced in vitro a proliferative stimulus of HP which represents 26% of the maximum stimulation obtained using IL-1, IL-3, IL-6, G-CSF, GM-CSF and stem cell factor in combination. In the patients treated with rhEPO plus rhG-CSF after chemotherapy, we observed a favourable trend for platelet and neutrophil recoveries compared with a control group treated with rhG-CSF alone and a significantly higher haematocrit nadir was observed in the rhEPO plus rhG-CSF series. In the patients treated with rhEPO plus rhG-CSF we observed a significant increase of circulating colony-forming unit granulocyte-macrophage (CFU-GM) and burst forming unit-erythroid (BFU-e) compared with the rhG-CSF series. Our results, in vitro and in vivo, encourage the in vivo use of rhEPO plus rhG-CSF to improve blood cell recoveries of patients who have undergone conventional or high-dose chemotherapy. Moreover, rhEPO plus rhG-CSF was demonstrated to be a good HP mobilising treatment for blood stem cell collection after chemotherapy.
Asunto(s)
Eritropoyetina/administración & dosificación , Factor Estimulante de Colonias de Granulocitos/administración & dosificación , Células Madre Hematopoyéticas/efectos de los fármacos , Adulto , Antineoplásicos/administración & dosificación , Recuento de Células Sanguíneas , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Ensayo de Unidades Formadoras de Colonias , Sinergismo Farmacológico , Femenino , Células Madre Hematopoyéticas/citología , Humanos , Técnicas In Vitro , Persona de Mediana Edad , Neoplasias Ováricas/sangre , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/patología , Proteínas Recombinantes/administración & dosificaciónRESUMEN
Six patients with advanced ovarian carcinoma (OvCa), and six patients with stage II or III resectable breast cancer (BrCa) were treated with low-dose CY (LD-CY, 1500 mg/m2) and cisplatin (CDDP) 100 mg/m2 (OvCa) or epirubicin (EPR) 120 mg/m2 (BrCa) plus recombinant human G-CSF (rhG-CSF). Twelve days after chemotherapy, all patients underwent PBSC collection on an outpatient basis. Following the completion of the induction programme, all patients underwent high-dose chemotherapy (HDC) with carboplatin 1200 mg/m2, etoposide 900 mg/m2 and melphalan 100 mg/m2 with the reinfusion of PBSC. LD-CY plus rhG-CSF in combination with CDDP or EPR mobilised a very large number of PBSC. After a median of 13 days from chemotherapy, the concentration of PBSC in the peripheral blood was 40-fold higher than the same patient's baseline value. Each collection yielded a median of 10.8 x 10(4)/kg colony-forming unit granulocyte-macrophage. Severe myelosuppression occurred in all patients following HDC, but the infusion of PBSC produced a rapid and sustained haemopoietic recovery. After a median of 11 days from reinfusion, haemopoietic engraftment was complete and 80% of the patients had platelets > 100 x 10(9)/l and PMN > 1 x 10(9)/l within 14 days after reinfusion. We can conclude that the present therapeutic approach is an excellent option for mobilisation, collection and transplantation of PBSC during intensive dose adjuvant polychemotherapy of high-risk cancer.
Asunto(s)
Neoplasias de la Mama/terapia , Cisplatino/administración & dosificación , Ciclofosfamida/administración & dosificación , Epirrubicina/administración & dosificación , Factor Estimulante de Colonias de Granulocitos/administración & dosificación , Trasplante de Células Madre Hematopoyéticas/métodos , Neoplasias Ováricas/terapia , Adulto , Terapia Combinada , Femenino , Hematopoyesis , Humanos , Persona de Mediana Edad , Trasplante AutólogoRESUMEN
Highly fluorescent reticulocyte (HFR) counts were evaluated in 13 consecutive patients affected by hematological malignancies and submitted to autologous selected CD34+ peripheral blood progenitor cell (PBPC) transplantation. Results were compared with a historical group of patients comparable for age, disease and conditioning regimen submitted to unfractionated PBPC transplantation. HFR counts of the CD34+ group declined to an undetectable level from day +4 to day +10 when they became detectable and reached 5% of total reticulocyte count by day +12. In the historical group, the nadir was identical but the recovery was faster (day +9). Total reticulocyte count > 1% was achieved at days +17 and +11, respectively. The absolute neutrophil count (ANC) recovery was identical in both groups, achieving a value > 0.5 x 10(9)/l at day +13 after reinfusion. Hence, in the historical group, HFR count gave advance notice of complete and stable hemopoietic engraftment while in the CD34+ group HFR and ANC count showed almost simultaneous recovery.
Asunto(s)
Antígenos CD34/metabolismo , Trasplante de Células Madre Hematopoyéticas , Reticulocitos/citología , Reticulocitos/inmunología , Adulto , Femenino , Colorantes Fluorescentes , Supervivencia de Injerto , Hematopoyesis , Enfermedad de Hodgkin/terapia , Humanos , Cinética , Linfoma no Hodgkin/terapia , Masculino , Persona de Mediana Edad , Mieloma Múltiple/terapia , Recuento de Reticulocitos , Trasplante AutólogoRESUMEN
We investigated whether modifications in noradrenergic neurotransmission occurred during the development of hippocampal kindling in rats. We measured the release of [3H]norepinephrine (NE) induced by field-electrical stimulation, NE-stimulation of inositol phosphates [( 3H]IP) accumulation in the presence of LiCl and isoproterenol-induced accumulation of cAMP in hippocampal slices taken from rats electrically kindled at stages 2 and 5 in the dorsal hippocampus. One week after the last of at least 3 consecutive stage 5 seizures or 48 h after the last stage 2 stimulation, 2 min electrical stimulation of stratum pyramidale CA1-CA3 or dentate gyrus (DG) slices from kindled and contralateral hippocampi induced frequency-dependent NE release (respectively 2, 4 and 8 times spontaneous release measured at 2, 5 and 10 Hz) which did not significantly differ from that observed in shams (implanted with electrodes but not stimulated). Basal release of NE from kindled and sham-treated rats did not differ either. Isoproterenol induced a dose-dependent increase above basal cAMP concentration ranging from 40% at 0.01 microM to 180% at 10 microM (P less than 0.01, Dunnett's test) which did not differ between stages 2 and 5 and sham-hippocampi. NE (1-1000 microM) induced a dose-dependent, prazosin-sensitive increase in [3H]IP accumulation in the hippocampal slices. A significantly higher increase was found at stages 2 (P less than 0.05, Tukey's test) and 5 (P less than 0.05 and P less than 0.01, Tukey's test) compared to shams at all doses studied.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Hipocampo/citología , Excitación Neurológica/fisiología , Norepinefrina/fisiología , Sinapsis/fisiología , Transmisión Sináptica/fisiología , Animales , Carbacol/farmacología , AMP Cíclico/metabolismo , Estimulación Eléctrica , Electroencefalografía , Hipocampo/efectos de los fármacos , Técnicas In Vitro , Isoproterenol/farmacología , Masculino , Norepinefrina/metabolismo , Fosfatidilinositoles/metabolismo , Tractos Piramidales/citología , Tractos Piramidales/fisiología , Ratas , Ratas Endogámicas , Receptores Adrenérgicos alfa/efectos de los fármacos , Receptores Adrenérgicos beta/efectos de los fármacosRESUMEN
Binding sites for excitatory amino acids have been determined by autoradiographical procedures in the rat hippocampus and striatum during hippocampal kindling. The binding sites measured were the N-methyl-D-aspartate (NMDA)-sensitive sites for L-[3H]glutamate and [3H]MK-801 sites (transmitter recognition site and ion channel of the NMDA receptor, respectively), [3H]alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) sites (quisqualate receptor), [3H]kainate sites (kainate receptor) and NMDA-insensitive sites for L-[3H]glutamate. In general, little change was apparent in the hippocampus or striatum for any of these binding sites when assessed 48 h after attaining stages 1/2, 3 or 5 of kindling. These results suggest that hippocampal kindling does not bring about a change in the excitatory amino acid receptor binding sites examined, and that the appearance of an NMDA receptor-mediated component to synaptic responses in the hippocampus produced by kindling, cannot be explained on this basis.