Comparative historical biogeography of three groups of Nearctic freshwater fishes across central Mexico.

Biogeographic patterns of the three main Nearctic groups of continental fishes inhabiting river drainages in central Mexico (livebearing goodeids, southern Mexican notropins and species of Algansea, the last two representing independent lineages of cyprinids) were obtained and compared by following two approaches: an estimate of divergence times and using a well-defined biogeographic method. Three concordant biogeographic events were identified among the three groups, showing some evidence of a partially congruent evolutionary history. The analysed groups show at least three independent colonization events into central Mexico: two western routes, followed by the Goodeinae and members of Algansea, and an early Plateau route followed by southern notropins. The most recent common ancestor (MRCA) of each of the three freshwater fish groups diversified in central Mexico in the Late Miocene. The lack of a strong congruence in their biogeographic patterns, and the differences in species richness among the three clades might be evidence for distinct patterns of diversification.

[Dispensing Service: An Essential Service Provided by Community Pharmacy to Enhance Patient Safety. A Case Study]. / Servicio de Dispensación: un servicio esencial brindado desde la Farmacia Comunitaria que permite reforzar la Seguridad del Paciente. A propósito de un caso.

A 38-year-old patient diagnosed with asthma and anxiety, who takes two medications (salbutamol 100 mcg inhaler (2 puffs every 6 hours), and diazepam 5 mg (0-0-1), visited the Community Pharmacy to pick up a treatment prescribed by the Primary Care Physician (PCP) following a diagnosis of anxious-depressive symptoms.During the Dispensing Service, a potential Drug-Related Problem (DRP) of prescription error is detected, which could be related with a Negative Outcomes Releated to Medicines (NOM) due to the concurrent use of desvenlafaxine and mirtazapine. Additionally, a Health Problem (HP)-related DRP was detected, as the proposal to discontinue the use of diazepam could result in an Insufficiently Treated HP, potentially leading to a NOM of Treatment Necessity due to the risk of worsening anxiety episodes.From de Community Pharmacy, a report was prepared with recommendations that were accepted by the PCP. Subsequent case monitoring revealed an improved management of the patient's health problems, as well as the resolution of the identified DRP and NOM. This ensured a rational, safe, and effective use of the medication.

Plasma membrane and vesicular glutamate transporter expression in chromaffin cells of bovine adrenal medulla.

The study of the functional expression of glutamate signaling molecules in peripheral tissues has received relatively little attention. However, evidence is increasing for a role of glutamate as an extracellular signal mediator in endocrine systems, in addition to having an excitatory amino acid neurotransmitter role in the CNS. Chromaffin cells are good models of catecholaminergic neurons, in which previous work from our group demonstrated the existence of both functional glutamate receptors and specific exocytotic and nonexocytotic glutamate release. In this work, the presence of specific plasma membrane (EAATs) and vesicular glutamate (VGLUTs) transporters has been investigated by using confocal microscopy, flow cytometric analysis, Western blot, and qRT-PCR techniques. We found specific expression of EAAT3, EAAT2, VGLUT1, and VGLUT3 in about 95%, 65%, 55%, and 25%, respectively, of the whole chromaffin cell population. However, chromaffin cells do not express VGLUT2 and have a very low expression of EAAT1. VGLUTs are localized mainly in the membrane fraction, and EAATs share their subcellular location between membrane and cytosolic fractions. Their estimated molecular weights were about 70 kDa for EAAT2, about 65 kDa for EAAT3, about 50 kDa for VGLUT1, and about 60 kDa for VGLUT3. RT-qPCR techniques confirm the expression of these glutamate transporters at the mRNA level and show a different regulation by cytokines and glucocorticoids between VGLUT1 and -3 and EAAT2 and -3 subfamilies. These interesting results support the participation of these glutamate transporters in the process of glutamate release in chromaffin cells and in the regulation of their neurosecretory function in adrenal medulla.

Servicio de Dispensación: un servicio esencial brindado desde la Farmacia Comunitaria que permite reforzar la Seguridad del Paciente. A propósito de un caso / Dispensing Service: An Essential Service Provided by Community Pharmacy to Enhance Patient Safety. A Case Study

Paciente de 38 años, diagnosticado de asma y ansiedad que utilizaba 2 medicamentos (salbutamol 100 mcg inhalador (2 pulv. cada 6 horas) y diazepam 5 mg (0-0-1)), acude a la Farmacia Comunitaria (FC) para retirar un tratamiento prescrito por el Médico de Atención Primaria (MAP) tras diagnóstico de un cuadro ansioso-depresivo. Desde el Servicio de Dispensación (SD) se detectó un Problema Relacionado con el Medicamento (PRM) de un potencial error en la prescripción, que podría tener asociado un Resultado Negativo asociado a la Medicación (RNM) ante uso concomitante de desvenlafaxina y mirtazapina, así como un PRM de Problema de Salud (PS) insuficientemente tratado ante una propuesta de suspensión de uso de diazepam, que podría derivar en un RNM de Necesidad de Tratamiento ante el riesgo de empeoramiento de episodios de ansiedad. Desde la FC, se elaboró un informe de derivación con recomendaciones que fueron aceptadas por el MAP. Se llevó a cabo un seguimiento del caso que permitió realizar un mejor control de los PS presentados por el paciente, así como la resolución de los PRM y RNM detectados, garantizando así un uso racional, seguro y eficaz del medicamento. (AU)

A 38-year-old patient diagnosed with asthma and anxiety, who takes two medications (salbutamol 100 mcg inhaler (2 puffs every 6 hours), and diazepam 5 mg (0-0-1), visited the Community Pharmacy to pick up a treatment prescribed by the Primary Care Physician (PCP) following a diagnosis of anxious-depressive symptoms. During the Dispensing Service, a potential Drug-Related Problem (DRP) of prescription error is detected, which could be related with a Negative Outcomes Releated to Medicines (NOM) due to the concurrent use of desvenlafaxine and mirtazapine. Additionally, a Health Problem (HP)-related DRP was detected, as the proposal to discontinue the use of diazepam could result in an Insufficiently Treated HP, potentially leading to a NOM of Treatment Necessity due to the risk of worsening anxiety episodes. From de Community Pharmacy, a report was prepared with recommendations that were accepted by the PCP. Subsequent case monitoring revealed an improved management of the patient’s health problems, as well as the resolution of the identified DRP and NOM. This ensured a rational, safe, and effective use of the medication. (AU)

Análisis integral de la colecistectomía en una unidad de cirugía ambulatoria y corta estancia. Experiencia de los últimos 7 años / Integral analysis of cholecystectomy in an outpatient surgery and short-stay unit. Experience of the last 7 years

Introducción: La colecistectomía laparoscópica ambulatoria (CLA) se considera en la actualidad un trazador representativo de la calidad de un servicio de cirugía general. La gran diversidad de unidades de cirugía ambulatoria dificulta la comparativa de los diferentes indicadores de calidad. Objetivo: Conocer los resultados del manejo de la CLA en un centro integrado y como afecta a sus indicadores de calidad. Pacientes y método: Estudio observacional prospectivo entre 2015 y 2021 de las colecistectomías programadas en unidad integrada. Resultados: Se intervinieron 887 pacientes, el 76,5 % (n = 679) programados en régimen ambulatorio. La pernocta no planificada (PNP) media fue del 25,2 % (n = 171), siendo el índice de sustitución del 57,8 %. Las principales causas de PNP fueron: intolerancia digestiva (48,5 %), cirugía compleja (29,2 %) y el dolor (12,8 %). Los tiempos quirúrgicos fueron superiores en los pacientes en régimen de ingreso (p < 0,001) y en aquellos que causaron PNP (p < 0,001). Un tiempo quirúrgico superior a los 45 minutos fue causa de PNP de forma significativa (p = 0,007). La tasa global de infección de sitio quirúrgico fue del 3,1 %,siendo la infección profunda del 0,59 %. Ningún paciente reingresó en las primeras 24 horas, siendo la asistencia a urgencias a 30 días del 8,2 % (n = 73),reingresando el 1,91 % (n = 17) de los pacientes, con una tasa de reintervención del 0,35 % (n = 3). La tasa de fístula biliar fue del 0,67 %. Conclusión: La CLA es una técnica segura y expansiva, aunque la obtención de parámetros de calidad estandarizados es complejo por la diversidad de unidades.(AU)

Introduction: Ambulatory laparoscopic cholecystectomy (ALC) is currently considered a representative tracer of the quality of a general surgery service. The great diversity of day surgery units makes it difficult to compare the different quality index. Objective: To know the results of the management of the CLA in an integrated center and how it affects its quality index. Patients and method: Prospective observational study between 2015 and 2021 of scheduled cholecystectomies in an integrated unit. Results: 887 patients were operated on, 76.5 % (n = 679) programmed on an outpatient basis. The average unplanned overnight stay (PNP) was 25.2 % (n = 171), with the replacement rate being 57.8 %. The main causes of PNP were: digestive intolerance (48.5 %), complex surgery (29.2 %) and pain (12.8 %). Surgical times were higher in patients on admission (p < 0.001) and in those who caused PNP (p < 0.001). Surgical time greater than 45 minutes was a significant cause of PNP (p = 0.007). The overall rate of surgical site infection was 3.1 %, with deep infection being 0.59 %. No patient was readmitted in the first 24 hours, with 30-day emergency care being 8.2 % (n = 73), readmission rate of 1.91 % (n = 17), with a reoperation rate of 0.35 % (n = 3). The biliary fistula rate was 0.67 %. Conclusion: CLA is a safe and expansive technique, although obtaining quality standard parameters is complex due to the diversity of units.(AU)

Servicio de Seguimiento Farmacoterapéutico prestado desde la farmacia comunitaria como práctica asistencial para la optimización de la farmacoterapia en paciente con urticaria y prurito / Pharmacotherapeutic Follow-Up Service provided by the community pharmacy as a care practice for the optimization of pharmacotherapy in patients with urticaria and pruritus

Se ofreció el Servicio de Seguimiento Farmacoterapéutico (SFT) a una mujer de 66 años, exfumadora, diagnosticada de hipertensión arterial, hipercolesterolemia, asma, ansiedad y migraña, que presentaba prurito y urticaria. Tomaba 5 medicamentos. Tras revisión de la farmacoterapia y realización de una entrevista en profundidad, se determinó la presencia de prurito y urticaria como Resultados Negativos asociados a la Medicación (RNM) de inseguridad derivado del uso de rosuvastatina/ezetimiba por posibles Problemas Relacionados con los Medicamentos (PRM) y, de probabilidad de efecto adverso y de error en la prescripción del fármaco. Se propuso a la paciente suspender el tratamiento y se realizó derivación al Médico de Atención Primaria (MAP) mediante informe de derivación que fue entregado por la paciente en cita médica, para valorar alternativa farmacológica para tratar la hipercolesterolemia. La propuesta fue aceptada por el MAP. Se realizó un seguimiento del caso, que permitió constatar la resolución de los PRM y RNM detectados, logrando una mejora en la salud del paciente y favoreciendo la adherencia al tratamiento (AU)

A 66-year-old woman, ex-smoker, diagnosed with hypertension, hypercholesterolemia, asthma, anxiety and migraine, who presented pruritus and urticaria, was given Medication Review with Follow-Up Service (MRF). She was taking 5 medications. After the review of the pharmacotherapy and conducting an in-depth interview, the presence of pruritus and urticaria was determined as a Negative Outcomes Releated to Medicines (NOM) and a Drug Related Problem (DRP) derived from the use of Rosuvastatin/Ezetimibe, and a possible DRPs of probability of adverse effects and prescription error. It was proposed to the patient to suspend the treatment and a referral was made to the Primary Care Physician (PCP) by means of a referral report that was submitted by the patient at the medical appoint ment to assess a pharmacological alternative to treat hypercholesterolemia. The proposal was accepted by the PCP. A follow-up of the case was carried out, which allowed verifying the resolution of the DRPs and NOMs detected, achieving an improvement in the patient’s health and favoring adherence to treatment (AU)

Seguridad del paciente durante el servicio de dispensación en farmacia comunitaria ante un error de prescripción. A propósito de un caso / Patient safety during the dispensing service in a community pharmacy in the event of a prescription error. About a case

Paciente de 63 años, diagnosticada de diabetes, hipertensión e hipercolesterolemia, con antecedentes de infarto de miocardio, que toma 9 medicamentos. Acude a la farmacia comunitaria (FC) para retirar el tratamiento prescrito desde el Servicio de Urgencias para tratar una conjuntivitis. Desde el servicio de dispensación se detecta un Problema Relacionado con el Medicamento (PRM) de un potencial error en la prescripción, que podría tener asociado un Resultado Negativo a la medicación (RNM) al prescribirse gotas óticas para uso oftálmico, comprometiendo la seguridad de la paciente. Se deriva al Servicio de Urgencias para modificación del tratamiento por considerarse un potencial riesgo, siguiendo indicaciones de Ficha Técnica. En el Servicio de Urgencias se rechaza la intervención propuesta y se remite de nuevo a la paciente a FC a retirar el fármaco. La paciente decide esperar dos días a una cita médica presencial con su Médico de Atención Primaria (MAP). La propuesta realizada desde la FC fue aceptada por el MAP. Se realiza seguimiento del caso, que permite constatar la resolución del problema de salud, así como los PRM y RNM detectados, logrando una mejora en la salud del paciente y garantizando así un uso racional, seguro y eficaz del medicamento (AU)

A 63-year-old patient, diagnosed with diabetes, hypertension, and hypercholesterolemia, with myocardial infarction background who takes 9 different medications. She goes to the Community Pharmacy (CP) to pick up the prescribed treatment from the Emergency Department to treat a diagnosed conjunctivitis. During the Dispensing Service, a potential Drug-Related Problem (DRP) of prescription error is detected, which could be related with a Negative Outcomes Related to Medicines (NOM) when prescribing ear drops for ophthalmic use, compromising patient safety . The patient was referred to the Emergency Department to modify the treatment due to a potential risk, following the indications of the Summary of products Characteristics (SmPC). In the Emergency Department, the proposed intervention was rejected and the patient was sent back to withdraw the drug again to the CP. The patient decides to wait two days for a face-to-face medical appointment with her Primary Care Physician (MAP). The proposal made by the FC was accepted by the General Practitioner (GP). Follow-up of the case is carried out, which allows verifying the resolution of the Health Problem as well as the DRP and NRM detected, achieving an improvement in the patient’s health and thus guaranteeing rational, safe and effective use of the medication (AU)

Estrategia para optimizar la seguridad del paciente en el servicio de dispensación en farmacia comunitaria. Propuesta de listados de verificación / Strategy to optimize patient safety in community pharmacy dispensing service. Checklists Proposed

La dispensación es el servicio más demandado e importante dentro de las labores que realiza el farmacéutico, en su día a día, en la farmacia comunitaria. El objetivo de la dispensación es garantizar el acceso del paciente al medicamento de manera adecuada y controlada. En dicho proceso, el farmacéutico comunitario tiene la obligación, como profesional de la salud, de participar de forma activa y comprometida en la detección de posibles errores de medicación o relacionados con la misma. El Grupo de Trabajo de Seguridad del Paciente de SEFAC ha elaborado, como comienzo de una serie de futuros proyectos, unos listados de verificación y decálogos de buenas prácticas sobre medicamentos formulados en forma de parches transdérmicos, comprimidos bucodispersables o formas de liberación modificada. Con esta iniciativa se pretende, mediante una entrevista previa con el paciente y el uso de estas herramientas, minimizar en la medida de lo posible, los errores o problemas que puedan surgir con dichos medicamentos velando finalmente por la seguridad del paciente. A lo largo del 2023 se espera comenzar un pilotaje en farmacias comunitarias colaboradoras con el Grupo de Trabajo de Seguridad del paciente de SEFAC y así poder recoger y notificar los resultados que se obtengan (AU)

Diagnostic efficacy and safety of 99mtc-labeled monoclonal antibody ior c5 in patients with colorectal and anal carcinomas: final report clinical trial phase I/II.

UNLABELLED: Monoclonal antibody (mAb) ior c5 is an IgG1, which recognizes a glycoprotein tumor specific antigen IOR C2 over expressed in the surface of colon and ovarian cancer cells. The aim of the present work was to evaluate the diagnostic efficacy of the 99mTc-labeled mAb ior c5 for the detection of colorectal tumors, its metastases and recurrences. METHODS: Eighty six patients with colorectal or anal cancer, mean age 57 +/- 13 yrs, were involved in a phase 1/11 multicentric, open clinical trial to assess the ability of Radioimmunoscintigraphy (RIS) with 99mTc- mAb ior c5 to detect those tumors. Seventy-four patients received 1 mg of c5 labeled with 1480-1850 MBq to determinate diagnosis efficacy and safety of murine mAb by intravenously (i.v.) bolus injection (group 1). In order to evaluate pharmacokinetic, bio distribution and dosimetry of this radiolabel molecule, 12 patients received 3 mg of labeled ior c5. Planar anterior and posterior images of the lesion sites and suspected metastases were acquired at 2, 4 and 18-24 hours after injection using a matrix size 128 x128 and 500 Kcounts per view. SPECT were scanned at 5 hr post-injection, using a 360' circular orbit with 64 images. HAMA response was measured in serum at 2, 4, 8 and 12 week post-administration. RESULTS: Labeling efficiency was (97.8 - 0.6) %. No adverse reactions or side effects were observed. Overall sensitivity, specificity, accuracy, positive and negative predictive values of the immunoscintigraphy were 95.80%, 100%, 96.51%, 100.0% and 82.35%, respectively. Unknown metastases were detected in 37 of 86 cases (43.02%). No HAMA response was found. CONCLUSIONS: Immunoscintigraphy with 99mTc-labeled mAb ior c5 could be a useful procedure for the diagnosis and follow-up of the patients with colorectal tumors, its metastasis and recurrences.

Nitric oxide donors induce calcium-mobilisation from internal stores but do not stimulate catecholamine secretion by bovine chromaffin cells in resting conditions.

The potential role of nitric oxide (NO) donors and peroxynitrites on both basal catecholamine (CA) secretion and modulation of calcium levels has been investigated in primary cultures of bovine chromaffin cells. NO donors did not modulate catecholamine secretion, while peroxynitrites induced a time dose-dependent increase in basal CA secretion. Two facts may explain the lack of these compounds on basal CA secretion. NO donors induce, on the one hand, an increase in intracellular calcium levels by depletion of internal IP3-stores from endoplasmic reticulum. On the other hand, a small calcium influx through N-type voltage-dependent calcium channels (VDCC), which seem not to be coupled to exocytosis of adrenaline and noradrenaline in chromaffin cells. Both effects, calcium-mobilisation from internal stores and calcium entry through N-type VDCC are mediated by cGMP synthesis. In contrast, peroxynitrites induce an increase in basal CA secretion by both a decrease of intracellular catecholamine content and a toxic effect on cellular membrane. All these results, taken together, could explain contradictory results in the literature on the role of NO on basal catecholamine secretion and on modulation of intracellular calcium in chromaffin cells.

Technetium-99m-antiepidermal growth factor-receptor antibody in patients with tumors of epithelial origin: part II. Pharmacokinetics and clearances.

UNLABELLED: Radiolabeled antitumor antibodies hold promise for diagnostic imaging and therapy in oncology. The purpose of this study was to investigate the pharmacokinetics, clearances and possible differences of two dosage administrations of the 99mTc-labeled antiepidermal growth factor (EGF)-receptor antibody and to predict the best dose and schedule for future clinical evaluations of this radiopharmaceutical. METHODS: Nine patients (4 women, 5 men; mean age 46.4 +/- 14.0 yr) were administered 1-3 mg 99mTc-labeled anti-EGF-receptor antibody (a murine IgG2a isotype) by intravenous bolus infusion. After administration, blood samples were collected from 7 patients from an antecubital vein opposite to the injection side at intervals from 2 min to 24 hr after injection, and plasma samples were obtained for pharmacokinetic analysis. Appropriate plasma samples were examined for isotope clearance (i.e., microCi/ml at various intervals) and 99mTc complexation to plasma proteins by fast protein liquid chromatography (FPLC) analysis. Urine was collected from each patient at 3 hr intervals up to 24 hr after monoclonal antibody administration to monitor 99mTc clearance. Plasma time-activity curves were fitted to a two-compartment model using nonlinear least-squares regression analysis by the method of flexible polyhedrals. RESULTS: Plasma disappearance curves of 99mTc-labeled anti-EGF-receptor antibody were best fit by biexponential equation with a distribution half-life (t(1/2alpha)) of 0.137 +/- 0.076 hr (n = 7) and elimination half-life (t(1/2beta)) of 20.3 +/- 8.0 hr. Analysis of urine showed that activity clearance by this route amounted to 4.9% +/- 0.6% of the injected dose in 24 hr, and FPLC analysis showed no evidence of decomposition, only 6%-7% of 99mTc was in a low molecular weight species. CONCLUSION: Plasma pharmacokinetics and urine clearance indicate comparability in both doses. The pharmacokinetic properties of the 99mTc-labeled anti-EGF-receptor antibody were found to be dose-independent. These findings provide an initial characterization of the radiopharmaceutical disposition in patients and may be used as the basis for calculating a better estimate of biodistribution and dosimetry for patients who will receive 188Re-labeled anti-EGF-receptor antibody (MAb ior egf/r3) injection for radioimmunotherapy and warrants further controlled clinical trials to define the efficacy of the radiopharmaceutical.

Factors affecting pharmacokinetics of monoclonal antibodies: A review article.

Factors affecting the successful therapy of malignant diseases include the antibody dose used and the schedule of administration, the half-life and fast blood clearance of the antibodies, the presence of circulating antigen, poor tumor penetration of the high/molecular-weight monoclonal antibody (mAb) and the way in which these molecules are catabolized. To circumvent these limitations and achieve higher uptake, increased tumor penetration, faster blood clearance and longer retention in the tumors, there is a need to generate mAbs suitable for diagnosis as well as therapy and to develop novel strategies to increase the efficacy of immunotherapeutic treatments. There is a lack of knowledge about many aspects of the physiological function and metabolism of antibodies. This paper is intended to discuss factors that affect the pharmacokinetics of mAbs in human subjects with the purpose of forming possible strategies to optimize this approach for tumor diagnosis and therapy.

188Re-labeled anti-epidermal growth factor receptor humanized monoclonal antibody h-R3: labeling conditions, in vitro and in vivo stability.

The use of antibodies as targeting agents for the delivery of radioisotopes to tumors is an appealing concept that has received widespread attention since the advent of monoclonal antibody (mAb) technology. The present study describes the (188)Re-direct labeling of anti-epidermal growth factor receptor (EGF-R) humanized mAb h-R3; the analytical methods for quality control of radiopharmaceuticals such as instant thin layer chromatography-silica gel (ITLC-SG); the immunoreactivity and biological recognition of the target antigen assessment of the radiolabeled molecule using flow cytometry analysis; in vitro stability studies using saline 0.9% solution, cysteine, diethylenetriaminepentaacetic acid (DTPA), human serum and human serum albumin (HSA) 1% challenge; and the assessment of in vivo stability through biodistribution studies in normal Balb/c mice. No fragmentation of the reduced molecules was found using 2-ME as a reducing agent. Labeling efficiency was greater than 98.5 +/- 0.6% of rhenium-188 (188Re) bound to IgG1 after 5 h, as determined by paper chromatography in saline 0.9% solution. Radiocolloids determined by albumin impregnated ITLC was 1.04 +/- 0.07% in all cases. The biological activity measured by flow cytometry analysis showed an immunoreactivity fraction and the biological recognition of the target antigen overexpressed on H-125 human lung adenocarcinoma cell line greater than 87%. Challenge studies with cysteine, DTPA, human serum and HSA 1% demonstrated no evidence of transcomplexation of 188Re to DTPA or HSA and showed that 30% and 85% of the 188Re-radiolabeled was transcomplexed to human serum and to 100 mM cysteine after 24 h for human serum and 1 h incubation for cysteine at 37 masculine C, respectively. Biodistribution studies indicated no accumulation of the radiolabeled antibodies in normal organs.

(99m)Tc-labeled murine ior C5 monoclonal antibody in colorectal carcinoma patients: pharmacokinetics, biodistribution, absorbed radiation doses to normal organs and tissues and tumor localization.

Monoclonal Antibody (mAb) ior C5 is a murine IgG(1) that recognizes the tumor associated antigen (TAA) ior C2, a cell surface O-linked glycoprotein carbohydrate chain not present in most normal tissues and homogeneously expressed in the cytoplasm of normal colon epithelium and heterogeneously expressed in more than 83% of primary colorectal carcinomas. This study was designed to investigate the pharmacokinetics, biodistribution and the absorbed radiation doses of (99m)Tc-labeled mAb ior C5 antibody in colorectal tumor patients. Ten patients were administered 3 mg of anti-O-linked glycoprotein carbohydrate chain TAA ior C2 murine monoclonal antibody ior C5 radiolabeled with (99m)Tc activity of 1435.0 +/- 123 MBq by intravenous (i.v.) bolus infusion. Blood and urine samples were collected from 4 out of 10 patients at timed intervals from 10 min and up to 24 h after injection of the (99m)Tc-labeled mAb ior C5 for pharmacokinetic studies. Whole body images were taken in 5 out of 10 patients for quantitative normal organ biodistribution and dosimetry studies and planar anterior and posterior and SPECT images were taken in 5 out of 10 patients for tumor localization. Mean absorbed doses were estimated using the methods developed by the Medical Internal Radiation Dose (MIRD) committee. The effective dose equivalent (EDE) and effective dose (ED) were calculated as prescribed in International Commission on Radiological Protection (ICRP) publications 30 and 60. Plasma disappearance curves of (99m)Tc-labeled murine antibody ior C5 were best fit by a two-compartment model in all patients with (t(1/2alpha)) of 4.32 +/- 2.18 h and (t(1/2beta) of 32.6 +/- 3.82 h. Among the main target organs, accumulation of the radiolabeled antibody was found in liver (9.38 +/- 0.80%), heart (8.92 +/- 0.94%) and spleen (1.37 +/- 0.30%) at 5 min post-administration. These values were reduced at 24 h to (5.91 +/- 0.73%) and (0.62 +/- 0.22%), respectively, for the heart and spleen and increased to (9.78 +/- 1.99%) for liver. Estimates of radiation absorbed dose to normal organs in rad/mCi administered were: whole body, 0.0181 +/- 0.0017; heart wall, 0.0768 +/- 0.0090; kidneys, 0.0530 +/- 0.0260; liver, 0.0565 +/- 0.0109 and spleen, 0.0540 +/- 0.0128. The effective dose equivalent and effective dose estimates for adults were 0.0314 +/- 0.0031 and 0.0249 +/- 0.0027 rem/mCi administered. This feasibility study indicates that the O-linked glycoprotein carbohydrate chain TAA ior C2 is expressed in primary and metastatic colorectal carcinomas and shows very limited expression in normal adult tissues. The very good pattern of biodistribution of (99m)Tc-labeled mAb ior C5 in patients will allow imaging of colorectal carcinoma lesions.

Preparation and comparative evaluation of 99mTc-labeled 2-iminothiolane modified antibodies and CITC-DTPA immunoconjugates of anti-EGF-receptor antibodies.

The use of antibodies as targeting agents for the delivery of radioisotopes to tumors is a promising concept that has received widespread attention since the advent of monoclonal antibody (mAb) technology. The following studies are described in this article: the 99mTc-randiolabeling of 2-iminothiolane (2-IT) modified antibodies and 6-p-isothiocyanatobenzyl- diethylene-triamine penta-acetic acid (CITC-DTPA) immunoconjugates of anti-EGF-receptor antibodies murine ior egf/r3 and humanized h-R3; the analytical methods for quality control of the radiopharmaceutical such as instant thin layer chromatography-silica gel (ITLC-SG); the biological assessment of the radiolabeled molecule using flow cytometry analysis; in vitro stability studies with cysteine and DTPA challenge and the biodistribution studies in 4NMRI xenografted nude mice with U-87 human glioblastoma multiforme and MDA-MB-468 breast cancer cell lines. Labeling efficency of (96.48 +/- 0.70%) (98.42 +/- 0.38%), (94.8 +/- 1.25%) and (96.41 +/- 0.89%) was achieved for 99mTC-2-IT ior efg/r3, 99mTc-CITC-DTPA- ior egf/r3, 99mTc-CITC-DTPA- h-R3 and 99mTc-DIACIM h-R3, respectively. Radiocolloids were less than 2.0% in all cases. The biological activity measured by flow cytometry analysis using the MDA-MB-468 breast cancer cell line showed an immunoreactivity fraction greater than 85% in all concentrations of each immunoconjugate. Challenge studies demonstrated no evidence of transcomplexation of 99mTc to 1.0 mM DTPA for 2-IT modified antibody ior egf/r3 and CITC-DTPA immunoconjugates and only 8.7%, 4.9% and 5.0% of the 99mTc-radiolabeled was transcomplexed to 1.0 mM cysteine after 1 h incubation at 37 degrees C for 2-IT modified antibody ior egf/r3, CITC-DTPA ior egf/r3 and CITC-DTPA h-R3, respectively. Biodistribution studies with 2-IT modified antibodies and CITC-DTPA immunoconjugates indicated high tumor uptake in both cell lines with both immunoconjugates and no accumulation of the radiolabeled antibodies in normal organs.

Glutamate triggers neurosecretion and apoptosis in bovine chromaffin cells through a mechanism involving NO production by neuronal NO synthase activation.

Previous work from our group stated that nitric oxide (NO), via cytokines, induces apoptosis in chromaffin cells by a mechanism involving iNOS, nNOS, and NF-κB. In this paper the involvement of glutamate as a possible intracellular trigger of neurosecretion and NO-mediated apoptosis has been evaluated. We show that chromaffin cells express different ionotropic and metabotropic glutamate receptors, this exerting different effects on the regulation of basal and glutamate-induced catecholamine secretion, via NO/cGMP. In addition, we studied the effects of endogenously generated NO, both basal and glutamate-stimulated, on apoptosis of chromaffin cells. Our results show that glutamate agonists are able to induce cell death and apoptosis in bovine chromaffin cells, parallel to an increase in NO production. Such effects were reversed by NOS inhibitors and glutamate receptor antagonists. Under basal conditions, iNOS inhibitors did not have any effect on apoptosis, whereas nNOS inhibitors induced apoptosis, indicating a neuroprotective effect of constitutive nNOS-generated NO. In contrast, glutamate-induced apoptosis was strongly reversed by nNOS inhibitors and weakly by iNOS inhibitors, thus indicating nNOS involvement in glutamate-mediated apoptosis. These results were confirmed by the fact that nNOS expression, but not iNOS, is specifically activated by glutamate. Finally, our results suggest the participation of PKG, PKA, PKC, and MAPK pathways in glutamate-mediated nNOS activation in chromaffin cells and point out the involvement of both PKA and PKC signaling pathways in the apoptotic effect of glutamate.

Nitric oxide and peroxynitrite induce cellular death in bovine chromaffin cells: evidence for a mixed necrotic and apoptotic mechanism with caspases activation.

Treatment of chromaffin cells with nitric oxide (NO) donors (SNP and SNAP) and peroxynitrite produces a time- and dose-dependent necrotic and apoptotic cell death. Necrotic cell death was characterized by both an increase in lactate dehydrogenase and ATP release and changes in nuclei and cell morphology (as seen with fluorescence microscopy analysis with propidium iodide and Hoechst 33342). Apoptotic cell death was characterized by nuclear fragmentation and presence of apoptotic cell bodies, by a decrease in DNA content, and by an increase in DNA fragmentation. Treatment of chromaffin cells with lipopolysaccharide (LPS) or cytokines (interferon-gamma, tumor necrosis factor-alpha) resulted only in apoptotic cell death. Apoptotic effects of NO-inducing compounds were specifically reversed, depending on the stimuli, by the NO scavenger carboxy-PTIO (CPTio) or by the NOS inhibitors L-NMA and thiocitrulline. NO-induced apoptotic death in chromaffin cells was concomitant to a cell cycle arrest in G0G1 phase and a decrease in the number of chromaffin cells in the G2M and S phases of cell cycle. All NO-producing compounds were able to induce activation of caspase 3 and cytochrome c release, and specific inhibitors of caspase 3 and 9, such as Ac-DEVD-CHO (CPP32) and Ac-Z-LEHD-FMK, respectively, prevented NO-induced apoptosis in chromaffin cells. These results suggest that chromaffin cells could be good models for investigating the molecular basis of degeneration in diseases showing death of catecholaminergic neurons, phenomenon in which NO plays an important role.

Evidence that hamster fibroblasts tumors emerge in nude mice through the process of two in vivo selections leading to growth factor "relaxation" and to immune resistance.

The Chinese hamster lung fibroblast cell line (CC139), anchorage- and highly serum-dependent for growth is tumorigenic in nude mice. Tumors arise after 4 to 8 weeks following the inoculation of 5 X 10(5) cells. We have shown that all the emerging tumoral clones (more than 20 analyzed) have lost the growth factor dependence of the parental cells (Pérez-Rodriguez et al., 1981 a). To mimic this selection which occurred in vivo, we selected in vitro growth-factor-independent variants. These variants, GFI304 and GFI461, can proliferate in a serum-free medium supplemented with transferrin alone. This character is stable since it is not lost after the GFI variants have been cultivated in non-selective medium (serum) for more than 20 generations. In spite of their "autonomous" growth and anchorage independence. GFI variants are poorly tumorigenic. The observation of nodule formation and subsequent regression in nude mice, immunosuppressed (irradiation or cyclophosphamide) or not, led us to the conclusion that at least two selections in vivo are required for the tumoral expression of CC139 cells. One leads to a loss of growth factor requirement, the second towards a resistance to the immune surveillance mechanisms of the nude mice. The 4- to 8-week lag period of tumor formation may be accounted for by the spontaneous emergence in vivo of the two new characters necessary to bypass host growth restraints. This report supports the concept of stepwise progression and clonal evolution of preneoplastic cells in vivo and also indicates that tumorigenicity tests in nude mice should be interpreted with caution.

Analysis of growth factor "relaxation" in Chinese hamster lung fibroblasts required for tumoral expression.

The Chinese hamster lung fibroblast line, CCl39, displays the properties characteristic of normal secondary cultures of Chinese hamster fibroblasts including: reversible G0 growth arrest (less than 2% labeled nuclei), anchorage dependence, and high serum-growth factor dependence. Injection of CCl39 cells, or anchorage-independent variants, in nude mice leads to tumor formation; however, as we have previously shown (Pérez-Rodriguez et al., 1981b), the resulting tumor clones no longer possess the high serum dependence of injected CCl39 cells. Hormonal growth restraints imposed by the host create an in vivo selection for diminished, or "relaxed," growth factor requirement. To characterize this growth factor "relaxation" further, we have analyzed the mitogenic response of parental CCl39 cells, anchorage-independent clones, and selected tumoral derivatives, to purified growth factors. Two highly purified growth factors, thrombin and insulin, together fulfill the growth factor requirements of CCl39 cells; thrombin (1 U/ml) stimulates the reinitiation of DNA synthesis in G0-arrested CCl39 cells, and insulin (10 micrograms/ml) maximally potentiates this stimulation to the level obtained with 10% fetal calf serum. First, we found no correlation between loss of anchorage dependence and growth factor relaxation. Second, we found that A71 (anchorage independent), a tumoral variant of CCl39 capable of growth arrest, and tumor-derived cells all display an increased sensitivity to thrombin and a diminished requirement for the potentiating action of insulin. Examination of thrombin binding to CCl39, A51 (nontumoral, anchorage independent), and A71 cells revealed that the increased sensitivity to thrombin of A71 cells is not attributable to an alteration in thrombin cell surface receptor number or affinity for thrombin. Rather, under standard conditions of serum or growth factor removal (30 hr), A71 cells maintain a metabolically elevated growth-arrested state, different from that of their nontumoral counterparts. Consequently, much lower concentrations of growth factors are needed to induce a proliferative response in these tumoral cells.