Analytical Determination of Serotonin Exocytosis in Human Platelets with BDD-on-Quartz MEA Devices.

Amperometry is arguably the most widely used technique for studying the exocytosis of biological amines. However, the scarcity of human tissues, particularly in the context of neurological diseases, poses a challenge for exocytosis research. Human platelets, which accumulate 90% of blood serotonin, release it through exocytosis. Nevertheless, single-cell amperometry with encapsulated carbon fibers is impractical due to the small size of platelets and the limited number of secretory granules on each platelet. The recent technological improvements in amperometric multi-electrode array (MEA) devices allow simultaneous recordings from several high-performance electrodes. In this paper, we present a comparison of three MEA boron-doped diamond (BDD) devices for studying serotonin exocytosis in human platelets: (i) the BDD-on-glass MEA, (ii) the BDD-on-silicon MEA, and (iii) the BDD on amorphous quartz MEA (BDD-on-quartz MEA). Transparent electrodes offer several advantages for observing living cells, and in the case of platelets, they control activation/aggregation. BDD-on-quartz offers the advantage over previous materials of combining excellent electrochemical properties with transparency for microscopic observation. These devices are opening exciting perspectives for clinical applications.

Multielectrode Arrays as a Means to Study Exocytosis in Human Platelets.

Platelets are probably the most accessible human cells to study exocytosis by amperometry. These cell fragments accumulate biological amines, serotonin in particular, using similar if not the same mechanisms as those employed by sympathetic, serotoninergic, and histaminergic neurons. Thus, platelets have been widely recognized as a model system to study certain neurological and psychiatric diseases. Platelets release serotonin by exocytosis, a process that entails the fusion of a secretory vesicle to the plasma membrane and that can be monitored directly by classic single cell amperometry using carbon fiber electrodes. However, this is a tedious technique because any given platelet releases only 4-8 secretory δ-granules. Here, we introduce and validate a diamond-based multielectrode array (MEA) device for the high-throughput study of exocytosis by human platelets. This is probably the first reported study of human tissue using an MEA, demonstrating that they are very interesting laboratory tools to assess alterations to exocytosis in neuropsychiatric diseases. Moreover, these devices constitute a valuable platform for the rapid testing of novel drugs that act on secretory pathways in human tissues.

Measurements of Calcium in Chromaffin Cell Organelles Using Targeted Aequorins.

The molecular mechanisms that mediate and regulate calcium (Ca2+) fluxes through the membranes of intracellular organelles play a key role in the generation and shaping of the local and global cytosolic Ca2+ signals triggering the process of regulated exocytosis in chromaffin cells. Beyond that role, intraorganellar Ca2+ homeostasis also regulates organelle-specific processes such as oxidative phosphorylation in mitochondria, maturation of secretory granules, or stress in the endoplasmic reticulum. In this chapter, we describe current methods to study mitochondrial, endoplasmic reticulum, and secretory vesicle calcium homeostasis in living chromaffin cells using engineered targeted aequorins.

Quantal Release Analysis of Electrochemically Active Molecules Using Single-Cell Amperometry.

Single-cell amperometry is a powerful technique that permits the detection of electrochemically active transmitters, such as catecholamines, histamine, or serotonin, released by exocytosis from secretory cells.Amperometry has two main characteristics that make it ideal for the study of exocytosis at the single-cell level with single-vesicle resolution quantal release. (i) It is noninvasive. The carbon fiber microelectrode can be carefully positioned on plasma membrane of a single cell, allowing the detection of the oxidation current of the secreted molecules. (ii) High temporal resolution and sensitivity. Exocytosis can be monitored with a real-time resolution that allows the determination of the kinetics release with an attomol detection sensitivity, which ensures an accurate calculation of the amount of transmitter released.Here, we compile some recommendations and advices to perform amperometry quantal analysis.

A Secretory Vesicle Failure in Parkinson's Disease Occurs in Human Platelets.

OBJECTIVE: The presence of elevated dopamine (DA) and its major metabolites in the cytosol of neurons has been associated with their vulnerability in Parkinson's disease (PD). Over 99% of the cell's amines are confined to secretory vesicles (SVs), making these structures fundamental in the regulation of cytosolic DA levels. SVs of platelets use similar, if not the same mechanisms to accumulate serotonin in SVs as dopaminergic neurons do to store DA. Hence, any functional defects in platelets probably mirrors events in DA neurons. METHODS: We have isolated fresh platelets from the blood of 75 PD patients, 116 matched controls and 24 patients with Parkinsonism, assaying serotonin handling (basal content, accumulation, secretion and spontaneous leakage). RESULTS: We found a dramatic decrease in the serotonin content and uptake by SVs, as well as decreased thrombin-induced release by platelets from PD patients but not in those from most Parkinsonism cases. Platelets from PD patients also failed to retain serotonin in SVs. INTERPRETATION: These findings indicate a functional impairment in the handling of amines by SVs in PD patients. This defect may serve as a biomarker of PD, and the approach described here may be potentially used for the subclinical detection of PD and to establish a platform to assay disease modifying drugs. ANN NEUROL 2022.

Phases of the exocytotic fusion pore.

Membrane fusion and fission are fundamental processes in living organisms. Membrane fusion occurs through the formation of a fusion pore, which is the structure that connects two lipid membranes during their fusion. Fusion pores can form spontaneously, but cells endow themselves with a set of proteins that make the process of fusion faster and regulatable. The fusion pore starts with a narrow diameter and dilates relatively slowly; it may fluctuate in size or can even close completely, producing a transient vesicle fusion (kiss-and-run), or can finally expand abruptly to release all vesicle contents. A set of proteins control the formation, dilation, and eventual closure of the fusion pore and, therefore, the velocity at which the contents of secretory vesicles are released to the extracellular medium. Thus, the regulation of fusion pore expansion or closure is key to regulate the release of neurotransmitters and hormones. Here, we review the phases of the fusion pore and discuss the implications in the modes of exocytosis.

Bisoprolol/amlodipine combination therapy improves blood pressure control in patients with essential hypertension following monotherapy failure.

OBJECTIVE: The efficacy of a bisoprolol/amlodipine fixed-dose combination (FDC) in patients with essential hypertension who had not responded to bisoprolol or amlodipine monotherapy was investigated. RESEARCH DESIGN AND METHODS: In an 18 week, multicenter, randomized, comparative phase III study (ClinicalTrials.gov identifier: NCT01977794), patients with blood pressure uncontrolled by bisoprolol or amlodipine monotherapy (5 mg OD) began treatment with bisoprolol/amlodipine FDC 5/5 mg OD. Patients with controlled blood pressure (BP) at week 6/12 continued at current FDC strength, and patients with uncontrolled BP received FDC dose uptitration (maximum dose: 10/10 mg). The primary efficacy endpoint was change in systolic blood pressure (SBP) at week 18 versus baseline (corresponding to SBP under monotherapy), and secondary endpoints included change from baseline in SBP after week 6/12 and percentage of BP-controlled patients at week 6, 12 and 18. Safety was assessed by number/types of adverse events (AEs). RESULTS: Two hundred patients were randomized to treatment (100 with uncontrolled BP under bisoprolol and 100 under amlodipine monotherapy). Overall, 196 patients were eligible for analysis. The patient groups displayed similar mean SBP reductions from baseline by study end (bisoprolol monotherapy failure: 25.9 ± 12.82 mmHg reduction; amlodipine monotherapy failure: 24.7 ± 11.67 mmHg reduction; p < 0.001 for both). Overall mean SBP decreased by 25.3 ± 12.25 mmHg (p < 0.001). Mean heart rate reductions were also observed (bisoprolol monotherapy failure: 6.6 ± 9.67 bpm reduction; amlodipine monotherapy failure: 11.5 ± 8.65 bpm reduction; p < 0.001 for both). Most patients (83.2%) displayed BP control with bisoprolol/amlodipine 5/5 mg at 6 weeks. Treatment was well tolerated at all dose levels; treatment-related AEs (mostly of mild/moderate intensity) were reported by 52.5% of patients, with no severe or serious treatment-related AEs reported. As the study focused on hypertension, total cardiovascular risk was not assessed. CONCLUSIONS: Bisoprolol/amlodipine FDC therapy is associated with significant BP improvements in patients with essential hypertension following monotherapy failure.

The dynamics of mitochondrial Ca2+ fluxes.

We have investigated the kinetics of mitochondrial Ca(2+) influx and efflux and their dependence on cytosolic [Ca(2+)] and [Na(+)] using low-Ca(2+)-affinity aequorin. The rate of Ca(2+) release from mitochondria increased linearly with mitochondrial [Ca(2+)] ([Ca(2+)](M)). Na(+)-dependent Ca(2+) release was predominant al low [Ca(2+)](M) but saturated at [Ca(2+)](M) around 400muM, while Na(+)-independent Ca(2+) release was very slow at [Ca(2+)](M) below 200muM, and then increased at higher [Ca(2+)](M), perhaps through the opening of a new pathway. Half-maximal activation of Na(+)-dependent Ca(2+) release occurred at 5-10mM [Na(+)], within the physiological range of cytosolic [Na(+)]. Ca(2+) entry rates were comparable in size to Ca(2+) exit rates at cytosolic [Ca(2+)] ([Ca(2+)](c)) below 7muM, but the rate of uptake was dramatically accelerated at higher [Ca(2+)](c). As a consequence, the presence of [Na(+)] considerably reduced the rate of [Ca(2+)](M) increase at [Ca(2+)](c) below 7muM, but its effect was hardly appreciable at 10muM [Ca(2+)](c). Exit rates were more dependent on the temperature than uptake rates, thus making the [Ca(2+)](M) transients to be much more prolonged at lower temperature. Our kinetic data suggest that mitochondria have little high affinity Ca(2+) buffering, and comparison of our results with data on total mitochondrial Ca(2+) fluxes indicate that the mitochondrial Ca(2+) bound/Ca(2+) free ratio is around 10- to 100-fold for most of the observed [Ca(2+)](M) range and suggest that massive phosphate precipitation can only occur when [Ca(2+)](M) reaches the millimolar range.

Artropatía de Jaccoud y fibrosis pulmonar en síndrome CREST / Jaccoud’s arthropathy and pulmonary fibrosis in CREST syndrome

Se describe el caso de una paciente de 48 años de edad con diagnóstico de síndrome CREST incompleto (variante de esclerosis sistémica limitada), en quien se documenta la presencia de artropatía de Jaccoud de las manos y compromiso pulmonar intersticial por fibrosis pulmonar tipo neumonía intersticial usual, con positividad para factor reumatoide y anticuerpos contra el péptido citrulinado.Palabras clave: síndrome CREST, artropatía, enfermedad pulmonar intersticial, fibrosis pulmonar.

We report a case of a 48 years old patient with diagnosis of incomplete CREST syndrome (variant limited systemic sclerosis) in who we documented the presence of Jaccoud’s arthropathy of the hands and pulmonary involvement by pulmonary fibrosis type usual interstitial pneumonia, with positivity for rheumatoid factor and anti-cyclic citrullinated peptide antibody.Key words: CREST syndrome, joint disease, interstitial lung disease, pulmonary fibrosis.

Estimation of health-care costs for work-related injuries in the Mexican Institute of Social Security.

BACKGROUND: Data on the economic consequences of occupational injuries is scarce in developing countries which prevents the recognition of their economic and social consequences. This study assess the direct heath care costs of work-related accidents in the Mexican Institute of Social Security, the largest health care institution in Latin America, which covered 12,735,856 workers and their families in 2005. METHODS: We estimated the cost of treatment for 295,594 officially reported occupational injuries nation wide. A group of medical experts devised treatment algorithms to quantify resource utilization for occupational injuries to which unit costs were applied. Total costs were estimated as the product of the cost per illness and the severity weighted incidence of occupational accidents. RESULTS: Occupational injury rate was 2.9 per 100 workers. Average medical care cost per case was $2,059 USD. The total cost of the health care of officially recognized injured workers was $753,420,222 USD. If injury rate is corrected for underreporting, the cost for formal injured workers is 791,216,460. If the same costs are applied for informal workers, approximately half of the working population in Mexico, the cost of healthcare for occupational injuries is about 1% of the gross domestic product. CONCLUSIONS: Health care costs of occupational accidents are similar to the economic direct expenditures to compensate death and disability in the social security system in Mexico. However, indirect costs might be as important as direct costs.

Proyecto de capacitación y transformación básica de derivados lacteos a diseño final formato FPS

El presente trabajo dirigido se realizó en la comunidad de Chaupisuyo jurisdicción de la provincia de Quillacollo en el Departamento de Cochabamba, bajo un convenio entre la UMSS y la ONG CETA con los siguientes términos de referencia: -Realizar un proyecto a diseño final, formato FPS de capacitación prpoductiva en transformación básica de derivados lacteos. -Transmitir a los beneficiarios de Chapisuyo los resultados de dicho proyecto. -Presentar informes bimensuales. De acuerdo a "exigencias del formato FPS" el estudio esta basado en una investigación del medio en cuanto a datos estadísticos socioeconómicos, ambientales, servicios básico, que son la base esencia para el requerimiento de la necesidad de elaborar este proyecto. En la situación con proyuecto se describe por módulos la capacitación en derivados lacteos que se llevara a cabo por módulos. Terminando con el estudio de fctibilidad, donde los indicadores económicos son: TIR = 76135.2 $Us. VAN = 21.2

[How much does the medical treatment of chronic Chagas cardiopathy cost? Direct costs in a cardiology hospital]. / Cuánto cuesta la atención de la cardiopatía chagásica crónica? Costos directos en un hospital de cardiología.

OBJECTIVE: To estimate health care costs of patients with chronic Chagasic cardiomyopathy (CCC) in a cardiovascular referral center (Instituto Nacional de Cardiología I. Chávez). MATERIAL AND METHODS: In a retrospective study, 13 clinical charts of CCC patients treated in the hospital during 1998 were reviewed. Diagnostic and therapeutic procedures and patients admission were identify as well as health care costs, which were drawn from the hospital costs system. RESULTS: 62% of the cases were admitted to the hospital through the out-patient facilities. All the patients had a health care subsidy of 15 to 55% allocated to the institutional budget. Health care costs were calculated for minimal, average, and maximum scenarios, according to the patient's clinical stage and the price of medical equipment used (low, medium, and high). Most of the health care costs are due to the use of high cost diagnostic equipment (33 a 58%) and hospital stay (including the emergency room) (19 a 28%). CONCLUSION: This is the first approximation to the economic study of Chagas disease in Mexico, providing foundations for further studies on health economics and quality care of CCC, and suggests that prevention should be enhanced.

Cuánto cuesta la atención de la cardiopatía chagásica crónica? Costos directos en un hospital de cardiología / How much does the medical treatment of chronic Chagas cardiopathy cost? Direct costs in a cardiology hospital

OBJECTIVE: To estimate health care costs of patients with chronic Chagasic cardiomyopathy (CCC) in a cardiovascular referral center (Instituto Nacional de CardiologÝa I. Chßvez). MATERIAL AND METHODS: In a retrospective study, 13 clinical charts of CCC patients treated in the hospital during 1998 were reviewed. Diagnostic and therapeutic procedures and patients admission were identify as well as health care costs, which were drawn from the hospital costs system. RESULTS: 62 of the cases were admitted to the hospital through the out-patient facilities. All the patients had a health care subsidy of 15 to 55 allocated to the institutional budget. Health care costs were calculated for minimal, average, and maximum scenarios, according to the patient's clinical stage and the price of medical equipment used (low, medium, and high). Most of the health care costs are due to the use of high cost diagnostic equipment (33 a 58) and hospital stay (including the emergency room) (19 a 28). CONCLUSION: This is the first approximation to the economic study of Chagas disease in Mexico, providing foundations for further studies on health economics and quality care of CCC, and suggests that prevention should be enhanced.

Lupus eritematoso sistémico: experiencia con 181 casos / Systemic lupus erythematosus: experience in 181 cases

Objetivo: Determinar las características clínicas y curso de 181 pacientes con Lupus Eritematoso Sistémico, diagnosticados y seguidos durante un período de 21 años. Metodología: Estudio retrospectivo de enero de 1973 a diciembre de 1983 realizado en pacientes del Hospital General San Juan de Dios y de la Clínica de Enfermedades Reumáticas (Privada). Resultados: Se analizaron 113 (62.4) pacientes del Hospital General y 68 (37.6) de la Clínica Privada. El 93.9 fueron del sexo femenino y el 80.1 fue del grupo adulto. La paciente de menor edad fue de 3 años y de 55 la de mayor edad. El 50 de los pacientes tuvieron más de 5 criterios diagnósticos, hubo 6 con sólo 3 criterios y 12 con más de 7 criterios. Al momento del diagnóstico la artropatía se presentó en el 91.7 de los casos, seguido por fiebre en 68.5, afección cutánea en el 57.4 y orina anormal en el 49.7El 51.4 de los pacientes experimentaron reactivaciones y la afección renal fue la manifestación más frecuente de las mismas. Los pacientes fueron seguidos por un período promedio de 4 años con 7.5 meses (rango de 1 mes a 19 años 9 meses). Se encuentran aún bajo tratamiento 91 (50.3) y se han perdido de seguimiento 41 (22.6). En remisión total, ya sin tratamiento, se encuentran 6 pacientes (3.3). Han fallecido 43 (23.8) y la infección fue la causa más frecuente de muerte, seguidos por la afección renal y la psicosis. Conclusiones:La enfermedad predomina en mujeres, con relación de 15:1 Las manifestaciones fueron similares para los distintos grupos de pacientes, pero la frecuencia de orina anormal fue mayor en pacientes del Hospital General (p= <0.01) y el hallazgo de glomerulonefritis proliferativa difusa lo fue en pacientes del grupo juve...

Infecciones en pacientes con lupus eritematoso sistémico: reporte de 107 pacientes, análisis de su etiología y factores de riesgo / Infections in patients with systemic lupus erythematosus, report of 107 cases: analysis of etiology and risk factors

Objetivo: Determinar la frecuencia de infecciones en pacientes con lupus eritematoso sistémico, su etiología y los factores de riesgo con relación a sexo, edad, procedencia, manifestaciones y tratamiento. Metodología: Estudio retrospectivo de 21 años (enero 1973 a febrero 1994) realizado en un grupo de 181 pacientes, 115 seguidos en el Hospital General San Juan de Dios y 66 en la Clínica de Enfermedades Reumáticas (privadas). Resultados: Se encontró infección en 107 (59.1) de los 181 pacientes, 37 de ellos tuvieron infección nosocomial. En 20 (46.5), de los 43 pacientes fallecidos durante el seguimiento, la infección fue parte de su cuadro final y en 10 de ellos de orgien nosocomial. Se detectó un total de 203 infecciones, de las cuales 50 (24.6) fueron nosocomiales. Hubo 114 bacterianas, 54 micóticas y 35 virales. Las bacterianas fueron divididas en: urinarias (38), de tejidos blandos (31), pulmonares (11), otitis media (10), tuberculosis (6), pioartritis (6) y misceláneas (12). Se aislaron 100 bacterias, 31 Staphylococcus aureus, 30 Escherichia coli, 12 Pseudomona, 10 Klebsiella, 7 Salmonella, 4 Enterobacter, 2 Proteus, 1 Acinetobacter, 1 Streptococcus, 1 Pneumococcus y 1 Mycobacterium. Conclusión: Los gérmenes gram negativos fueron los más frecuentes de las infeccciones bacterianas (66), la Cándida albicans el de las infecciones micóticas (88) y el Herpes zoster el de las virales (86). El riesgo relativo de infección, fue mayor en pacientes masculinos, del grupo juvenil, de la Clínica Privada, cuando la creatinina al ingreso fue mayor de 3 mg/dl, pero significante sólo en quienes fueron tratados con medicamentos antimaláricos (rr=1.37, p=0.01) y citotóxicos (rr=1.45, p=0.002). Los pacientes con infección nosocomial tuvieron más riesgo de morir por infección (rr=3.57, p=0.001)