COVID-19 y tiroides. Revisión de la literatura y recomendaciones para pacientes con enfermedad tiroidea / Thyroid and COVID-19. Review and recommendations for patients with thyroid disease

Resumen Las enfermedades de la tiroides, tanto benignas como malignas, son altamente prevalentes a nivel mundial, por lo que es muy probable que durante la pandemia por SARS-CoV-2 veamos pacientes con ambas enfermedades. Esto exige el conocimiento de las implicaciones potenciales de este nuevo virus en el funcionamiento de la glándula, en los tratamientos usuales para estas enfermedades y en consideraciones especiales para este grupo poblacional. A la fecha no hay evidencia que soporte que las enfermedades tiroideas aumenten el riesgo de infección o severidad de la enfermedad; sin embargo, es posible que durante infecciones severas por SARS-CoV-2 en personas con o sin antecedente de enfermedad tiroidea puedan presentar alteración de las pruebas tiroideas, aunque transitoriamente y sin requerimiento de tratamiento específico. Es fundamental que los pacientes continúen con sus tratamientos ambulatorios y se difiera, en la medida de lo posible, los procedimientos quirúrgicos o la administración de yodo radioactivo hasta que se considere seguro realizarlos.

Abstract Both benign and malignant thyroid diseases are highly prevalent worldwide, so it is highly likely that during the COVID-19 pandemic we will see patients with this comorbidity. This requires knowledge of the potential implications of this new virus in the functioning of the gland, the usual treatments for these diseases and special recommendations in this population. To date, there is no evidence to support that thyroid diseases increase the risk of infection or the disease severity. However, it is possible that during severe SARS-CoV-2 infections in people with or without history of thyroid disease, the thyroid tests may be altered, although transitory and does not require specific treatment. It is essential for patients to continue with their outpatient treatments and defer as far as possible surgical procedures or administration of radioactive iodine until it is considered safe to perform.

COVID-19 y diabetes. Retos, Implicaciones y manejo durante la pandemia / COVID-19 and Diabetes. Challenges, Implications and treatment during the pandemic

Resumen Desde finales del año 2019 un nuevo coronavirus, SARS-CoV-2, se extendió desde China hacia el resto del mundo, causando la pandemia de una enfermedad denominada COVID-19. Una enfermedad sistémica que en algunos casos produce neumonía severa que incluso puede progresar a falla respiratoria aguda y finalmente la muerte. Entre las comorbilidades que se han asociado con un aumento en la mortalidad por SARS-CoV-2 se encuentra la diabetes. En general, se estima que tener diabetes aumenta un 18 % el riesgo de infecciones respiratorias, en parte por el impacto que genera sobre la inmunidad innata o adquirida, lo que estaría contribuyendo a una presentación clínica más severa del SARS-CoV-2 al comparar con población sin diabetes. Considerando que existe una asociación entre mal control glucémico y mayor severidad clínica de la infección por COVID-19, se deben hacer importantes consideraciones sobre el manejo farmacológico brindado a los pacientes; el perfilamiento dependerá de las condiciones de cada paciente, de la severidad de la enfermedad y del tipo de manejo instaurado ya sea ambulatorio o intrahospitalario.

Abstract Since the end of the year 2019 a new coronavirus called Severe Acute Respiratory Syndrome (SARS-CoV-2) has spread from China to the rest of the world, causing the pandemic of the disease called COVID-19. A systemic disease that in some cases produces severe pneumonia that can even progress to acute respiratory failure and eventually death. Among the comorbidities that have been associated with an increase in mortality from SARS-CoV-2, diabetes is one of them. In general, it is estimated that having diabetes increases the risk of respiratory infections by 18 %, in part, due to the impact on innate and acquired immunity, which would be contributing to a more severe clinical presentation of SARS-CoV-2 when compared with population without diabetes. Considering that there is an association between worse glycemic control and higher clinical severity of COVID-19 infection, important considerations must be made regarding the type of pharmacological management that is provided to patients; the profiling will depend on the conditions of each patient, the severity of the disease, and the type of management either as outpatient or in-hospital.

Impacto clínico del desescalamiento a ertapenem en pacientes críticos con infecciones por Enterobacteriaceae / Clinical impact of ertapenem de-escalation in critically-ill patients with Enterobacteriaceae infections

Resumen Introducción: Ertapenem ha demostrado eficacia frente a Enterobacteriaceae productoras de β-lactamasas de espectro extendido, pero carece de actividad contra bacterias no fermentadoras; el desescalamiento a este antimicrobiano cuando no existe la presencia de P. aeruginosa podría reducir la presión selectiva contra esta bacteria y mejorar los resultados clínicos. Objetivo: Evaluar el impacto clínico del desescalamiento de antimicrobianos con cobertura anti-pseudomonas a ertapenem, un agente sin este espectro, en pacientes críticos con infecciones por Enterobacteriaceae. Métodos: Se realizó un estudio de cohorte prospectivo en adultos admitidos a Unidades de Cuidado Intensivo (UCI) con infecciones por Enterobacteriaceae, que habían sido desescalados de una cobertura anti-pseudomonas, a un antimicrobiano sin la misma (ertapenem). Se realizó un modelo de riesgo proporcional de Cox comparando mortalidad por cualquier causa y duración de estancia hospitalaria entre aquellos pacientes que permanecieron con cobertura anti-pseudomonas versus aquellos que fueron desescalados a ertapenem. Resultados: 105 pacientes en el grupo anti-pseudomonas fueron comparados con 148 pacientes del grupo de desescalamiento a ertapenem. El desescalamiento estuvo asociado con una menor mortalidad por cualquier causa comparado con los pacientes que permanecieron con cobertura anti-pseudomonas (hazard ratio ajustado 0,24; IC 95%: 0,12-0,46). La estancia hospitalaria en UCI fue similar en ambos grupos. Discusión: Los pacientes de UCI con infecciones por Enterobacteriaceae desescalados a terapia con ertapenem, tuvieron mejores resultados clínicos comparados con aquellos que permanecieron en terapia anti-pseudomonas, sugiriendo que el desescalamiento es una práctica segura en esta población.

Background: Ertapenem has proven to be effective for extended-spectrum beta-lactamases-producing Enterobacteriaceae but lacks activity against non-fermenters; de-escalation to this antibiotic may reduce the selection of resistance to Pseudomonas aeruginosa and improve clinical outcomes. Aim: To evaluate the clinical impact of de-escalation from broad-spectrum anti-pseudomonal agents to ertapenem, a non-pseudomonal antibiotics for Enterobacteriaceae infections in critically-ill patients. Methods: We conducted a prospective cohort study in adult patients admitted to intensive care units (ICUs) who had Enterobacteriaceae infections and were de-escalated from empiric anti-pseudomonal coverage to non-pseudomonal antibiotics. Cox proportional hazards models were performed comparing all-cause mortality and length of hospital stay between patients who remained on anti-pseudomonal coverage versus those who were de-escalated to ertapenem. Results: 105 patients in the anti-pseudomonal group were compared to 148 patients in the ertapenem de-escalation group. De-escalation was associated with lower all-cause mortality compared to patients who remained on anti-pseudomonal coverage (adjusted Hazard Ratio 0.24; 95% CI: 0.12-0.46). The length of ICU stay was similar between the groups. Discussion: ICU patients with Enterobacteriaceae infections de-escalated to ertapenem therapy had better outcomes compared to patients who remained on broad-spectrum, anti-pseudomonal therapy, suggesting that de-escalation is a safe approach amongst ICU patients.

Optical and nuclear imaging of glioblastoma with phosphatidylserine-targeted nanovesicles.

Multimodal tumor imaging with targeted nanoparticles potentially offers both enhanced specificity and sensitivity, leading to more precise cancer diagnosis and monitoring. We describe the synthesis and characterization of phenol-substituted, lipophilic orange and far-red fluorescent dyes and a simple radioiodination procedure to generate a dual (optical and nuclear) imaging probe. MALDI-ToF analyses revealed high iodination efficiency of the lipophilic reporters, achieved by electrophilic aromatic substitution using the chloramide 1,3,4,6-tetrachloro-3α,6α-diphenyl glycoluril (Iodogen) as the oxidizing agent in an organic/aqueous co-solvent mixture. Upon conjugation of iodine-127 or iodine-124-labeled reporters to tumor-targeting SapC-DOPS nanovesicles, optical (fluorescent) and PET imaging was performed in mice bearing intracranial glioblastomas. In addition, tumor vs non-tumor (normal brain) uptake was compared using iodine-125. These data provide proof-of-principle for the potential value of SapC-DOPS for multimodal imaging of glioblastoma, the most aggressive primary brain tumor.

Variation in human cancer cell external phosphatidylserine is regulated by flippase activity and intracellular calcium.

Viable cancer cells expose elevated levels of phosphatidylserine (PS) on the exoplasmic face of the plasma membrane. However, the mechanisms leading to elevated PS exposure in viable cancer cells have not been defined. We previously showed that externalized PS may be used to monitor, target and kill tumor cells. In addition, PS on tumor cells is recognized by macrophages and has implications in antitumor immunity. Therefore, it is important to understand the molecular details of PS exposure on cancer cells in order to improve therapeutic targeting. Here we explored the mechanisms regulating the surface PS exposure in human cancer cells and found that differential flippase activity and intracellular calcium are the major regulators of surface PS exposure in viable human cancer cells. In general, cancer cell lines with high surface PS exhibited low flippase activity and high intracellular calcium, whereas cancer cells with low surface PS exhibited high flippase activity and low intracellular calcium. High surface PS cancer cells also had higher total cellular PS than low surface PS cells. Together, our results indicate that the amount of external PS in cancer cells is regulated by calcium dependent flippase activity and may also be influenced by total cellular PS.

Imaging and Therapy of Pancreatic Cancer with Phosphatidylserine-Targeted Nanovesicles.

Pancreatic cancer remains one of the most intractable cancers, with a dismal prognosis reflected by a 5-year survival of ~6%. Since early disease symptoms are undefined and specific biomarkers are lacking, about 80% of patients present with advanced, inoperable tumors that represent a daunting challenge. Despite many clinical trials, no single chemotherapy agent has been reliably associated with objective response rates above 10% or median survival longer than 5 to 7 months. Although combination chemotherapy regimens have in recent years provided some improvement, overall survival (8-11 months) remains very poor. There is therefore a critical need for novel therapies that can improve outcomes for pancreatic cancer patients. Here, we present a summary of the current therapies used in the management of advanced pancreatic cancer and review novel therapeutic strategies that target tumor biomarkers. We also describe our recent research using phosphatidylserine-targeted saposin C-coupled dioleoylphosphatidylserine nanovesicles for imaging and therapy of pancreatic cancer.

Introduction of software tools for epidemiological surveillance in infection control in Colombia / Introducción de herramientas informáticas para la vigilancia epidemiológica en el control de infecciones en Colombia

Introduction: Healthcare-Associated Infections (HAI) are a challenge for patient safety in the hospitals. Infection control committees (ICC) should follow CDC definitions when monitoring HAI. The handmade method of epidemiological surveillance (ES) may affect the sensitivity and specificity of the monitoring system, while electronic surveillance can improve the performance, quality and traceability of recorded information. Objective: To assess the implementation of a strategy for electronic surveillance of HAI, Bacterial Resistance and Antimicrobial Consumption by the ICC of 23 high-complexity clinics and hospitals in Colombia, during the period 2012-2013. Methods: An observational study evaluating the introduction of electronic tools in the ICC was performed; we evaluated the structure and operation of the ICC, the degree of incorporation of the software HAI Solutions and the adherence to record the required information. Results: Thirty-eight percent of hospitals (8/23) had active surveillance strategies with standard criteria of the CDC, and 87% of institutions adhered to the module of identification of cases using the HAI Solutions software. In contrast, compliance with the diligence of the risk factors for device-associated HAIs was 33%.

Introducción: Las infecciones asociadas a la atención en salud (IAAS) son un reto para la seguridad del paciente. Los comités de infecciones hospitalarios (CIH) deben realizar una vigilancia epidemiológica (VE) de las IAAS siguiendo los criterios de los Centros para el Control y Prevención de Enfermedades - EE.UU (CDC). La VE manual afecta la sensibilidad y especificidad del sistema de vigilancia, mientras que la VE electrónica mejora el desempeño, calidad y trazabilidad de la información registrada. Objetivo: Evaluar la implementación de una estrategia para la VE electrónica de las IAAS, resistencia bacteriana, consumo de antimicrobianos y características de los CIH en 23 clínicas y hospitales de alta complejidad en Colombia, en el periodo 2012-2013. Métodos: Se realizó un estudio observacional descriptivo de la introducción de herramientas informáticas en los CIH, evaluando la estructura y funcionamiento de los CIH, el grado de incorporación del software HAI Solutions y la cumplimiento al registro de la información requerida. Resultados: El 38% de las clínicas y hospitales (8/23) presentaron estrategias de vigilancia epidemiológica activa con criterios estándar del CDC. El 87% de las instituciones se adhirieron al módulo de captación de casos del software HAI Solutions, y el cumplimiento del diligenciamiento de los factores de riesgo de las IAAS asociadas a dispositivos fue del 33%. Conclusiones: La introducción del modelo de VE electrónica podría lograr un mayor cumplimiento a un modelo de vigilancia epidemiológica activo, estandarizado y prospectivo, contribuyendo al mejoramiento en la validez y calidad de la información registrada.

SapC-DOPS nanovesicles induce Smac- and Bax-dependent apoptosis through mitochondrial activation in neuroblastomas.

BACKGROUND: High toxicity, morbidity and secondary malignancy render chemotherapy of neuroblastoma inefficient, prompting the search for novel compounds. Nanovesicles offer great promise in imaging and treatment of cancer. SapC-DOPS, a stable nanovesicle formed from the lysosomal protein saposin C and dioleoylphosphatidylserine possess strong affinity for abundantly exposed surface phosphatidylserine on cancer cells. Here, we show that SapC-DOPS effectively targets and suppresses neuroblastoma growth and elucidate the molecular mechanism of SapC-DOPS action in neuroblastoma in vitro. METHODS: In vivo targeting of neuroblastoma was assessed in xenograft mice injected intravenously with fluorescently-labeled SapC-DOPS. Xenografted tumors were also used to demonstrate its therapeutic efficacy. Apoptosis induction in vivo was evaluated in tumor sections using the TUNEL assay. The mechanisms underlying the induction of apoptosis by SapC-DOPS were addressed through measurements of cell viability, mitochondrial membrane potential (ΔΨM), flow cytometric DNA fragmentation assays and by immunoblot analysis of second mitochondria-derived activator of caspases (Smac), Bax, Cytochrome c (Cyto c) and Caspase-3 in the cytosol or in mitochondrial fractions of cultured neuroblastoma cells. RESULTS: SapC-DOPS showed specific targeting and prevented the growth of human neuroblastoma xenografts in mice. In neuroblastoma cells in vitro, apoptosis occurred via a series of steps that included: (1) loss of ΔΨM and increased mitochondrial superoxide formation; (2) cytosolic release of Smac, Cyto c, AIF; and (3) mitochondrial translocation and polymerization of Bax. ShRNA-mediated Smac knockdown and V5 peptide-mediated Bax inhibition decreased cytosolic Smac and Cyto c release along with caspase activation and abrogated apoptosis, indicating that Smac and Bax are critical mediators of SapC-DOPS action. Similarly, pretreatment with the mitochondria-stabilizing agent bongkrekic acid decreased apoptosis indicating that loss of ΔΨM is critical for SapC-DOPS activity. Apoptosis induction was not critically dependent on reactive oxygen species (ROS) production and Cyclophilin D, since pretreatment with N-acetyl cysteine and cyclosporine A, respectively, did not prevent Smac or Cyto c release. CONCLUSIONS: Taken together, our results indicate that SapC-DOPS acts through a mitochondria-mediated pathway accompanied by an early release of Smac and Bax. Specific tumor-targeting capacity and anticancer efficacy of SapC-DOPS supports its potential as a dual imaging and therapeutic agent in neuroblastoma therapy.

SapC-DOPS nanovesicles: a novel targeted agent for the imaging and treatment of glioblastoma.

Glioblastoma multiforme (GBM) is the most common malignant primary brain tumor. Classified by the World Health Organization (WHO) as grade IV astrocytoma, GBMs are extremely aggressive, almost always recur, and despite our best efforts, remain incurable. This review describes the traditional treatment approaches that led to moderate successes in GBM patients, discusses standard imaging modalities, and presents data supporting the use of SapC-DOPS, a novel proteoliposomal formulation with tumoricidal activity, as a promising diagnostic imaging tool and an innovative anti-cancer agent against GBM.

SapC-DOPS nanovesicles as targeted therapy for lung cancer.

Lung cancer is the deadliest type of cancer for both men and women. In this study, we evaluate the in vitro and in vivo efficacy of a biotherapeutic agent composed of a lysosomal protein (Saposin C, SapC) and a phospholipid (dioleoylphosphatidylserine, DOPS), which can be assembled into nanovesicles (SapC-DOPS) with selective antitumor activity. SapC-DOPS targets phosphatidylserine, an anionic phospholipid preferentially exposed in the surface of cancer cells and tumor-associated vasculature. Because binding of SapC to phosphatidylserine is favored at acidic pHs, and the latter characterizes the milieu of many solid tumors, we tested the effect of pH on the binding capacity of SapC-DOPS to lung tumor cells. Results showed that SapC-DOPS binding to cancer cells was more pronounced at low pH. Viability assays on a panel of human lung tumor cells showed that SapC-DOPS cytotoxicity was positively correlated with cell surface phosphatidylserine levels, whereas mitochondrial membrane potential measurements were consistent with apoptosis-related cell death. Using a fluorescence tracking method in live mice, we show that SapC-DOPS specifically targets human lung cancer xenografts, and that systemic therapy with SapC-DOPS induces tumor apoptosis and significantly inhibits tumor growth. These results suggest that SapC-DOPS nanovesicles are a promising treatment option for lung cancer.

Phosphatidylserine-selective targeting and anticancer effects of SapC-DOPS nanovesicles on brain tumors.

Brain tumors, either primary (e.g., glioblastoma multiforme) or secondary (metastatic), remain among the most intractable and fatal of all cancers. We have shown that nanovesicles consisting of Saposin C (SapC) and dioleylphosphatidylserine (DOPS) are able to effectively target and kill cancer cells both in vitro and in vivo. These actions are a consequence of the affinity of SapC-DOPS for phosphatidylserine, an acidic phospholipid abundantly present in the outer membrane of a variety of tumor cells and tumor-associated vasculature. In this study, we first characterize SapC-DOPS bioavailability and antitumor effects on human glioblastoma xenografts, and confirm SapC-DOPS specificity towards phosphatidylserine by showing that glioblastoma targeting is abrogated after in vivo exposure to lactadherin, which binds phosphatidylserine with high affinity. Second, we demonstrate that SapC-DOPS selectively targets brain metastases-forming cancer cells both in vitro, in co-cultures with human astrocytes, and in vivo, in mouse models of brain metastases derived from human breast or lung cancer cells. Third, we demonstrate that SapC-DOPS have cytotoxic activity against metastatic breast cancer cells in vitro, and prolong the survival of mice harboring brain metastases. Taken together, these results support the potential of SapC-DOPS for the diagnosis and therapy of primary and metastatic brain tumors.

Evolución de la resistencia antimicrobiana de bacilos Gram negativos en unidades de cuidados intensivos en Colombia / Evolution of antimicrobial resistance in Gram negative bacilli from intensive care units in Colombia

Introducción. La evolución de la resistencia bacteriana constituye una amenaza para la salud pública mundial. Los sistemas de vigilancia epidemiológica han integrado técnicas de biología molecular para mejorar las estrategias de control. Objetivo. Describir los perfiles moleculares y fenotípicos de los bacilos Gram negativos en unidades de cuidados intensivos de 23 hospitales de Colombia entre 2009 y 2012. Materiales y métodos. Se diseñó un estudio descriptivo en 23 hospitales del Grupo para el Estudio de la Resistencia Nosocomial (sic.) en Colombia. Se analizaron 38.048 aislamientos usando WHONET durante el periodo descrito. Se describieron perfiles de resistencia para Escherichia coli , Klebsiella pneumoniae , Pseudomonas aeruginosa y Acinetobacter baumannii. En 1.248 cepas se realizó reacción en cadena de la polimerasa (PCR) para detectar las carbapenemasas clínicamente más relevantes. Resultados. Escherichia coli fue el microorganismo más frecuente (promedio=14,8 %); la frecuencia de aislamientos de K. pneumoniae aumentó de 11 % en 2009 a 15 % en 2012 (p<0,001). La tendencia de los perfiles de multirresistencia aumentó en todas las especies estudiadas. De los aislamientos de K. pneumoniae evaluados, 68,4 % fue positivo para KPC ( Klebsiella pneumoniae Carbapenemase ), mientras que la VIM ( Verona Integron-encoded Metallo-betalactamase ) en P. aeruginosa se observó en 46,5 %. Conclusiones. Se observó un incremento en la tendencia de los microorganismos hacia la multirresistencia y una amplia distribución de las carbapenemasas. La articulación de la biología molecular con los sistemas de vigilancia permitió integrar el análisis del fenotipo con los mecanismos de resistencia involucrados en las bacterias estudiadas. Este análisis permitirá la elaboración de guías para el uso adecuado de antimicrobianos y contribuirá a la contención de estas bacterias multirresistentes en Colombia.

Introduction: The continuous evolution of antimicrobial resistance poses a major threat to public health worldwide. Molecular biology techniques have been integrated to epidemiological surveillance systems to improve the control strategies of this phenomenon. Objective: To describe the phenotypic and molecular profiles of the most important Gram negative bacilli from intensive care units in 23 Colombian hospitals during the study period 2009-2012. Materials and methods: A descriptive study was conducted in 23 hospitals belonging to the Colombian Nosocomial Resistance Study Group. A total of 38.048 bacterial isolates were analyzed using WHONET over a four-year period. The antimicrobial resistant profiles were described for Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii . Polymerase chain reaction was performed in 1.248 strains to detect the most clinically relevant carbapenemases. Results: Escherichia coli was the most frequently isolated organism (mean=14.8%). Frequency of K. pneumoniae increased significantly from 11% in 2009 to 15% in 2012 (p<0.001). All screened isolates had rising trends of multidrug-resistant profiles. KPC ( Klebsiella pneumoniae carbapenemase) was detected in 68.4% of K. pneumoniae isolates while VIM (Verona integron-encoded metallo-betalactamase) was present in 46.5% of them. Conclusion: In this study, an increase in the trend of multidrug-resistant organisms and a wide distribution of carbapenemases was observed. The integration of molecular biology to surveillance systems allowed the compilation of this data, which will aid in the construction of guidelines on antimicrobial stewardship for prevention in Colombia.

CD14 directs adventitial macrophage precursor recruitment: role in early abdominal aortic aneurysm formation.

BACKGROUND: Recruitment of macrophage precursors to the adventitia plays a key role in the pathogenesis of abdominal aortic aneurysms (AAAs), but molecular mechanisms remain undefined. The innate immune signaling molecule CD14 was reported to be upregulated in adventitial macrophages in a murine model of AAA and in monocytes cocultured with aortic adventitial fibroblasts (AoAf) in vitro, concurrent with increased interleukin-6 (IL-6) expression. We hypothesized that CD14 plays a crucial role in adventitial macrophage precursor recruitment early during AAA formation. METHODS AND RESULTS: CD14(-/-) mice were resistant to AAA formation induced by 2 different AAA induction models: aortic elastase infusion and systemic angiotensin II (AngII) infusion. CD14 gene deletion led to reduced aortic macrophage infiltration and diminished elastin degradation. Adventitial monocyte binding to AngII-infused aorta in vitro was dependent on CD14, and incubation of human acute monocytic leukemia cell line-1 (THP-1) monocytes with IL-6 or conditioned medium from perivascular adipose tissue (PVAT) upregulated CD14 expression. Conditioned medium from AoAf and PVAT induced CD14-dependent monocyte chemotaxis, which was potentiated by IL-6. CD14 expression in aorta and plasma CD14 levels were increased in AAA patients compared with controls. CONCLUSIONS: These findings link CD14 innate immune signaling via a novel IL-6 amplification loop to adventitial macrophage precursor recruitment in the pathogenesis of AAA.

Parallel changes in intracellular water volume and pH induced by NH(3)/NH(4)(+) exposure in single neuroblastoma cells.

BACKGROUND: Increased blood levels of ammonia (NH3) and ammonium (NH4(+)), i.e. hyperammonemia, leads to cellular brain edema in humans with acute liver failure. The pathophysiology of this edema is poorly understood. This is partly due to incomplete understanding of the osmotic effects of the pair NH3/NH4(+) at the cellular and molecular levels. Cell exposure to solutions containing NH3/NH4(+) elicits changes in intracellular pH (pHi), which can in turn affect cell water volume (CWV) by activating transport mechanisms that produce net gain or loss of solutes and water. The occurrence of CWV changes caused by NH3/NH4(+) has long been suspected, but the mechanisms, magnitude and kinetics of these changes remain unknown. METHODS: Using fluorescence imaging microscopy we measured, in real time, parallel changes in pHi and CWV caused by brief exposure to NH3/NH4(+) of single cells (N1E-115 neuroblastoma or NG-108 neuroblastoma X glioma ) loaded with the fluorescent indicator BCECF. Changes in CWV were measured by exciting BCECF at its intracellular isosbestic wavelength (∼438 nm), and pHi was measured ratiometrically. RESULTS: Brief exposure to isosmotic solutions (i.e. having the same osmolality as that of control solutions) containing NH4Cl (0.5- 30 mM) resulted in a rapid, dose-dependent swelling, followed by isosmotic regulatory volume decrease (iRVD). NH4Cl solutions in which either extracellular [NH3] or [NH4(+)] was kept constant while the other was changed by varying the pH of the solution, demonstrated that [NH3]o rather than [NH4(+)]o is the main determinant of the NH4Cl-induced swelling. The iRVD response was sensitive to the anion channel blocker NPPB, and partly dependent on external Ca(2+). Upon removal of NH4Cl, cells shrank and displayed isosmotic regulatory volume increase (iRVI). Regulatory volume responses could not be activated by comparable CWV changes produced by anisosmotic solutions, suggesting that membrane stretch or contraction by themselves are not sufficient to trigger these responses. Inhibition of glutamine synthetase partially blocked the NH4Cl-induced swelling. CONCLUSIONS: A quantitative description of the osmotic changes produced by exposure to NH3/NH4(+) in single neurons and glial cells shows that ∼35 to 45% of the initial cell swelling can be explained by intracellular accumulation of NH4(+) due to rapid permeation and protonation of NH3. Another∼23% of the swelling can be accounted for by rapid glutamine accumulation. The results are discussed in terms of basic cell physiology and their potential relevance to the pathophysiology of hyperammonemic cellular brain edema.

Role of epoxyeicosatrienoic acids as autocrine metabolites in glutamate-mediated K+ signaling in perivascular astrocytes.

Epoxyeicosatrienoic acids (EETs), synthesized and released by astrocytes in response to glutamate, are known to play a pivotal role in neurovascular coupling. In vascular smooth muscle cells (VSMC), EETs activate large-conductance, Ca(2+)-activated K(+) (BK) channels resulting in hyperpolarization and vasodilation. However, the functional role and mechanism of action for glial-derived EETs are still to be determined. In this study, we evaluated the effect of the synthetic EET analog 11-nonyloxy-undec-8(Z)-enoic acid (NUD-GA) on outward K(+) currents mediated by calcium-activated K(+) channels. Addition of NUD-GA significantly increased intracellular Ca(2+) and outward K(+) currents in perivascular astrocytes. NUD-GA-induced currents were significantly inhibited by BK channel blockers paxilline and tetraethylammonium (TEA) (23.4 ± 2.4%; P < 0.0005). Similarly, NUD-GA-induced currents were also significantly inhibited in the presence of the small-conductance Ca(2+)-activated K(+) channel inhibitor apamin along with a combination of blockers against glutamate receptors (12.8 ± 2.70%; P < 0.05). No changes in outward currents were observed in the presence of the channel blocker for intermediate-conductance K(+) channels TRAM-34. Blockade of the endogenous production of EETs with N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide (MS-PPOH) significantly blunted (dl)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (t-ACPD)-induced outward K(+) currents (P < 0.05; n = 6). Both NUD-GA and t-ACPD significantly increased BK channel single open probability; the later was blocked following MS-PPOH incubation. Our data supports the idea that EETs are potent K(+) channel modulators in cortical perivascular astrocytes and further suggest that these metabolites may participate in NVC by modulating the levels of K(+) released at the gliovascular space.

Simultaneous measurement of water volume and pH in single cells using BCECF and fluorescence imaging microscopy.

Regulation and maintenance of cell water volume and intracellular pH (pHi) are vital functions that are interdependent; cell volume regulation affects, and is in turn affected by, changes in pHi. Disruption of either function underlies various pathologies. To study the interaction and kinetics of these two mechanisms, we developed and validated a quantitative fluorescence imaging microscopy method to measure simultaneous changes in pHi and volume in single cells loaded with the fluorescent probe BCECF. CWV is measured at the excitation isosbestic wavelength, whereas pHi is determined ratiometrically. The method has a time resolution of <1 s and sensitivity to osmotic changes of approximately 1%. It can be applied in real time to virtually any cell type attached to a coverslip, independently of cellular shape and geometry. Calibration procedures and algorithms developed to transform fluorescence signals into changes in cell water volume (CWV) and examples of applications are presented.