High-Dose Rate Brachytherapy Combined with PD-1 Blockade as a Treatment for Metastatic Adrenocortical Carcinoma - A Single Center Case Series.

The response rate of advanced adrenocortical carcinoma (ACC) to standard chemotherapy with mitotane and etoposide/doxorubicin/cisplatin (EDP-M) is unsatisfactory, and benefit is frequently short lived. Immune checkpoint inhibitors (CPI) have been examined in patient's refractory to EDP-M, but objective response rates are only approximately 15%. High-dose rate brachytherapy (HDR-BT) is a catheter-based internal radiotherapy and expected to favorably combine with immunotherapies. Here we describe three cases of patients with advanced ACC who were treated with HDR-BT and the CPI pembrolizumab. None of the tumors were positive for established response markers to CPI. All patients were female, had progressed on EDP-M and received external beam radiation therapy for metastatic ACC. Pembrolizumab was initiated 7 or 23 months after brachytherapy in two cases and prior to brachytherapy in one case. Best response of lesions treated with brachytherapy was complete (n=2) or partial response (n=1) that was ongoing at last follow up after 23, 45 and 4 months, respectively. Considering all sites of tumor, response was complete and partial remission in the two patients with brachytherapy prior to pembrolizumab. The third patient developed progressive disease with severe Cushing's syndrome and died due to COVID-19. Immune-related adverse events of colitis (grade 3), gastroduodenitis (grade 3), pneumonitis (grade 2) and thyroiditis (grade 1) occurred in the two patients with systemic response. HDR-BT controlled metastases locally. Sequential combination with CPI therapy may enhance an abscopal antitumoral effect in non-irradiated metastases in ACC. Systematic studies are required to confirm this preliminary experience and to understand underlying mechanisms.

Identification and evaluation of cell- growth-inhibiting bDtBPP-analogue degradation products from phosphite antioxidants used in polyolefin bioprocessing materials.

The inhibiting effect of the secondary phosphite antioxidant degradation product bis(2,4-di-tert-butylphenyl)phosphate (bDtBPP) on cell growth is well-known. The present study describes structurally related compounds which are likely to be formed from similar widely used phosphite antioxidants used in materials for the manufacturing of single-use (SU) equipment. Two potential candidates of such compounds-3,3',5,5'-tetra-tert-butyl-2,2'-dihydroxybiphenylphosphate (TtBBP) and bis(p-nonylphenyl)phosphate (bNPP)-were identified by chromatography and mass spectrometry followed by synthesis and X-ray structure elucidation. Additionally, the formation of TtBBP was confirmed in an analytical degradation study and its migration from SU bioprocessing material was estimated. The cytotoxicity evaluation by means of cell culture spiking experiments and flow cytometry analysis revealed that' even if cell growth was inhibited by all the compounds to some extent, bDtBPP showed the most severe effect and stoods out from the other two degradants investigated. Graphical abstract.

Challenges of metabolomics in human gut microbiota research.

The review highlights the role of metabolomics in studying human gut microbial metabolism. Microbial communities in our gut exert a multitude of functions with huge impact on human health and disease. Within the meta-omics discipline, gut microbiome is studied by (meta)genomics, (meta)transcriptomics, (meta)proteomics and metabolomics. The goal of metabolomics research applied to fecal samples is to perform their metabolic profiling, to quantify compounds and classes of interest, to characterize small molecules produced by gut microbes. Nuclear magnetic resonance spectroscopy and mass spectrometry are main technologies that are applied in fecal metabolomics. Metabolomics studies have been increasingly used in gut microbiota related research regarding health and disease with main focus on understanding inflammatory bowel diseases. The elucidated metabolites in this field are summarized in this review. We also addressed the main challenges of metabolomics in current and future gut microbiota research. The first challenge reflects the need of adequate analytical tools and pipelines, including sample handling, selection of appropriate equipment, and statistical evaluation to enable meaningful biological interpretation. The second challenge is related to the choice of the right animal model for studies on gut microbiota. We exemplified this using NMR spectroscopy for the investigation of cross-species comparison of fecal metabolite profiles. Finally, we present the problem of variability of human gut microbiota and metabolome that has important consequences on the concepts of personalized nutrition and medicine.

Aquagenic wrinkling of the palms: morphological changes in reflectance confocal microscopy and high-definition optical coherence tomography.

Aquagenic wrinkling of the palms (AWP) is a rare condition, which is characterized by appearance of whitish papules and plaques, and an excessive wrinkling and swelling of the palmar skin after exposure to water. In most cases, young women are affected, and an association of AWP with cystic fibrosis (CF) has been surmised. We report on two cases of AWP, which were not related to CF, in whom we used two innovative imaging techniques, namely high-definition optical coherence tomography and reflectance confocal microscopy, to show in vivo skin changes occurring after exposure of the skin to tap water in comparison to the findings in a healthy control person.

Noninvasive monitoring of basal cell carcinomas treated with systemic hedgehog inhibitors: pseudocysts as a sign of tumor regression.

BACKGROUND: Oral hedgehog inhibitors (HHIs) have shown significant efficacy in the treatment of basal cell carcinoma (BCC). The evaluation of tumor regression has been performed using clinical photography and radiographic scans. Noninvasive imaging techniques, such as reflectance confocal microscopy (RCM) and high-definition optical coherence tomography (HD-OCT), have been shown to be valuable in detecting BCC in the skin. OBJECTIVE: We monitored HHI-treated BCC using RCM and HD-OCT in vivo and correlated morphologic changes seen on imaging to changes in traditional histopathology. METHODS: Six BCCs in 5 patients receiving HHIs (vismodegib or sonidegib) were examined by RCM and HD-OCT before and during treatment. Characteristic features were compared to histopathologic findings, including immunohistochemical analysis. RESULTS: Characteristic features of BCC in RCM and HD-OCT decreased or disappeared completely during HHI treatment. Half of the clinically complete responding tumors still featured tumor residue. Pseudocystic structures ("empty" tumor nests in imaging) and widespread fibrosis (coarse bright fibers) were new findings and could be confirmed by histopathology. LIMITATIONS: Our study was limited by the number of tumor samples and imaging timepoints. CONCLUSION: Using RCM and HD-OCT, HHI-induced regression of BCC can be visualized noninvasively in the skin. The formation of pseudocysts and fibrosis were characteristic signs of BCC response to HHIs.

Parylene C Coating Efficacy Studies: Enhancing Biocompatibility of 3D Printed Polyurethane Parts for Biopharmaceutical and CGT Applications.

Additive manufacturing, particularly Vat photopolymerization, presents a promising technique for producing complex, tailor-made structures, making it an attractive option for generating single-use components used in biopharmaceutical manufacturing equipment or cell culture devices. However, the potential leaching of cytotoxic compounds from Vat photopolymer resins poses a significant concern, especially regarding cell growth and viability in cell culture applications. This study explores the potential of parylene C coating to enhance the inertness of a polyurethane-based photopolymer resin, aiming to prevent cytotoxicity and improve biocompatibility. The study includes an analysis of extractables from the resin and photoinitiator to evaluate the resin's composition and to define selected marker compounds for investigating the coating efficiency. The time-dependent accumulation of relevant extractable compounds over a 70-day period are assessed to address the long-term use of the coated components. The impact of irradiation on the material and the coating was evaluated, along with an accelerated aging study to address the long-term performance of the coating. Biocompatibility in terms of in vitro cell growth studies is evaluated using Chinese hamster ovary cells, a standard cell line in biopharmaceutical manufacturing. Results demonstrate that parylene C coating significantly reduces the release of cytotoxic compounds, such as the photoinitiator diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide (TPO). Although accelerated aging indicates a reduction in the barrier properties of the coating over time, the parylene C coating still effectively slows the release of extractables and significantly improves cell compatibility of the 3D printed parts. The findings suggest that parylene C-coated components can be safely integrated into biopharmaceutical manufacturing processes, with recommendations to minimize storage times between coating application and use to ensure optimal performance.

Equivalence study of extractables from single-use biopharmaceutical manufacturing equipment after X-ray or gamma irradiation.

Single-use (SU) devices and assemblies used as manufacturing equipment in the biopharmaceutical industry require comprehensive qualifications. These qualifications include the assessment of compounds released from SU devices in contact with the process fluids, and how these leachable compounds potentially influence process performance, drug product quality, and patient safety. SU suppliers need to provide comprehensive qualification data for several parameters, for both new products and product changes, such as changes in the sterilization process applied to the SU device. The introduction of X-ray irradiation as an alternative to the currently used and established gamma irradiation of SU devices represents a situation where robust data is required to demonstrate equivalency between these two radiation technologies. Here, we present the results of a comprehensive extractables study for three SU components, bags, tubing, and sterilizing grade filters, evaluated after X-ray and gamma-ray irradiation. The selected study conditions were set up to allow a direct comparison of the results from the two sterilization methods, and to allow conclusions to be made on the impact of irradiation type on the polymers and their additives. Orthogonal analytical methods are applied to identify and quantify all organic compounds present. The data package provided here supports risk assessments for application of irradiated SU equipment in biopharmaceutical manufacturing. The formation of reaction products and the fundamental chemical pathways are discussed and found to be independent of the irradiation type. The results demonstrate the equivalency of both irradiation methods for extractables from plastic components used in pharmaceutical and biopharmaceutical manufacturing.

Dimethylsilanediol from silicone elastomers: Analysis, release from biopharmaceutical process equipment, and clearance studies.

Polysiloxanes are considered one of the most important commercial families of synthetic elastomers. They are frequently employed in biopharmaceutical manufacturing equipment as flexible single-use solutions due to superior material properties and compatibility with diverse sterilization methods. Extractables and leachables (E&L) testing is essential in qualifying such equipment, involving extraction studies to assess the potential release of compounds from plastic components for risk assessment. Silicone releases oligomeric siloxanes and small hydrolysis products, with dimethylsilanediol (DMSD) being the main hydrolysis product found in significant concentrations in aqueous process solutions. DMSD presents challenges for analysis, requiring specifically tailored analytical methods to detect it, which are commonly not applied in standard E&L screening tests. In biopharmaceutical manufacturing, it is relevant to consider the potential of DMSD to repolymerize into silicone oil when specific process parameters are altered. This may lead to interactions with drug ingredients, including proteins, resulting in the formation of aggregates. We synthesized and characterized DMSD using X-ray structure analysis and established an HPLC method with a refractive index detector to investigate the release of DMSD from commercially available silicone tubing used in drug manufacturing following autoclaving and irradiation. Subsequently, we assessed typical biopharmaceutical downstream operations for effectively removing this compound from the process stream.

X-ray sterilization of biopharmaceutical manufacturing equipment-Extractables profile of a film material and copolyester Tritan™ compared to gamma irradiation.

The biopharmaceutical industry gains enormous flexibility in production processes by using sterilized preassembled single-use devices. Gamma irradiation is an established sterilization technology that may be restricted in the future by the availability of 60 Co as irradiation source and irradiation capacities. X-ray technology is considered an alternative type of radiation for sterilizing SU equipment. In the context of extractables and leachables-one concern connected with the use of single-use process equipment-the effect of X-ray irradiation on the extractables profile of the materials needs to be compared to established gamma irradiation to qualify this alternative technology. An approach is presented to obtain robust and comprehensive extractables data for materials used in SU devices after sterilization either using X-ray or gamma irradiation. A careful selection of the test items and the test design allows a one-to-one comparison of data obtained from a combination of orthogonal analytical techniques. The extractables of a modern SU film material and the copolyester Tritan™ are evaluated. The data presented allow a risk evaluation on the safety of this new sterilization modality for biopharmaceutical applications. It is demonstrated that the extractables profile of a polymer is not affected by the type of irradiation used for sterilization.

Mental but no bio-physiological long-term habituation to repeated social stress: A study on soldiers and the influence of mission abroad.

Soldiers regularly participate in missions abroad and subjectively adapt to this situation. However, they have an increased lifetime cardiovascular risk compared to other occupational groups. To test the hypothesis that foreign deployment results in different stress habituation patterns, we investigated long-term psychological and bio-physiological stress responses to a repeated social stress task in healthy soldiers with and without foreign deployment. Ninety-one female and male soldiers from the BEST study (German armed forces deployment and stress) participated three times in the Trier Social Stress Test for groups (TSST-G) prior to, 6-8 weeks after and 1 year after the mission abroad and were compared to a control group without foreign deployment during the study period. They completed the State-Trait-Anxiety Inventory scale (STAI), the Primary Appraisal Secondary Appraisal questionnaire (PASA) and the Multidimensional Mood State Questionnaire (MDBF). Salivary cortisol and α-amylase, blood pressure, heart rate and heart rate variability were determined. Soldiers showed mental habituation over the three times with a significant decrease after the TSST-G in anxiousness (STAI) and cognitive stress appraisal (PASA), they were calmer and reported better mood (MDBF). Prior to the social stress part, the mood (MDBF) declined significantly. None of the biological and physiological markers showed any adaptation to the TSST-G. Mission abroad did not significantly influence any measured psychobiological marker when compared to soldiers without foreign deployment. Foreign deployment does not result in alterations in psychobiological social stress response patterns over 1 year after mission abroad which indicates that adaptation to acute social stress is highly maintained in healthy soldiers. The discrepancy between subjective perception and objective stress response has numerous clinical implications and should receive more attention.

Longitudinal Profiles of Dietary and Microbial Metabolites in Formula- and Breastfed Infants.

The early-life metabolome of the intestinal tract is dynamically influenced by colonization of gut microbiota which in turn is affected by nutrition, i.e. breast milk or formula. A detailed examination of fecal metabolites was performed to investigate the effect of probiotics in formula compared to control formula and breast milk within the first months of life in healthy neonates. A broad metabolomics approach was conceptualized to describe fecal polar and semi-polar metabolites affected by feeding type within the first year of life. Fecal metabolomes were clearly distinct between formula- and breastfed infants, mainly originating from diet and microbial metabolism. Unsaturated fatty acids and human milk oligosaccharides were increased in breastfed, whereas Maillard products were found in feces of formula-fed children. Altered microbial metabolism was represented by bile acids and aromatic amino acid metabolites. Elevated levels of sulfated bile acids were detected in stool samples of breastfed infants, whereas secondary bile acids were increased in formula-fed infants. Microbial co-metabolism was supported by significant correlation between chenodeoxycholic or lithocholic acid and members of Clostridia. Fecal metabolites showed strong inter- and intra-individual behavior with features uniquely present in certain infants and at specific time points. Nevertheless, metabolite profiles converged at the end of the first year, coinciding with solid food introduction.

Formulation Development of Sublingual Cyclobenzaprine Tablets Empowered by Standardized and Physiologically Relevant Ex Vivo Permeation Studies.

Suitable ex vivo models are required as predictive tools of oromucosal permeability between in vitro characterizations and in vivo studies in order to support the development of novel intraoral formulations. To counter a lack of clinical relevance and observed method heterogenicity, a standardized, controlled and physiologically relevant ex vivo permeation model was established. This model combined the Kerski diffusion cell, process automation, novel assays for tissue integrity and viability, and sensitive LC-MS/MS analysis. The study aimed to assess the effectiveness of the permeation model in the sublingual formulation development of cyclobenzaprine, a promising agent for the treatment of psychological disorders. A 4.68-fold enhancement was achieved through permeation model-led focused formulation development. Here, findings from the preformulation with regard to pH and microenvironment-modulating excipients proved supportive. Moreover, monitoring of drug metabolism during transmucosal permeation was incorporated into the model. In addition, it was feasible to assess the impact of dosage form alterations under stress conditions, with the detection of a 33.85% lower permeation due to salt disproportionation. Integrating the coherent processes of disintegration, dissolution, permeation, and metabolization within a physiological study design, the model enabled successful formulation development for cyclobenzaprine sublingual tablets and targeted development of patient-oriented drugs for the oral cavity.

Structural Equation Modeling of a Global Stress Index in Healthy Soldiers.

Accumulation of stress is a prognostic trigger for cardiovascular disease. Classical scores for cardiovascular risk estimation typically do not consider psychosocial stress. The aim of this study was to develop a global stress index (GSI) from healthy participants by combining individual measures of acute and chronic stress from childhood to adult life. One-hundred and ninety-two female and male soldiers completed the Perceived Stress Scale (PSS4), Trier Inventory for Chronic Stress (TICS), Hospital Anxiety and Depression Scale (HADS), Childhood Trauma Questionnaire (CTQ), Posttraumatic Diagnostic Scale Checklist (PDS), and the Deployment Risk and Resilience Inventory (DRRI-2). The underlying structure for the GSI was examined through structural equation modeling. The final hierarchical multilevel model revealed fair fit by taking modification indices into account. The highest order had a g-factor called the GSI. On a second level the latent variables stress, HADS and CTQ were directly loading on the GSI. A third level with the six CTQ subscales was implemented. On the lowest hierarchical level all manifest variables and the DRRI-2/PDS sum scores were located. The presented GSI serves as a valuable and individual stress profile for soldiers and could potentially complement classical cardiovascular risk factors.

Exploring the transmucosal permeability of cyclobenzaprine: A comparative preformulation by standardized and controlled ex vivo and in vitro permeation studies.

As part of early drug development, preformulation studies are used to comprehensively explore the properties of new drugs. In particular, this includes the biopharmaceutical characterization and evaluation of impacting factors (e.g. excipients, microenvironmental conditions etc.) by permeation studies. To overcome the limitations of current studies, a novel standardized ex vivo procedure using esophageal mucosa as surrogate has been established successfully and applied to preformulation studies for oromucosal delivery of cyclobenzaprine hydrochloride, a tricyclic muscle relaxant with potential for psychopharmacotherapeutic use. By using the standardized ex vivo permeation process, a twofold enhancement of permeability (0.98 ± 0.16 to 1.96 ± 0.10 * 10-5 cm/s) was observed by adjustment and controlling of microenvironmental pH, empowering a targeted and effective development of sublingual formulations. Predictivity and suitability were superior compared to in vitro experiments using artificial biomimetic membranes, revealing a determination coefficient (R2) of 0.995 vs. 0.322 concerning pH-dependent permeability of cyclobenzaprine. In addition, diffusion properties were extensively examined (e.g. influence of mucosal thicknesses, tissue freezing etc.). The alignment of the study design regarding physiologically/clinically relevant conditions resulted in ex vivo data that allowed for the estimation of plasma AUC levels in the extend of reported in vivo ranges.

Comparative analysis of rabies virus reverse transcription-PCR and virus isolation using samples from a patient infected with rabies virus.

Definite and rapid diagnosis of rabies is required for individual case management as well as for public health. For the first time, a direct comparison of virus isolation with quantitative real-time reverse transcription (RT)-PCR on human rabies samples was conducted. RT-PCR was found to be more sensitive than virus isolation.

Nestin and SOX9 and SOX10 transcription factors are coexpressed in melanoma.

Nestin is an intermediate filament expressed in proliferating neural progenitor cells and has been considered as a stem cell marker. Nestin is also found in melanoma and we recently demonstrated that its expression in melanoma cell lines is regulated by the transcription factors SOX9 and SOX10, but not BRN2. In this study, the expression levels of nestin, BRN2, SOX9 and SOX10 were analysed in tissues of melanoma (n = 78) and melanocytic nevi (n = 26) by immunohistochemistry. All proteins were highly expressed in primary and metastatic melanomas and, apart from BRN2, showed much lower levels in melanocytic nevi. Significant coexpression of nestin with SOX9 and SOX10 was found in primary melanoma confirming our in vitro data. Correlation analysis with clinicopathological data revealed that nestin was significantly associated with presence of ulceration in primary tumors and SOX9 with more advanced stage of disease. Our data reveal that SOX9 and SOX10 are highly expressed in melanoma and seem to have a regulatory role in nestin expression. The association with ulceration and advanced-stage tumors, respectively, suggests that nestin and SOX9 may be negative prognostic markers in melanoma.

Quantitative characterization of leachables sinks in biopharmaceutical downstream processing.

This article demonstrates, on a quantitative level, that leachables - potentially accumulated during a biopharmaceutical manufacturing process - will be significantly reduced/removed during four key downstream process steps: cell removal using centrifugation or depth filtration, sterile filtration and virus filtration. Eight common leachables model compounds (LMCs) were spiked into typical feed solutions containing buffer and proteins and were analyzed post-processing in the supernatant or filtrates by HPLC-UV. The clearance rates were calculated as the quotient between the scavenged and initially spiked amount of each leachable. High clearance rates were found for hydrophobic LMCs for all investigated downstream operation steps. It is shown that the removal of cells and cell debris from a culture broth reduces the amount of LMCs almost completely after centrifugation or depth filtration. Also, sterilizing-grade and virus filtration provided a high scavenger effect to most of the LMCs. In contrast, only one hydrophilic acid was not significantly scavenged by the described operations. The possibility to include leachables sinks to a process qualification and risk mitigation concept is explained.

Social Stress-Induced Oxidative DNA Damage Is Related to Prospective Cardiovascular Risk.

Psychosocial stress increases cardiovascular risk, which coincides with enhanced oxidative DNA damage. Increased sympathetic tone-related catecholamine release causes oxidative stress, which contributes to catecholamine-related cardiotoxicity. Therefore, we tested the hypothesis whether acute psychosocial stress induces oxidative DNA damage, its degree being related to the cardiovascular risk profile and depending on the sympathetic stress response. After assessment of the prospective cardiovascular Münster score (PROCAM) to determine the risk of acute myocardial infarction, 83 male and 12 female healthy volunteers underwent the Trier social stress test for groups (TSST-G). Heart rate variability was quantified by measuring the standard deviation (SDNN) and root mean square of successive differences (RMSSD) between normal-to-normal inter-beat intervals. Salivary α-amylase (sAA) activity was assessed as a surrogate for noradrenaline plasma concentrations. Oxidative DNA damage was determined using whole-blood single-cell gel electrophoresis ("tail moment" in the "comet assay"). A total of 33 subjects presented with a prospective risk of myocardial infarction (risk+) vs. 59 subjects without risk (risk-). The TSST-G stress significantly increased blood pressure, heart rate, and sAA in both groups, while oxidative DNA damage was only increased in the risk+ group. Immediately after the TSST-G, the "tail moment" showed significant inverse linear relations with both SDNN and RMSSD. Acute psychosocial stress may cause oxidative DNA damage, the degree of which is directly related to the individual cardiovascular risk profile and depends on the stress-induced increase in the sympathetic tone.

Osteopontin and 'melanoma inhibitory activity': comparison of two serological tumor markers in metastatic uveal melanoma patients.

BACKGROUND: Evaluation of the protein osteopontin (OPN) as a potential new marker in comparison to melanoma inhibitory activity (MIA) for screening and detection of metastatic uveal melanoma. METHODS: Plasma levels of 32 patients with uveal melanoma were analyzed for OPN and MIA by enzyme-linked immunosorbent assay (ELISA). Fourteen of these patients had clinically detectable liver metastases. RESULTS: Median plasma concentration of OPN in patients with metastatic disease was 152.01 ng/ml compared to 47.39 ng/ml in patients without clinically detectable metastases (p < 0.001). The difference between the median MIA plasma levels in patients with (13.11 ng/ml) and patients without (5.64 ng/ml) metastatic disease was also statistically significant (p < 0.001). No correlation could be found between MIA or OPN levels and tumor height in patients without clinically detectable metastases. CONCLUSION: The proteins MIA and OPN seem to be promising tumor markers for the metastasis screening in patients with uveal melanoma.

Impaired endothelium-derived hyperpolarizing factor-mediated dilations and increased blood pressure in mice deficient of the intermediate-conductance Ca2+-activated K+ channel.

The endothelium plays a key role in the control of vascular tone and alteration in endothelial cell function contributes to several cardiovascular disease states. Endothelium-dependent dilation is mediated by NO, prostacyclin, and an endothelium-derived hyperpolarizing factor (EDHF). EDHF signaling is thought to be initiated by activation of endothelial Ca(2+)-activated K(+) channels (K(Ca)), leading to hyperpolarization of the endothelium and subsequently to hyperpolarization and relaxation of vascular smooth muscle. In the present study, we tested the functional role of the endothelial intermediate-conductance K(Ca) (IK(Ca)/K(Ca)3.1) in endothelial hyperpolarization, in EDHF-mediated dilation, and in the control of arterial pressure by targeted deletion of K(Ca)3.1. K(Ca)3.1-deficient mice (K(Ca)3.1(-/-)) were generated by conventional gene-targeting strategies. Endothelial K(Ca) currents and EDHF-mediated dilations were characterized by patch-clamp analysis, myography and intravital microscopy. Disruption of the K(Ca)3.1 gene abolished endothelial K(Ca)3.1 currents and significantly diminished overall current through K(Ca) channels. As a consequence, endothelial and smooth muscle hyperpolarization in response to acetylcholine was reduced in K(Ca)3.1(-/-) mice. Acetylcholine-induced dilations were impaired in the carotid artery and in resistance vessels because of a substantial reduction of EDHF-mediated dilation in K(Ca)3.1(-/-) mice. Moreover, the loss of K(Ca)3.1 led to a significant increase in arterial blood pressure and to mild left ventricular hypertrophy. These results indicate that the endothelial K(Ca)3.1 is a fundamental determinant of endothelial hyperpolarization and EDHF signaling and, thereby, a crucial determinant in the control of vascular tone and overall circulatory regulation.