Cytochalasin B Treatment and Osmotic Pressure Enhance the Production of Extracellular Vesicles (EVs) with Improved Drug Loading Capacity

Extracellular vesicles (EVs) have been highlighted as novel drug carriers due to their unique structural properties and intrinsic features, including high stability, biocompatibility, and cell-targeting properties. Although many efforts have been made to harness these features to develop a clinically effective EV-based therapeutic system, the clinical translation of EV-based nano-drugs is hindered by their low yield and loading capacity. Herein, we present an engineering strategy that enables upscaled EV production with increased loading capacity through the secretion of EVs from cells via cytochalasin-B (CB) treatment and reduction of EV intravesicular contents through hypo-osmotic stimulation. CB (10 µg/mL) promotes cells to extrude EVs, producing ~three-fold more particles than through natural EV secretion. When CB is induced in hypotonic conditions (223 mOsm/kg), the produced EVs (hypo-CIMVs) exhibit ~68% less intravesicular protein, giving 3.4-fold enhanced drug loading capacity compared to naturally secreted EVs. By loading doxorubicin (DOX) into hypo-CIMVs, we found that hypo-CIMVs efficiently deliver their drug cargos to their target and induce up to ~1.5-fold more cell death than the free DOX. Thus, our EV engineering offers the potential for leveraging EVs as an effective drug delivery vehicle for cancer treatment.

Validity of different copeptin assays in the differential diagnosis of the polyuria-polydipsia syndrome

The aim of this study was to correlate three commercially available copeptin assays and their diagnostic accuracy in the differential diagnosis of the polyuria-polydipsia syndrome. Analyzed data include repeated copeptin measures of 8 healthy volunteers and 40 patients with polyuria-polydipsia syndrome undergoing osmotic stimulation and of 40 patients hospitalized with pneumonia. Copeptin was measured using the automated Brahms KRYPTOR, the manual Brahms LIA and the manual Cloud Clone ELISA assay. Primary outcome was the interrater correlation coefficient (ICC) and diagnostic accuracy in the polyuria-polydipsia syndrome of the three assays. In healthy volunteers, there was a moderate correlation for the KRYPTOR and LIA (ICC 0.74; 95% CI 0.07 to 0.91), and a poor correlation for the KRYPTOR and ELISA (ICC 0.07; 95% CI − 0.06 to 0.29), as for the LIA and ELISA (ICC 0.04; 95% CI − 0.04 to 0.17). The KRYPTOR had the highest diagnostic accuracy (98% (95% CI 83 to100)), comparable to the LIA (88% (95% CI 74 to 100)), while the ELISA had a poor diagnostic accuracy (55% (95% CI 34 to 68)) in the differential diagnosis of the polyuria-polydipsia syndrome. The KRYPTOR and LIA yield comparable copeptin concentrations and high diagnostic accuracy, while the ELISA correlates poorly with the other two assays and shows a poor diagnostic accuracy for polyuria-polydipsia patients. The current copeptin cut-off is valid for the KRYPTOR and LIA assay. Our results indicate that interpretation with other assays should be performed with caution and separate validation studies are required before their use in differentiating patients with polyuria-polydipsia syndrome.Trial registration: NCT02647736 January 6, 2016/NCT01940614 September 12, 2013/NCT00973154 September 9, 2009.

Validity of Different Copeptin Assays in the Differential Diagnosis of the Polyuria-Polydipsia Syndrome

Background: Copeptin is used in the differential diagnosis of diabetes insipidus. Different copeptin immunoassays exist but inter-assay comparability is unclear. The aim of this study was to correlate three commercially available copeptin assays and their diagnostic accuracy in the differential diagnosis of the polyuria-polydipsia-syndrome. Methods: Analyzed data include three different studies: repeated copeptin measures of 8 healthy volunteers undergoing osmotic stimulation; copeptin measures of 40 patients hospitalized with pneumonia; osmotically stimulated copeptin measures of 40 patients with polyuria-polydipsia-syndrome. Copeptin was measured using the automated B.R.A.H.M.S. KRYPTOR, the manual B.R.A.H.M.S. LIA and the manual Cloud Clone ELISA assay. Primary outcome was the diagnostic accuracy in the polyuria-polydipsia-syndrome. Results: In total, 150 copeptin measurements were analyzed. In healthy volunteers, there was a moderate correlation for the KRYPTOR and LIA (interrater correlation coefficient (ICC) 0.74; 95%-CI 0.07-0.91), and a poor correlation for the KRYPTOR and ELISA (ICC 0.07; 95%-CI -0.06-0.29), as for the LIA and ELISA (ICC 0.04; 95%-CI -0.04-0.17). The KRYPTOR had the highest diagnostic accuracy (98% (95%-CI: 83-100)), comparable to the LIA (88% (95%-CI: 74-100)), while the ELISA had a poor diagnostic accuracy (55% (95%-CI: 34-68)) in the differential diagnosis of the polyuria-polydipsia-syndrome. Conclusion: The KRYPTOR and LIA yield comparable copeptin levels and a high diagnostic accuracy, while the ELISA correlates poorly with the other two assays and shows a poor diagnostic accuracy between polyuria-polydipsia patients. Redefining cut-off levels for copeptin assays other than KRYPTOR and LIA must take place before their use in the differential diagnosis of diabetes insipidus.

Osmotic stimulation of vasopressin acutely impairs glucose regulation: a counterbalanced, crossover trial

ABSTRACT Background Epidemiological studies in humans show increased concentrations of copeptin, a surrogate marker of arginine vasopressin (AVP), to be associated with increased risk for type 2 diabetes. Objectives To examine the acute and independent effect of osmotically stimulated AVP, measured via the surrogate marker copeptin, on glucose regulation in healthy adults. Methods Sixty subjects (30 females) participated in this crossover design study. On 2 trial days, separated by ≥7 d (males) or 1 menstrual cycle (females), subjects were infused for 120 min with either 0.9% NaCl [isotonic (ISO)] or 3.0% NaCl [hypertonic (HYPER)]. Postinfusion, a 240-min oral-glucose-tolerance test (OGTT; 75 g) was administered. Results During HYPER, plasma osmolality and copeptin increased (P < 0.05) and remained elevated during the entire 6-h protocol, whereas renin-angiotensin-aldosterone system hormones were within the lower normal physiological range at the beginning of the protocol and declined following infusion. Fasting plasma glucose did not differ between trials (P > 0.05) at baseline and during the 120 min of infusion. During the OGTT the incremental AUC for glucose from postinfusion baseline (positive integer) was greater during HYPER (401.5 ± 190.5 mmol/L·min) compared with the ISO trial (354.0 ± 205.8 mmol/L·min; P < 0.05). The positive integer of the AUC for insulin during OGTT did not differ between trials (HYPER 55,850 ± 36,488 pmol/L·min compared with ISO 57,205 ± 31,119 pmol/L·min). Baseline values of serum glucagon were not different between the 2 trials; however, the AUC of glucagon during the OGTT was also significantly greater in HYPER (19,303 ± 3939 ng/L·min) compared with the ISO trial (18,600 ± 3755 ng/L·min; P < 0.05). Conclusions The present data indicate that acute osmotic stimulation of copeptin induced greater hyperglycemic responses during the oral glucose challenge, possibly due to greater glucagon concentrations. This study was registered at clinicaltrials.gov as NCT02761434.

The challenges of sodium measurements: indirect versus direct ion-selective method

Background Diagnosis and treatment of dysnatremia is challenging and further complicated by the pitfalls of different sodium measurement methods. Routinely used sodium measurements are the indirect (plasma/serum) and direct (whole blood) ion-selective electrode (ISE) method, showing discrepant results especially in the setting of acute illness. Few clinicians are aware of the differences between the methods in clinically stable patients or healthy volunteers. Methods Data of 140 patients and 91 healthy volunteers undergoing osmotic stimulation with hypertonic saline infusion were analyzed. Sodium levels were measured simultaneously by indirect and direct ISE method before and at different time points during osmotic stimulation up to a sodium threshold of ≥150 mmol/L. The primary outcome was the difference in sodium levels between the indirect and direct ISE method. Results 878 sodium measurements were analyzed. Mean (s.d.) sodium levels ranged from 141 mmol/L (2.9) to 151 mmol/L (2.1) by the indirect ISE compared to 140 mmol/L (3) to 149 mmol/L (2.8) by the direct ISE method. The interclass correlation coefficient between the two methods was 0.844 (95% CI: 0.823–0.863). On average, measurements by the indirect ISE were 1.9 mmol/L (95% CI limits: −3.2 to 6.9) higher than those by the direct ISE method (P < 0.001). The tendency of the indirect ISE method resulting in higher levels increased with increasing sodium levels. Conclusion Intra-individual sodium levels differ significantly between the indirect and direct ISE method also in the absence of acute illness. It is therefore crucial to adhere to the same method in critical situations to avoid false decisions due to measurement differences.

Osmotic Stimulation of Thirst in Men and Women (P15-017-19)

Objectives Examine potential sex differences in osmotically stimulated thirst. Methods 60 healthy adults (30 female - measured in early follicular phase, age: 39 ± 8 y, weight: 78.2 ± 15.2 kg, body mass index: 26.9 ± 4.0 kg·m−2) were infused intravenously with 3% (HYPER) or 0.9% (ISO) NaCl for 120 min (0.1 ml·kg−1·min−1) in a counterbalanced, cross-over design. The same volume of saline was infused in both trials. Blood samples and thirst responses were collected prior to infusion and every 30 minutes. Plasma osmolality (POsm) and sodium (PNa) were measured. Thirst was assessed via a 180-mm visual analog scale consisting of a line with an anchor on the left side (0 mm, “not at all”) and a second anchor on the 125-mm mark with the label “extremely”. Results From 0 min to 120 min in the HYPER trial, PNa increased from 136 ± 2 mmol ·L−1 to 146.5 ± 3 mmol ·l−1 (P < 0.001). Similarly, POsm and thirst increased significantly from 286 ± 3 mmol·kg−1 to 305 ± 4 mmol·kg−1 (P < 0.001) and from 38 ± 29 mm to 88 ± 35 mm (P < 0.001), respectively. During the ISO trial, PNa (0 min: 135.9 ± 1.9; 120 min: 137.6 ± 1.6 mmol·L−1), POsm (0 min: 285 ± 4 mmol·kg−1; 120 min: 287 ± 3 mmol·kg−1), and thirst (0 min: 30 ± 27 mm; 120 min: 44 ± 32 mm) remained unchanged (P > 0.05). POsm at the end of the infusion did not differ between men and women for the HYPER (men: 306 ± 5 mmol·kg−1; women: 305 ± 3 mmol·kg−1) or ISO (men: 288 ± 3 mmol·kg−1; women: 288 ± 3 mmol·kg−1) trials. When thirst responses were split by sex women exhibited greater thirst (98 ± 27 mm) than men (77 ± 38 mm) at the end of the HYPER trial (P < 0.0001), but not at the end of the ISO trial (women: 43 ± 32 mm, men 44 ± 31 mm; P > 0.05). Conclusions These data suggest that similar hypertonic stimulus may lead to greater thirst stimulation in women. Funding Sources This study was funded by Danone Research.

FGF-21 levels in polyuria-polydipsia syndrome

The pathomechanism of primary polydipsia is poorly understood. Recent animal data reported a connection between fibroblast growth factor 21 (FGF-21) and elevated fluid intake independently of hormonal control by the hormone arginine-vasopressin (AVP) and osmotic stimulation. We therefore compared circulating FGF-21 levels in patients with primary polydipsia to patients with AVP deficiency (central diabetes insipidus) and healthy volunteers. In this prospective cohort study, we analyzed FGF-21 levels of 20 patients with primary polydipsia, 20 patients with central diabetes insipidus and 20 healthy volunteers before and after stimulation with hypertonic saline infusion targeting a plasma sodium level ≥150 mmol/L. The primary outcome was the difference in FGF-21 levels between the three groups. Baseline characteristics were similar between the groups except for patients with central diabetes insipidus being heavier. There was no difference in baseline FGF-21 levels between patients with primary polydipsia and healthy volunteers (122 pg/mL (52,277) vs 193 pg/mL (48,301), but higher levels in patients with central diabetes insipidus were observed (306 pg/mL (114,484); P = 0.037). However, this was not confirmed in a multivariate linear regression analysis after adjusting for age, sex, BMI and smoking status. Osmotic stimulation did not affect FGF-21 levels in either group (difference to baseline: primary polydipsia −23 pg/mL (−43, 22); central diabetes insipidus 17 pg/mL (−76, 88); healthy volunteers −6 pg/mL (−68, 22); P = 0.45). To conclude, FGF-21 levels are not increased in patients with primary polydipsia as compared to central diabetes insipidus or healthy volunteers. FGF-21 therefore does not seem to be causal of elevated fluid intake in these patients.