Abstract Background and purpose The optimal choice of antidiabetic medication in patients that need combined therapy is under debate. The aim of this study was to analyze whether beyond glucose control the combination of empagliflozin (E) and linagliptin (L) improves blood pressure (BP) and vascular function in patients with type 2 diabetes (T2DM) as opposed to the combination of metformin (M) and insulin glargine (I). Methods This was a prospective, randomized, controlled, single center study including 101 patients with T2DM, who were randomized 1:1 to E 10–25mg combined with L 5mg once daily or M 850 or 1000mg twice daily combined with I once daily. All patients underwent BP measurement and vascular function analysis by validated systems at baseline and after 12 weeks of treatment. Results In comparison to baseline, office, 24-hour ambulatory BP as well as central blood and pulse pressure (PP) values decreased significantly after 12 weeks of treatment with E+L, whereas there was no change in the M+I group (see table). Twenty-four-hour peripheral systolic (mean difference: −5.2±1.5mmHg, p=0.004) and diastolic BP (−1.9±1.0mmHg, p=0.036), central clinical systolic BP (−5.56±1.9mmHg, p=0.009), forward pressure pulse height (−2.0±0.9mmHg, p=0.028), 24-h central systolic BP (−3.6±1.4mmHg, p=0.045) and 24-h pulse wave velocity (−0.14±0.05m/s, p=0.043) were reduced to a greater extent in the E+L than in the M+I group. Empagliflozin+Linagliptin Metformin+Insulin Baseline 12 weeks p value Baseline 12 weeks p value Peripheral ambulatory BP values 24-h SBP [mmHg] 131.0±10.9 127.0±8.8 <0.001 131.0±9.7 131.0±8.6 0.438 24-h DBP [mmHg] 81.5±7.1 79.7±7.0 0.013 81.0±7.1 81.0±7.5 0.976 Clinical (laboratory) central vascular parameters Central SBP [mmHg] 123.0±9.6 117.0±10.4 <0.001 121.0±9.9 121.0±8.3 0.944 Central PP [mmHg] 44.4±8.0 41.4±6.6 0.004 43.5±8.6 42.8±7.5 0.471 Forward pressure pulse height [mmHg] 33.0±5.7 30.2±4.6 <0.001 32.5±5.6 31.8±4.5 0.216 Central office PWV [m/s] 8.2±1.6 8.0±1.5 0.039 8.4±1.3 8.3±1.2 0.400 24-h ambulatory central vascular parameters Central 24-h SBP [mmHg] 120.5±9.3 117.3±7.9 0.007 121.0±9.1 121.0±8.0 0.608 Central 24-h DBP [mmHg] 83.2±7.3 81.1±6.9 0.016 82.4±7.1 82.4±7.7 0.928 Central 24-h PWV [m/s] 8.9±1.3 8.8±1.3 0.010 9.0±1.4 9.0±1.3 0.349 SBP, systolic blood pressure; DBP, diastolic blood pressure; PP, pulse pressure; PWV, pulse wave velocity. Conclusion The combination of E+L significantly improves BP and vascular function in contrast to the combination of M+I. Acknowledgement/Funding This IIS was supported by a research grant from Boehringer Ingelheim International GmBH

Objective: Arginine vasopressin (ADH) is released from the neurohypophysis and regulates intravascular volume status. ADH activity is reflected by copeptin, the C-terminal peptide of pro-vasopressin. Elevated copeptin levels are associated with increased cardiovascular and all-cause mortality. The aim of this study is to compare copeptin levels in patients with different cardiovascular diseases. Design and method: In this cross-sectional analysis we measured copeptin concentrations in 69 patients with diabetes mellitus type 2 (T2DM), 30 patients with primary hypertension stage 1 or 2 (HT1–2), 34 patients with treatment resistant hypertension (TRH) (21 of them with T2DM), and 28 healthy individuals, who participated in clinical trials. In 2 study groups we analyzed changes after therapeutic interventions. Patients with T2DM received 6 weeks of treatment with 25 mg empagliflozin or placebo. Patients with TRH underwent full four quadrant renal denervation (RDN) by an experienced interventionalist. Copeptin concentrations were measured before and after treatment using Time Resolved Amplified Cryptate Emision method. Results: Patients with TRH showed higher copeptin levels than patients with HT1-2 (median 8.4 [interquartile range 3.6-14] vs. 4.2 [2.8–6.3]pmol/l, p = 0.039), patients with T2DM (4.5 [3.3–7.2]pmol/l, p = 0.020) and healthy individuals (5.7 [2.9–9.2]pmol/l, p = 0.024). There was no significant change in copeptin levels in patients with TRH before and 6 month after RDN (8.4 [3.6–14] vs 8.5 [4.5–13]pmol/l, p = 0.334), even though 24 h ambulatory blood pressure decreased from 154 ± 15/ 87 ± 12 mmHg (p = 0.001) to 146 ± 13/ 83 ± 7.9 mmHg (p = 0.034). In patients with T2DM (double blind randomized cross-over trial), no significant change in copeptin levels was observed in the placebo group compared to baseline (5.08 ± 2.83 vs 5.76 ± 4.05 pmol/l, p = 0.09), whereas treatment with empagliflozin increased copeptin levels compared to baseline (6.87 ± 3.89 pmol/l, p = 0.001). Patients receiving empagliflozin showed higher copeptin levels (p < 0.001) compared to placebo. Conclusions: Patients with TRH showed higher copeptin levels than patients with HT1-2, T2DM and healthy individuals. RDN did not lead to any change of copeptin levels in patients with TRH, but empagliflozin, as expected induced an increase in copeptin levels due to volume contraction in patients with T2DM. Copeptin emerged as a valuable research marker in cardiovascular disease.

Objective: Epidemiological studies have found a link between aircraft noise exposure and increased incidence of arterial hypertension and cardiovascular disease. The underlying pathophysiological mechanisms are not yet fully understood. The kidney acts as a long-term regulator of blood pressure. Clinical studies show that mental stress affects the systemic and renal hemodynamic. Design and method: We analysed cardiovascular and renal effects of 30 minutes standardized aircraft noise with a maximal sound pressure level of 80 dB in a sham controlled clinical study including 80 healthy normotensive subjects and 34 patients with hypertension stage 1 or 2. Systemic hemodynamic was measured using impedance cardiography and renal hemodynamic using steady state input clearance with infusion of paraaminohippuric-acid and inulin. Results: In the renal circulation of hypertensive patients change in renal plasma flow (30 ± 29 vs 30 ± 29 ml/min, p = 0.92) and glomerular filtration rate (16 ± 11 vs 18 ± 10 ml/min, p = 0.41) did not differ between aircraft noise exposure and sham procedure. The same was true in normotensive individuals (change in renal plasma flow: 37 ± 34 vs 32 ± 36 ml/min, p = 0.22, change in glomerular filtration rate: 16 ± 11 vs 15 ± 9 ml/min, p = 0.62). In the systemic circulation change in heart rate (−7.2 ± 5.2 vs −6.6 ± 13.2 bpm, p = 0.79) and stroke volume (−2.1 ± 8.9 vs 2.4 ± 8.3 ml, p = 0.78) 15 seconds after aircraft noise exposure did also not differ compared to sham procedure in hypertensive patients. The same was true for normotensive subjects (change in heart rate: −4.7 ± 9.0 vs −3.7 ± 6.7 bpm, p = 0.364, change in stroke volume: (−2.8 ± 11.4 vs 3.1 ± 9.6 ml, p = 0.83). However, in hypertensive patients there was an increase in total peripheral resistance after aircraft noise exposure (1416 ± 388 vs 1605 ± 435 dyn·sec·cm-5, p = 0.001, which was not present after sham procedure (1379 ± 401 vs 1476 ± 586 dyn·sec·cm-5, p = 0.24). Change in total peripheral resistance in normotensive subjects was not different after aircraft noise exposure compared to sham procedure (91.4 ± 237 vs 66.1 ± 177, p = 0.361). Conclusions: In hypertensive patients we did not observe an immediate increase in heart rate or cardiac output after aircraft noise exposure, but we found a vasoconstrictive response in the systemic circulation. No changes in renal hemodynamic were observed after aircraft noise exposure.

The approval of anti-CTLA-4 and anti-PD-1 antibodies underscores the relevance of stimulating T cells to treat melanoma. T cell-directed immunotherapy highly depends on recognition of antigen expressed in the context of HLA molecules on the melanoma cell surface. However, melanoma can escape T cell-driven antitumor immunity through a variety of mechanisms, including the down-regulation of HLA. NK cells show the ability to respond to melanoma in an HLA-unrestricted fashion. Their activity is controlled by a repertoire of surface receptors, which upon engagement signal either an inhibitory or activating response. From a clinical perspective, treatment of melanoma with IL-2, a cytokine known to expand NK cells in vitro and in vivo, induces responses in 15% of patients. However, systemic IL-2 treatment is associated with severe side-effects, and not approved as melanoma treatment option in Europe. An adoptive NK cell transfer is an attractive strategy to boost innate immunity against autologous melanoma without incurring the systemic toxicity through IL-2. The purpose of this study was twofold. We defined if NK cells could be ex vivo expanded from melanoma patients, and subsequently tested their cytotoxic potential against autologous melanoma cells. We were able to expand patient-derived NK cells in the presence of exogenous IL-2 and an NK cell-sensitive tumor cell line transfected with IL-15 and 4-1BBLigand. As shown by flow cytometry and chromium-release assays, expanded NK cells up-regulated cytotoxicity receptors, which mediated autologous melanoma killing in a dose-dependent fashion. These findings provide a proof-of-concept for the adoptive transfer of expanded NK cells in melanoma patients and complement immunotherapeutic strategies solely geared towards the activation of T cell-specific anti-melanoma responses.

Current methods for the detection and isolation of antigen-specific CD4+ and CD8+ T cells require the availability of peptide/MHC multimers or are restricted to cells that produce cytokines after antigen contact. We have recently reported that de novo cell surface expression of the TNF receptor family member CD137 (4-1BB) identifies currently activated, but not resting, human alloreactive CD8+ T cells. This observation allowed us to develop a CD137-based technology for the depletion of alloreactive CD8+ T cells in vitro (Wehler et al. Blood 2007; 109:365–373). More recently, a similar approach has been described that uses activation-induced CD137 expression for the detection and enrichment of antigen-specific CD8+ T cells (Wolfl et al. Blood 2007; 110:201–210). In the current study we complement this work and demonstrate the transient up-regulation of CD137 directly on activated cytomegalovirus (CMV) or Epstein-Barr virus specific CD8+ T cells using peptide/HLA tetramer staining of PBMC from seropositive healthy individuals. Antigen-triggered CD137 expression was first detectable upon 6h of stimulation, and reached peak intensity at 24h, allowing the determination of a clear-cut population of CD137+ T cells at this time point. Most importantly, we also observed a similar CD137 expression kinetics (i.e. low baseline, maximum at 24h) on virus-specific CD4+ T cells upon activation with CMVpp65 peptides. The median frequencies of CMVpp65-reactive CD137+ cells measured ex vivo in 4 different CMV+ healthy donors after 24h of stimulation were 3.6% (range, 1.1–6.7) in CD8+ T cells and 2.7% (range, 0.8–6.4) in CD4+ T cells, respectively. We also analyzed PBMC derived from the same donors and left unstimulated, as well as PBMC from CMV-seronegative donors (n=3) stimulated with CMV peptides. None of these samples contained more than 0.3% CD137+ cells per total CD4+ and CD8+ T cells, thereby confirming the specificity of antigen-induced CD137 expression. We next established a two-step in vitro approach allowing the activation and subsequent CD137-based immunomagnetic cell sorting of virus-reactive CD4+ and CD8+ T cells at the same time. We demonstrated the suitability of this assay to isolate CMVpp65-reactive CD4+ and CD8+ T cells from PBMC of 6 CMV+ healthy individuals. Enriched fractions had a median purity of CD137+ cells of 69.4% (range 12.7–94.1) among CD4+ T cells and 70.6% (range, 28.5–93.4) among CD8+ T cells, respectively. The CD137+ populations could be expanded in vitro and showed CMVpp65-specific cytokine production by CD4+ and CD8+ T cells as well as a strong enrichment of CMVpp65/HLA tetramer-binding CD8+ T cells. We finally compared the efficiency of the CD137 assay with the IFN-γ secretion assay to isolate CMVpp65-specific CD4+ and CD8+ T cells from PBMC. Although both methods were performed at optimal conditions, the numbers of CD137+ T cells measured before and after enrichment were approximately 2-fold higher than those of IFN-γ+ cells (n=5), suggesting that CD137 might detect a broader repertoire of virus-reactive T cells. In conclusion, activation-induced CD137 expression provides a means for the rapid detection and isolation of viable virus-reactive CD4+ and CD8+ T cells. The CD137 assay is most attractive for the simultaneous targeting of both T-cell subsets in monitoring studies and adoptive immunotherapy trials.