Direct in vitro and in vivo comparison of 161Tb and 177Lu using a tumour-targeting folate conjugate

Purpose The radiolanthanide 161Tb (T 1/2 = 6.90 days, Eβ− av = 154 keV) was recently proposed as a potential alternative to 177Lu (T 1/2 = 6.71 days, Eβ− av = 134 keV) due to similar physical decay characteristics but additional conversion and Auger electrons that may enhance the therapeutic efficacy. The goal of this study was to compare 161Tb and 177Lu in vitro and in vivo using a tumour-targeted DOTA-folate conjugate (cm09). Methods 161Tb-cm09 and 177Lu-cm09 were tested in vitro on folate receptor (FR)-positive KB and IGROV-1 cancer cells using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) viability assay. In vivo 161Tb-cm09 and 177Lu-cm09 (10 MBq, 0.5 nmol) were investigated in two different tumour mouse models with regard to the biodistribution, the possibility for single photon emission computed tomography (SPECT) imaging and the antitumour efficacy. Potentially undesired side effects were monitored over 6 months by determination of plasma parameters and examination of kidney function with quantitative SPECT using 99mTc-dimercaptosuccinic acid (DMSA). Results To obtain half-maximal inhibition of tumour cell viability a 4.5-fold (KB) and 1.7-fold (IGROV-1) lower radioactivity concentration was required for 161Tb-cm09 (IC50 ~0.014 MBq/ml and ~2.53 MBq/ml) compared to 177Lu-cm09 (IC50 ~0.063 MBq/ml and ~4.52 MBq/ml). SPECT imaging visualized tumours of mice with both radioconjugates. However, in therapy studies 161Tb-cm09 reduced tumour growth more efficiently than 177Lu-cm09. These findings were in line with the higher absorbed tumour dose for 161Tb-cm09 (3.3 Gy/MBq) compared to 177Lu-cm09 (2.4 Gy/MBq). None of the monitored parameters indicated signs of impaired kidney function over the whole time period of investigation after injection of the radiofolates. Conclusion Compared to 177Lu-cm09 we demonstrated equal imaging features for 161Tb-cm09 but an increased therapeutic efficacy for 161Tb-cm09 in both tumour cell lines in vitro and in vivo. Further preclinical studies using other tumour-targeting radioconjugates are clearly necessary to draw final conclusions about the future clinical perspectives of 161Tb.

Butyrate increases intracellular calcium levels and enhances growth hormone release from rat anterior pituitary cells via the G-protein-coupled receptors GPR41 and 43

Butyrate is a short-chain fatty acid (SCFA) closely related to the ketone body ß-hydroxybutyrate (BHB), which is considered to be the major energy substrate during prolonged exercise or starvation. During fasting, serum growth hormone (GH) rises concomitantly with the accumulation of BHB and butyrate. Interactions between GH, ketone bodies and SCFA during the metabolic adaptation to fasting have been poorly investigated to date. In this study, we examined the effect of butyrate, an endogenous agonist for the two G-protein-coupled receptors (GPCR), GPR41 and 43, on non-stimulated and GH-releasing hormone (GHRH)-stimulated hGH secretion. Furthermore, we investigated the potential role of GPR41 and 43 on the generation of butyrate-induced intracellular Ca2+ signal and its ultimate impact on hGH secretion. To study this, wt-hGH was transfected into a rat pituitary tumour cell line stably expressing the human GHRH receptor. Treatment with butyrate promoted hGH synthesis and improved basal and GHRH-induced hGH-secretion. By acting through GPR41 and 43, butyrate enhanced intracellular free cytosolic Ca2+. Gene-specific silencing of these receptors led to a partial inhibition of the butyrate-induced intracellular Ca2+ rise resulting in a decrease of hGH secretion. This study suggests that butyrate is a metabolic intermediary, which contributes to the secretion and, therefore, to the metabolic actions of GH during fasting.

Stimulation of monocytes by placental microparticles involves toll-like receptors and nuclear factor kappa-light-chain-enhancer of activated B cells.

Human pregnancy is accompanied by a mild systemic inflammatory response, which includes the activation of monocytes circulating in maternal blood. This response is exaggerated in preeclampsia, a placental-dependent disorder specific to human pregnancies. We and others showed that placental syncytiotrophoblast membrane microparticles (STBM) generated in vitro from normal placentas stimulated peripheral blood monocytes, which suggest a contribution of STBM to the systemic maternal inflammation. Here, we analyzed the inflammatory potential of STBM prepared from preeclamptic placentas on primary monocytes and investigated the mode of action in vitro. STBM generated in vitro by placental villous explants of normal or preeclamptic placentas were co-incubated with human peripheral blood monocytes. In some cases, inhibitors of specific cellular functions or signaling pathways were used. The analysis of the monocytic response was performed by flow cytometry, enzyme-linked immunoassays, real-time PCR, and fluorescence microscopy. STBM derived from preeclamptic placentas up-regulated the cell surface expression of CD54, and stimulated the secretion of the pro-inflammatory interleukin (IL)-6 and IL-8 in a similar, dose-dependent manner as did STBM prepared from normal placentas. STBM bound to the cell surface of monocytes, but phagocytosis was not necessary for activation. STBM-induced cytokine secretion was impaired in the presence of inhibitors of toll-like receptor (TLR) signaling or when nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation was blocked. Our results suggest that the inflammatory reaction in monocytes may be initiated by the interaction of STBM with TLRs, which in turn signal through NF-κB to mediate the transcription of genes coding for pro-inflammatory factors.

Circulating FABP4 Is a Prognostic Biomarker in Patients With Acute Coronary Syndrome but Not in Asymptomatic Individuals.

OBJECTIVE Blood-borne biomarkers reflecting atherosclerotic plaque burden have great potential to improve clinical management of atherosclerotic coronary artery disease and acute coronary syndrome (ACS). APPROACH AND RESULTS Using data integration from gene expression profiling of coronary thrombi versus peripheral blood mononuclear cells and proteomic analysis of atherosclerotic plaque-derived secretomes versus healthy tissue secretomes, we identified fatty acid-binding protein 4 (FABP4) as a biomarker candidate for coronary artery disease. Its diagnostic and prognostic performance was validated in 3 different clinical settings: (1) in a cross-sectional cohort of patients with stable coronary artery disease, ACS, and healthy individuals (n=820), (2) in a nested case-control cohort of patients with ACS with 30-day follow-up (n=200), and (3) in a population-based nested case-control cohort of asymptomatic individuals with 5-year follow-up (n=414). Circulating FABP4 was marginally higher in patients with ST-segment-elevation myocardial infarction (24.9 ng/mL) compared with controls (23.4 ng/mL; P=0.01). However, elevated FABP4 was associated with adverse secondary cerebrovascular or cardiovascular events during 30-day follow-up after index ACS, independent of age, sex, renal function, and body mass index (odds ratio, 1.7; 95% confidence interval, 1.1-2.5; P=0.02). Circulating FABP4 predicted adverse events with similar prognostic performance as the GRACE in-hospital risk score or N-terminal pro-brain natriuretic peptide. Finally, no significant difference between baseline FABP4 was found in asymptomatic individuals with or without coronary events during 5-year follow-up. CONCLUSIONS Circulating FABP4 may prove useful as a prognostic biomarker in risk stratification of patients with ACS.

Human skin is protected by four functionally and phenotypically discrete populations of resident and recirculating memory T cells

The skin of an adult human contains about 20 billion memory T cells. Epithelial barrier tissues are infiltrated by a combination of resident and recirculating T cells in mice, but the relative proportions and functional activities of resident versus recirculating T cells have not been evaluated in human skin. We discriminated resident from recirculating T cells in human-engrafted mice and lymphoma patients using alemtuzumab, a medication that depletes recirculating T cells from skin, and then analyzed these T cell populations in healthy human skin. All nonrecirculating resident memory T cells (TRM) expressed CD69, but most were CD4(+), CD103(-), and located in the dermis, in contrast to studies in mice. Both CD4(+) and CD8(+) CD103(+) TRM were enriched in the epidermis, had potent effector functions, and had a limited proliferative capacity compared to CD103(-) TRM. TRM of both types had more potent effector functions than recirculating T cells. We observed two distinct populations of recirculating T cells, CCR7(+)/L-selectin(+) central memory T cells (TCM) and CCR7(+)/L-selectin(-) T cells, which we term migratory memory T cells (TMM). Circulating skin-tropic TMM were intermediate in cytokine production between TCM and effector memory T cells. In patients with cutaneous T cell lymphoma, malignant TCM and TMM induced distinct inflammatory skin lesions, and TMM were depleted more slowly from skin after alemtuzumab, suggesting that TMM may recirculate more slowly. In summary, human skin is protected by four functionally distinct populations of T cells, two resident and two recirculating, with differing territories of migration and distinct functional activities.

Gender-Specific Associations between Circulating T-Cadherin and High Molecular Weight-Adiponectin in Patients with Stable Coronary Artery Disease.

Close relationships exist between presence of adiponectin (APN) within vascular tissue and expression of T-cadherin (T-cad) on vascular cells. APN and T-cad are also present in the circulation but here their relationships are unknown. This study investigates associations between circulating levels of high molecular weight APN (HMW-APN) and T-cad in a population comprising 66 women and 181 men with angiographically proven stable coronary artery disease (CAD). Plasma HMW-APN and T-cad were measured by ELISA and analysed for associations with baseline clinical characteristics and with each other. In multivariable analysis BMI and HDL were independently associated with HMW-APN in both genders, while diabetes and extent of coronary stenosis were independently associated with T-cad in males only. Regression analysis showed no significant association between HMW-APN and T-cad in the overall study population. However, there was a negative association between HMW-APN and T-cad (P=0.037) in a subgroup of young men (age <60 years, had no diabetes and no or 1-vessel CAD) which persisted after multivariable analysis with adjustment for all potentially influential variables (P=0.021). In the corresponding subgroup of women there was a positive association between HMW-APN and T-cad (P=0.013) which disappeared after adjustment for HDL. After exclusion of the young men, a positive association (P=0.008) between HMW-APN and T-cad was found for the remaining participants of the overall population which disappeared after adjustment for HDL and BMI. The existence of opposing correlations between circulating HMW-APN and T-cad in male and female patient populations underscores the necessity to consider gender as a confounding variable when evaluating biomarker potentials of APN and T-cad.

Endothelial progenitor cells as a biological marker of peripheral artery disease.

The role of endothelial progenitor cells (EPCs) in peripheral artery disease (PAD) remains unclear. We hypothesized that EPC mobilization and function play a central role in the development of endothelial dysfunction and directly influence the degree of atherosclerotic burden in peripheral artery vessels. The number of circulating EPCs, defined as CD34(+)/KDR(+) cells, were assessed by flow cytometry in 91 subjects classified according to a predefined sample size of 31 non-diabetic PAD patients, 30 diabetic PAD patients, and 30 healthy volunteers. Both PAD groups had undergone endovascular treatment in the past. As a functional parameter, EPC colony-forming units were determined ex vivo. Apart from a broad laboratory analysis, a series of clinical measures using the ankle-brachial index (ABI), flow-mediated dilatation (FMD) and carotid intima-media thickness (cIMT) were investigated. A significant reduction of EPC counts and proliferation indices in both PAD groups compared to healthy subjects were observed. Low EPC number and pathological findings in the clinical assessment were strongly correlated to the group allocation. Multivariate statistical analysis revealed these findings to be independent predictors of disease appearance. Linear regression analysis showed the ABI to be a predictor of circulating EPC number (p=0.02). Moreover, the functionality of EPCs was correlated by linear regression (p=0.017) to cIMT. The influence of diabetes mellitus on EPCs in our study has to be considered marginal in already disease-affected patients. This study demonstrated that EPCs could predict the prevalence and severity of symptomatic PAD, with ABI as the determinant of the state of EPC populations in disease-affected groups.