Pregnancy induces numerical and functional changes of CD4+CD25 high regulatory T cells in patients with rheumatoid arthritis

OBJECTIVE: In a prospective study we investigated whether numerical and functional changes of CD4+CD25(high) regulatory T cells (Treg) were associated with changes of disease activity observed during pregnancy and post partum in patients with rheumatoid arthritis (RA). METHODS: The frequency of CD4+CD25(high) T cells was determined by flow cytometry in 12 patients with RA and 14 healthy women during and after pregnancy. Fluorescence-activated cell sorting (FACS) was used to sort CD4+CD25(high) T cells and CD4+CD25- T cells were stimulated with anti-CD3 and anti-CD28 monoclonal antibodies alone or in co-culture to investigate proliferation and cytokine secretion. RESULTS: Frequencies of CD4+CD25(high) Treg were significantly higher in the third trimester compared to 8 weeks post partum in patients and controls. Numbers of CD4+CD25(high) Treg inversely correlated with disease activity in the third trimester and post partum. In co-culture experiments significantly higher amounts of IL10 and lowered levels of tumour necrosis factor (TNF)alpha and interferon (IFN)gamma were found in supernatants of the third trimester compared to postpartum samples. These findings were independent from health or disease in pregnancy, however postpartum TNFalpha and IFN gamma levels were higher in patients with disease flares. CONCLUSION: The amelioration of disease activity in the third trimester corresponded to the increased number of Treg that induced a pronounced anti-inflammatory cytokine milieu. The pregnancy related quantitative and qualitative changes of Treg suggest a beneficial effect of Treg on disease activity.

Soluble tumour necrosis factor receptors sTNFR1 and sTNFR2 are produced at sites of inflammation and are markers of arthritis activity in Behçet's disease

OBJECTIVE: We analysed the production of soluble tumour necrosis factor receptors sTNFR1 and sTNFR2 at sites of inflammation and measured their plasma concentrations to evaluate them as biological markers of disease activity. METHODS: Plasma samples of 35 patients with Behçet's disease (BD) were collected prospectively at monthly intervals and grouped for inactive disease, active BD without arthritis, and active BD with arthritis. sTNFR1 and sTNFR2 concentrations were measured using immunoassays and compared with other biological disease activity parameters. Plasma sTNFR levels were compared to synovial fluid (SF) levels in seven patients. Sixteen tissue samples of mucocutaneous lesions were stained for TNFR2 expression by immunohistochemistry. RESULTS: sTNFR1 and sTNFR2 were found at increased plasma concentrations in active BD, with the highest concentration in active BD with arthritis (p<0.001). Concentrations of both sTNFRs were at least three times higher in SF of arthritic joints than in the corresponding plasma samples (p = 0.025). A change of more than 1 ng/mL of sTNFR2 plasma concentrations correlated with a concordant change in arthritic activity (96% confidence interval). Sensitivity to change was superior to that of sTNFR1, and other biological disease activity parameters such as erythrocyte sedimentation rate (ESR), immunoglobulin (Ig)G, IgA, and interleukin (IL)-10 plasma concentrations. A strong staining for TNFR2 was found in mucocutaneous lesions, where mast cells were identified as the major source for this receptor. CONCLUSIONS: This longitudinal study demonstrates that sTNFR2 plasma concentrations are closely linked with active BD, and especially with arthritis. Taken together with the expression of TNFR molecules in mast cells of mucocutaneous lesions, our results indicate a fundamental role for the TNF/TNFR pathway in BD.

CD39 is incorporated into plasma microparticles where it maintains functional properties and impacts endothelial activation

Plasma microparticles (MPs, <1.5 mum) originate from platelet and cell membrane lipid rafts and possibly regulate inflammatory responses and thrombogenesis. These actions are mediated through their phospholipid-rich surfaces and associated cell-derived surface molecules. The ectonucleotidase CD39/ecto-nucleoside triphosphate diphosphohydrolase1 (E-NTPDase1) modulates purinergic signalling through pericellular ATP and ADP phosphohydrolysis and is localized within lipid rafts in the membranes of endothelial- and immune cells. This study aimed to determine whether CD39 associates with circulating MPs and might further impact phenotype and function. Plasma MPs were found to express CD39 and exhibited classic E-NTPDase ecto-enzymatic activity. Entpd1 (Cd39) deletion in mice produced a pro-inflammatory phenotype associated with quantitative and qualitative differences in the MP populations, as determined by two dimensional-gel electrophoresis, western blot and flow cytometry. Entpd1-null MPs were also more abundant, had significantly higher proportions of platelet- and endothelial-derived elements and decreased levels of interleukin-10, tumour necrosis factor receptor 1 and matrix metalloproteinase 2. Consequently, Cd39-null MP augment endothelial activation, as determined by inflammatory cytokine release and upregulation of adhesion molecules in vitro. In conclusion, CD39 associates with circulating MP and may directly or indirectly confer functional properties. Our data also suggest a modulatory role for CD39 within MP in the exchange of regulatory signals between leucocytes and vascular cells.

Increased circulating placental growth factor during percutaneous coronary intervention is associated with applied radiocontrast agent

BACKGROUND AND AIM OF THE STUDY: Recent studies have suggested placental growth factor (PlGF) and vascular endothelial growth factor (VEGF) as promising new biomarkers for risk stratification in acute coronary syndromes (ACS). However, little is known about the influence of percutaneous coronary intervention (PCI) on circulating PlGF and VEGF levels. METHODS: Thirty-five patients with ACS, 27 patients with stable coronary artery disease (sCAD), and nine healthy controls were enrolled in the study. Although all patients with ACS and 14 patients with stable angina pectoris underwent PCI, 13 patients with coronary artery disease required no revascularization (sCAD). PlGF and VEGF plasma concentrations were measured by immunoassay during and at the end of PCI and coronary angiography. RESULTS: Plasma PlGF levels were comparable in patients with ACS and sCAD on admission. Although coronary angiography or heparin alone did not alter PlGF and VEGF levels, immediately after PCI a dramatic increase was seen in circulating PlGF and a decrease in VEGF, which was independent of the clinical presentation of the patients, heparin administration, or the angiographic procedure itself, but was associated with the extent of coronary artery disease and the amount of the injected contrast media. In-vitro experiments revealed that radiocontrast agents induced the release of PlGF from endothelial cells without altering PlGF mRNA expression. CONCLUSION: Patients undergoing PCI exhibit an increase in circulating PlGF, probably caused by posttranslational modifications of radiocontrast agents in endothelial cells. Therefore, analysis of plasma PlGF and VEGF levels may consider the timing of blood sampling with respect to PCI and contrast media exposure.

Hypoxia inducible factor-1 alpha induction by tumour necrosis factor-alpha, but not by toll-like receptor agonists, modulates cellular respiration in cultured human hepatocytes

BACKGROUND/AIMS: Genes encoding for some of the mitochondrial proteins are under the control of the transcriptional factor hypoxia inducible factor-1 alpha (HIF-1 alpha), which can accumulate under normoxic conditions in inflammatory states. The aim of this study was to evaluate the effects of cobalt chloride (CoCl(2), a hypoxia mimicking agent), tumour necrosis factor-alpha (TNF-alpha) and toll-like receptor (TLR) -2, -3 and -4 agonists on HIF-1 alpha accumulation, and further on HIF-1 alpha-mediated modulation of mitochondrial respiration in cultured human hepatocytes. METHODS: The human hepatoma cell line HepG2 was used in this study. Cells were treated with CoCl(2), TNF-alpha and TLR-2, -3 and -4 agonists. HIF-1 alpha was determined by Western blotting and mitochondrial respiration in stimulated cells by high-resolution respirometry. RESULTS: CoCl(2), TNF-alpha and TLR agonists induced the expression of HIF-1 alpha in a time-dependent fashion. TNF-alpha and CoCl(2), but not TLR agonists, induced a reduction in complex I-, II- and IV-dependent mitochondrial oxygen consumption. TNF-alpha-associated reduction of cellular oxygen consumption was abolished through inhibition of HIF-1 alpha activity by chetomin (CTM). Pretreatment with cyclosporine A prevented CoCl(2)-induced reduction of complex I- and II-dependent mitochondrial oxygen consumption and TNF-alpha-induced reduction of complex-I-dependent respiration, implicating the involvement of the mitochondrial permeability transition pore openings. TNF-alpha and TLR-2, -3 and -4 agonists induced the expression of vascular endothelial growth factor, which was partially abolished by the blockage of HIF-1 alpha with CTM. CONCLUSIONS: The data suggest that HIF-1 alpha modulates mitochondrial respiration during CoCl(2) and TNF-alpha stimulation, whereas it has no effect when induced with TLR-2, -3 and -4 agonists.

Class-switzched B cells display response to therapeutic B-cell depletion in rheumatoid arthritis

Introduction Reconstitution of peripheral blood (PB) B cells after therapeutic depletion with the chimeric anti-CD20 antibody rituximab (RTX) mimics lymphatic ontogeny. In this situation, the repletion kinetics and migratory properties of distinct developmental B-cell stages and their correlation to disease activity might facilitate our understanding of innate and adaptive B-cell functions in rheumatoid arthritis (RA). Methods Thirty-five 'RTX-naïve' RA patients with active arthritis were treated after failure of tumour necrosis factor blockade in an open-label study with two infusions of 1,000 mg RTX. Prednisone dose was tapered according to clinical improvement from a median of 10 mg at baseline to 5 mg at 9 and 12 months. Conventional disease-modifying antirheumatic drugs were kept stable. Subsets of CD19+ B cells were assessed by flow cytometry according to their IgD and CD27 surface expression. Their absolute number and relative frequency in PB were followed every 3 months and were determined in parallel in synovial tissue (n = 3) or synovial fluid (n = 3) in the case of florid arthritis. Results Six of 35 patients fulfilled the European League Against Rheumatism criteria for moderate clinical response, and 19 others for good clinical response. All PB B-cell fractions decreased significantly in number (P < 0.001) after the first infusion. Disease activity developed independently of the total B-cell number. B-cell repopulation was dominated in quantity by CD27-IgD+ 'naïve' B cells. The low number of CD27+IgD- class-switched memory B cells (MemB) in the blood, together with sustained reduction of rheumatoid factor serum concentrations, correlated with good clinical response. Class-switched MemB were found accumulated in flaring joints. Conclusions The present data support the hypothesis that control of adaptive immune processes involving germinal centre-derived, antigen, and T-cell-dependently matured B cells is essential for successful RTX treatment.

Primary splenic peripheral nerve sheath tumour in a dog

An 8-year-old crossbred dog was presented with a one-month history of progressive weakness, respiratory impairment and abdominal distension. Surgical exploration revealed the presence of a splenic mass that infiltrated the mesentery and was adherent to the stomach and pancreas. The mass was composed of highly cellular areas of spindle-shaped cells arranged in interlacing bundles, streams, whorls and storiform patterns (Antoni A pattern) and less cellular areas with more loosely arranged spindle to oval cells (Antoni B pattern). The majority of neoplastic cells expressed vimentin, S-100 and glial fibrillary acidic protein (GFAP), but did not express desmin, alpha-smooth muscle actin or factor VIII. These morphological and immunohistochemical findings characterized the lesion as a malignant peripheral nerve sheath tumour (PNST). Primary splenic PNST has not been documented previously in the dog.

The PDZ protein MPP2 interacts with c-Src in epithelial cells

c-Src is a non-receptor tyrosine kinase involved in regulating cell proliferation, cell migration and cell invasion and is tightly controlled by reversible phosphorylation on regulatory sites and through protein-protein interactions. The interaction of c-Src with PDZ proteins was recently identified as novel mechanism to restrict c-Src function. The objective of this study was to identify and characterise PDZ proteins that interact with c-Src to control its activity. By PDZ domain array screen, we identified the interaction of c-Src with the PDZ protein Membrane Protein Palmitoylated 2 (MPP2), a member of the Membrane-Associated Guanylate Kinase (MAGUK) family, to which also the Discs large (Dlg) tumour suppressor protein belongs. The function of MPP2 has not been established and the functional significance of the MPP2 c-Src interaction is not known. We found that in non-transformed breast epithelial MCF-10A cells, endogenous MPP2 associated with the cytoskeleton in filamentous structures, which partially co-localised with microtubules and c-Src. MPP2 and c-Src interacted in cells, where c-Src kinase activity promoted increased interaction of c-Src with MPP2. We furthermore found that MPP2 was able to negatively regulate c-Src kinase activity in cells, suggesting that the functional significance of the MPP2-c-Src interaction is to restrict Src activity. Consequently, the c-Src-dependent disorganisation of the cortical actin cytoskeleton of epithelial cells expressing c-Src was suppressed by MPP2. In conclusion we demonstrate here that MPP2 interacts with c-Src in cells to control c-Src activity and morphological function.

Stimulation of MMP-1 and CCL2 by NAMPT in PDL cells

Periodontitis is an inflammatory disease caused by pathogenic microorganisms and characterized by the destruction of the periodontium. Obese individuals have an increased risk of periodontitis, and elevated circulating levels of adipokines, such as nicotinamide phosphoribosyltransferase (NAMPT), may be a pathomechanistic link between both diseases. The aim of this in vitro study was to examine the regulation of periodontal ligament (PDL) cells by NAMPT and its production under inflammatory and infectious conditions. NAMPT caused a significant upregulation of 9 genes and downregulation of 3 genes, as analyzed by microarray analysis. Eight of these genes could be confirmed by real-time PCR: NAMPT induced a significant upregulation of EGR1, MMP-1, SYT7, ITPKA, CCL2, NTM, IGF2BP3, and NRP1. NAMPT also increased significantly the MMP-1 and CCL2 protein synthesis. NAMPT was significantly induced by interleukin-1 β and the periodontal microorganism P. gingivalis. NAMPT may contribute to periodontitis through upregulation of MMP-1 and CCL2 in PDL cells. Increased NAMPT levels, as found in obesity, may therefore represent a mechanism whereby obesity could confer an increased risk of periodontitis. Furthermore, microbial and inflammatory signals may enhance the NAMPT synthesis in PDL cells and thereby contribute to the increased gingival and serum levels of this adipokine, as found in periodontitis.

Characteristics and functional relevance of apolipoprotein-A1 and cholesterol binding in mammary gland tissues and epithelial cells

Cholesterol in milk is derived from the circulating blood through a complex transport process involving the mammary alveolar epithelium. Details of the mechanisms involved in this transfer are unclear. Apolipoprotein-AI (apoA-I) is an acceptor of cellular cholesterol effluxed by the ATP-binding cassette (ABC) transporter A1 (ABCA1). We aimed to 1) determine the binding characteristics of (125)I-apoA-I and (3)H-cholesterol to enriched plasma membrane vesicles (EPM) isolated from lactating and non-lactating bovine mammary glands (MG), 2) optimize the components of an in vitro model describing cellular (3)H-cholesterol efflux in primary bovine mammary epithelial cells (MeBo), and 3) assess the vectorial cholesterol transport in MeBo using Transwell(®) plates. The amounts of isolated EPM and the maximal binding capacity of (125)I-apoA-I to EPM differed depending on the MG's physiological state, while the kinetics of (3)H-cholesterol and (125)I-apoA-I binding were similar. (3)H-cholesterol incorporated maximally to EPM after 25±9 min. The time to achieve the half-maximum binding of (125)I-apoA-I at equilibrium was 3.3±0.6 min. The dissociation constant (KD) of (125)I-apoA-I ranged between 40-74 nmol/L. Cholesterol loading to EPM increased both cholesterol content and (125)I-apoA-I binding. The ABCA1 inhibitor Probucol displaced (125)I-apoA-I binding to EPM and reduced (3)H-cholesterol efflux in MeBo. Time-dependent (3)H-cholesterol uptake and efflux showed inverse patterns. The defined binding characteristics of cholesterol and apoA-I served to establish an efficient and significantly shorter cholesterol efflux protocol that had been used in MeBo. The application of this protocol in Transwell(®) plates with the upper chamber mimicking the apical (milk-facing) and the bottom chamber corresponding to the basolateral (blood-facing) side of cells showed that the degree of (3)H-cholesterol efflux in MeBo differed significantly between the apical and basolateral aspects. Our findings support the importance of the apoA-I/ABCA1 pathway in MG cholesterol transport and suggest its role in influencing milk composition and directing cholesterol back into the bloodstream.

PLASMACYTOID DENDRITIC CELLS SENSE SKIN INJURY AND PROMOTE WOUND HEALING THROUGH TYPE I INTERFERONS

Plasmacytoid dendritic cells (pDCs) are a rare population of circulating cells, which selectively express intracellular Toll-like receptors (TLR)-7 and TLR-9 and have the capacity to produce large amounts of type I IFNs (IFN-a/b) in response to viruses or host derived nucleic acid containing complexes. pDCs are normally absent in skin but accumulate in the skin of psoriasis patients where their chronic activation to produce IFN-a/b drives the disease formation. Whether pDCs and their activation to produce IFN-a/b play a functional role in healthy skin is unknown. Here we show that pDCs are rapidly and transiently recruited into healthy human and mouse skin upon epidermal injury. Infiltrating pDCs were found to sense nucleic acids in wounded skin via TLRs, leading to the production of IFN-a/b. The production of IFN-a/b was paralleled by a short lived expression of cathelicidins, which form complexes with extracellular nucleic acids and activated pDCs to produce IFN-a/b in vitro. In vivo, cathelicidins were sufficient but not necessary for the induction of IFN-a/b in wounded skin, suggesting redundancy of this pathway. Depletion of pDCs or inhibition of IFN-a/bR signaling significantly impaired the inflammatory response and delayed re-epithelialization of skin wounds. Thus we uncover a novel role of pDCs in sensing skin injury via TLR mediated recognition of nucleic acids and demonstrate their involvement in the early inflammatory process and wound healing response through the production of IFN-a/b.

Osteopontin (OPN) and neoplastic disease: Circulating levels and tissue distribution

OPN is a secreted phosphate containing protein which is expressed by osteoblasts and a variety of other cells in vivo. Data from in vitro studies has accumulated which relates OPN to cellular transformation. We hypothesize that OPN expression is associated with neoplastic disease in humans as suggested by cell culture models. The overall objective of the current study was to determine the tissue distribution of OPN in human malignancy and to determine whether or not a correlation exists between OPN serum levels and malignancy. At the inception of this project, no study had been made demonstrating the relevance of OPN expression with naturally occurring neoplastic disease in humans. To date, few studies have reported OPN distribution in human neoplasia and are limited by either the number of specimens analyzed or the technique used in analysis. In this dissertation study, OPN was purified from human milk and $\alpha$-OPN antiserum developed and characterized. Following antibody development, the distribution and prevalence of OPN in human oral squamous cell carcinoma and human prostate carcinoma was evaluated using immunohistochemical localization. OPN immunolocalization was found in a high percentage of oral epithelial dysplasia and oral squamous cell carcinoma in humans. One oral squamous cell carcinoma cells line, UMSCC-1, was found to express OPN mRNA using Northern blotting. OPN localized to a high percentage of primary prostate adenocarcinomas. OPN localized to 52% of androgen dependent cases and 100% of androgen independent cases. Androgen dependent cell lines such as LNCap and NbE showed minimal OPN mRNA expression while the androgen independent lines C4-2 and PC3 produced ample OPN mRNA. An OPN sandwich assay was developed and used to determine the serum level of OPN in normal males, patients with BPH (benign prostate hypertrophy), and patients with prostate carcinoma. No statistically significant difference was found in OPN serum levels among the three groups. However, a trend of increasing OPN in the serum was noted in patients with BPH and prostate cancer. ^

Influence of natural killer cells and perforin-mediated cytolysis on the development of chemically induced lung cancer in A/J mice

One alternative approach for the treatment of lung cancer might be the activation of the immune system using vaccination strategies. However, most of clinical vaccination trials for lung cancer did not reach their primary end points, suggesting that lung cancer is of low immunogenicity. To provide additional experimental information about this important issue, we investigated which type of immune cells contributes to the protection from lung cancer development. Therefore, A/J mice induced for lung adenomas/adenocarcinomas by the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were depleted of CD4(+) or CD8(+) T cells, CD11b(+) macrophages, Gr-1(+) neutrophils and asialo GM1(+) natural killer (NK) cells. Subsequent analysis of tumour growth showed an increase in tumour number only in mice depleted of NK cells. Further asking by which mechanism NK cells suppressed tumour development, we neutralized several death ligands of the tumour necrosis factor (TNF) family known to be involved in NK cell-mediated cytotoxicity. However, neither depletion of TNF-α, TNF-related apoptosis-inducing ligand, TNF-like weak inducer of apoptosis or FasL alone nor in combination induced an augmentation of tumour burden. To show whether an alternative cell death pathway is involved, we next generated A/J mice deficient for perforin. After challenging with NNK, mice deficient for perforin showed an increase in tumour number and volume compared to wild-type A/J mice. In summary, our data suggest that NK cells and perforin-mediated cytolysis are critically involved in the protection from lung cancer giving promise for further immunotherapeutic strategies for this disease.

Increased FOXP3 expression in tumour-associated tissues of horses affected with equine sarcoid disease.

Recent studies suggest that regulatory T cells (Tregs) are associated with disease severity and progression in papilloma virus induced neoplasia. Bovine papilloma virus (BPV) is recognised as the most important aetiological factor in equine sarcoid (ES) disease. The aim of this study was to compare expression levels of Treg markers and associated cytokines in tissue samples of ES-affected equids with skin samples of healthy control horses. Eleven ES-affected, and 12 healthy horses were included in the study. Expression levels of forkhead box protein 3 (FOXP3), interleukin 10 (IL10), interleukin 4 (IL4) and interferon gamma (IFNG) mRNA in lesional and tumour-distant samples from ES-affected horses, as well as in dermal samples of healthy control horses were measured using quantitative reverse transcription polymerase chain reaction (PCR). Expression levels were compared between lesional and tumour-distant as well as between tumour-distant and control samples. Furthermore, BPV-1 E5 DNA in samples of ES-affected horses was quantified using quantitative PCR, and possible associations of viral load, disease severity and gene expression levels were evaluated. Expression levels of FOXP3, IL10 and IFNG mRNA and BPV-1 E5 copy numbers were significantly increased in lesional compared to tumour-distant samples. There was no difference in FOXP3 and cytokine expression in tumour-distant samples from ES- compared with control horses. In tumour-distant samples viral load was positively correlated with IL10 expression and severity score. The increased expression of Treg markers in tumour-associated tissues of ES-affected equids indicates a local, Treg-induced immune suppression.

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.