Amperometric Biosensor for Quantitative Measurement Using Sandwich Immunoassays.

State-of-the-art clinical detection methods typically involve standard immunoassay methods, requiring specialized equipment and trained personnel. This impedes their use in the Point-of-Care (PoC) environment, where ease of operation, portability, and cost efficiency are prioritized. Small, robust electrochemical biosensors provide a means with which to analyze biomarkers in biological fluids in PoC environments. Optimized sensing surfaces, immobilization strategies, and efficient reporter systems are key to improving biosensor detection systems. The signal transduction and general performance of electrochemical sensors are determined by surface properties that link the sensing element to the biological sample. We analyzed the surface characteristics of screen-printed and thin-film electrodes using scanning electron microscopy and atomic force microscopy. An enzyme-linked immunosorbent assay (ELISA) was adapted for use in an electrochemical sensor. The robustness and reproducibility of the developed electrochemical immunosensor were investigated by detecting Neutrophil Gelatinase-Associated Lipocalin (NGAL) in urine. The sensor showed a detection limit of 1 ng/mL, a linear range of 3.5-80 ng/mL, and a CV% of 8%. The results demonstrate that the developed platform technology is suitable for immunoassay-based sensors on either screen-printed or thin-film gold electrodes.

Influence of Microgravity on Apoptosis in Cells, Tissues, and Other Systems In Vivo and In Vitro.

All life forms have evolved under the constant force of gravity on Earth and developed ways to counterbalance acceleration load. In space, shear forces, buoyance-driven convection, and hydrostatic pressure are nullified or strongly reduced. When subjected to microgravity in space, the equilibrium between cell architecture and the external force is disturbed, resulting in changes at the cellular and sub-cellular levels (e.g., cytoskeleton, signal transduction, membrane permeability, etc.). Cosmic radiation also poses great health risks to astronauts because it has high linear energy transfer values that evoke complex DNA and other cellular damage. Space environmental conditions have been shown to influence apoptosis in various cell types. Apoptosis has important functions in morphogenesis, organ development, and wound healing. This review provides an overview of microgravity research platforms and apoptosis. The sections summarize the current knowledge of the impact of microgravity and cosmic radiation on cells with respect to apoptosis. Apoptosis-related microgravity experiments conducted with different mammalian model systems are presented. Recent findings in cells of the immune system, cardiovascular system, brain, eyes, cartilage, bone, gastrointestinal tract, liver, and pancreas, as well as cancer cells investigated under real and simulated microgravity conditions, are discussed. This comprehensive review indicates the potential of the space environment in biomedical research.

Soluble Klotho: a possible predictor of quality of life in acromegaly patients.

PURPOSE: Although quality of life (QoL) is improved in patients with acromegaly after disease control, QoL correlates only weakly with traditional biomarkers. Our objective is to investigate a potential relation between the new serum biomarker soluble Klotho (sKlotho), GH and insulin-like growth factor 1 (IGF-1) levels, and QoL. METHODS: In this prospective cohort study, we investigated 54 acromegaly patients biochemically well-controlled on combination treatment with first-generation somatostatin receptor ligands (SRLs) and pegvisomant (PEGV) at baseline and 9 months after switching to pasireotide LAR (PAS-LAR; either as monotherapy, n = 28; or in combination with PEGV, n = 26). QoL was measured by the Patient-Assessed Acromegaly Symptom Questionnaire (PASQ) and Acromegaly Quality of Life (AcroQoL) questionnaire. RESULTS: Switching to PAS-LAR treatment significantly improved QoL without altering IGF-1 levels. QoL did not correlate with GH or IGF-1 levels, but sKlotho correlated with the observed improvements in QoL by the AcroQoL global (r = -0.35, p = 0.012) and physical subdimension (r = -0.34, p = 0.017), and with PASQ headache (r = 0.28, p = 0.048), osteoarthralgia (r = 0.46, p = 0.00080) and soft tissue swelling score (r = 0.29, p = 0.041). Parallel changes in serum sKlotho and IGF-1 (r = 0.31, p = 0.023) suggest sKlotho and IGF-1 to be similarly dependent on GH. Comparing the PAS-LAR combination therapy and the monotherapy group we did not observe a significant difference in improvement of QoL. CONCLUSIONS: Patients experienced improved QoL during PAS-LAR, either as monotherapy or in combination with PEGV. Soluble Klotho concentrations appear to be a useful marker of QoL in acromegaly patients but the underlying mechanisms remain to be investigated.

Simulated Microgravity Influences VEGF, MAPK, and PAM Signaling in Prostate Cancer Cells.

Prostate cancer is one of the leading causes of cancer mortality in men worldwide. An unusual but unique environment for studying tumor cell processes is provided by microgravity, either in space or simulated by ground-based devices like a random positioning machine (RPM). In this study, prostate adenocarcinoma-derived PC-3 cells were cultivated on an RPM for time periods of 3 and 5 days. We investigated the genes associated with the cytoskeleton, focal adhesions, extracellular matrix, growth, survival, angiogenesis, and metastasis. The gene expression of signaling factors of the vascular endothelial growth factor (VEGF), mitogen-activated protein kinase (MAPK), and PI3K/AKT/mTOR (PAM) pathways was investigated using qPCR. We performed immunofluorescence to study the cytoskeleton, histological staining to examine the morphology, and a time-resolved immunofluorometric assay to analyze the cell culture supernatants. When PC-3 cells were exposed to simulated microgravity (s-µg), some cells remained growing as adherent cells (AD), while most cells detached from the cell culture flask bottom and formed multicellular spheroids (MCS). After 3-day RPM exposure, PC-3 cells revealed significant downregulation of the VEGF, SRC1, AKT, MTOR, and COL1A1 gene expression in MCS, whereas FLT1, RAF1, MEK1, ERK1, FAK1, RICTOR, ACTB, TUBB, and TLN1 mRNAs were not significantly changed. ERK2 and TLN1 were elevated in AD, and FLK1, LAMA3, COL4A5, FN1, VCL, CDH1, and NGAL mRNAs were significantly upregulated in AD and MCS after 3 days. After a 5-day culture in s-µg, the PC-3 cells showed significant downregulations of VEGF mRNA in AD and MCS, and FN1, CDH1, and LAMA3 in AD and SCR1 in MCS. In addition, we measured significant upregulations in FLT1, AKT, ERK1, ERK2, LCN2, COL1A1, TUBB, and VCL mRNAs in AD and MCS, and increases in FLK1, FN1, and COL4A5 in MCS as well as LAMB2, CDH1, RAF1, MEK1, SRC1, and MTOR mRNAs in AD. FAK1 and RICTOR were not altered by s-µg. In parallel, the secretion rate of VEGFA and NGAL proteins decreased. Cytoskeletal alterations (F-actin) were visible, as well as a deposition of collagen in the MCS. In conclusion, RPM-exposure of PC-3 cells induced changes in their morphology, cytoskeleton, and extracellular matrix protein synthesis, as well as in their focal adhesion complex and growth behavior. The significant upregulation of genes belonging to the PAM pathway indicated their involvement in the cellular changes occurring in microgravity.

Key Proteins Involved in Spheroid Formation and Angiogenesis in Endothelial Cells After Long-Term Exposure to Simulated Microgravity.

BACKGROUND/AIMS: Cardiovascular complications are common in astronauts returning from a prolonged spaceflight. These health problems might be driven by complex modulations of gene expression and protein synthesis in endothelial cells (ECs). Studies on the influence of microgravity on phenotype, growth pattern and biological processes of ECs can help to understand these complications. METHODS: We exposed ECs (EA.hy926) to a Random Positioning Machine (RPM). Proteins associated with cell structure, angiogenesis and endothelial dysfunction were investigated in distinct pools of multicellular spheroids (MCS), adherent cells (AD) and tubular structures (TS) formed after a 35-day RPM-exposure. RESULTS: Combining morphological and molecular approaches, we found AD, MCS and TS with changes in the synthesis and release of proteins involved in three-dimensional growth. Fibronectin and monocyte chemoattractant protein-1 (MCP-1) mRNAs and protein contents were elevated along with an increased secretion of vascular endothelial growth factor (VEGF), interleukin (IL)-6, IL-8, MCP-1, intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), neutrophil gelatinase-associated lipocalin (NGAL) and regulated on activation, normal T cell expressed and secreted (RANTES) proteins in the culture supernatant as determined by multianalyte profiling technology. Together they form a network of interaction. CONCLUSIONS: These results show that a prolonged RPM-exposure of ECs induced TS and MCS formation. The factors VEGF, NGAL, IL-6, IL-8, MCP-1, VCAM-1, ICAM-1, fibronectin and RANTES seem to be affected when gravity is omitted.

Hypertension Caused by Lenvatinib and Everolimus in the Treatment of Metastatic Renal Cell Carcinoma.

Multikinase inhibitors (MKI) and mammalian target of rapamycin (mTOR) inhibitors prolong progression-free (PFS) and overall survival (OS) in the treatment of metastatic renal cell carcinoma (mRCC) by reducing angiogenesis and tumor growth. In this regard, the MKI lenvatinib and the mTOR inhibitor everolimus proved effective when applied alone, but more effective when they were administered combined. Recently, both drugs were included in clinical trials, resulting in international clinical guidelines for the treatment of mRCC. In May 2016, lenvatinib was approved by the American Food and Drug Administration (FDA) for the use in combination with everolimus, as treatment of advanced renal cell carcinoma following one prior antiangiogenic therapy. A major problem of treating mRCC with lenvatinib and everolimus is the serious adverse event (AE) of arterial hypertension. During the treatment with everolimus and lenvatinib combined, 42% of the patients developed hypertension, while 10% of the patients treated with everolimus alone and 48% of the of the lenvatinib only treated patients developed hypertension. Lenvatinib carries warnings and precautions for hypertension, cardiac failure, and other adverse events. Therefore, careful monitoring of the patients is necessary.

Effects and Side Effects of Using Sorafenib and Sunitinib in the Treatment of Metastatic Renal Cell Carcinoma.

In recent years, targeted therapies have proven beneficial in terms of progression-free survival (PFS) and overall survival (OS) in the treatment of metastatic renal cell carcinoma (mRCC). The tyrosine kinase inhibitors (TKIs) sorafenib and sunitinib are included in international clinical guidelines as first-line and second-line therapy in mRCC. Hypertension is an adverse effect of these drugs and the degree of hypertension associates with the anti-tumour effect. Studies have compared newer targeted drugs to sorafenib and sunitinib in terms of PFS, OS, quality of life and safety profiles. Phase III studies presented promising response rates and acceptable safety profiles of axitinib and tivozanib compared to sorafenib, and a phase II study reported greater efficacy using a combination of bevacizumab and IFN-α compared to sunitinib. Treatment with nintedanib exhibited a notably low prevalence of hypertension compared to sunitinib. The use of sorafenib and sunitinib are challenged by new drugs, but do not appear likely to be substituted in the near future. To clarify whether newer targeted drugs should replace sorafenib and sunitinib, more research is needed. This manuscript reviews the current utility and adverse effects of sorafenib and sunitinib and newer targeted therapies in the treatment of mRCC.

Reduced Expression of Cytoskeletal and Extracellular Matrix Genes in Human Adult Retinal Pigment Epithelium Cells Exposed to Simulated Microgravity.

BACKGROUND/AIMS: Microgravity (µg) has adverse effects on the eye of humans in space. The risk of visual impairment is therefore one of the leading health concerns for NASA. The impact of µg on human adult retinal epithelium (ARPE-19) cells is unknown. METHODS: In this study we investigated the influence of simulated µg (s-µg; 5 and 10 days (d)), using a Random Positioning Machine (RPM), on ARPE-19 cells. We performed phase-contrast/fluorescent microscopy, qRT-PCR, Western blotting and pathway analysis. RESULTS: Following RPM-exposure a subset of ARPE-19 cells formed multicellular spheroids (MCS), whereas the majority of the cells remained adherent (AD). After 5d, alterations of F-actin and fibronectin were observed which reverted after 10d-exposure, suggesting a time-dependent adaptation to s-µg. Gene expression analysis of 12 genes involved in cell structure, shape, adhesion, migration, and angiogenesis suggested significant changes after a 10d-RPM-exposure. 11 genes were down-regulated in AD and MCS 10d-RPM-samples compared to 1g, whereas FLK1 was up-regulated in 5d- and 10d-RPM-MCS-samples. Similarly, TIMP1 was up-regulated in 5d-RPM-samples, whereas the remaining genes were down-regulated in 5d-RPM-samples. Western blotting revealed similar changes in VEGF, ß-actin, laminin and fibronectin of 5d-RPM-samples compared to 10d, whereas different alterations of ß-tubulin and vimentin were observed. The pathway analysis showed complementing effects of VEGF and integrin ß-1. CONCLUSIONS: These findings clearly show that s-µg induces significant alterations in the F-actin-cytoskeleton and cytoskeleton-related proteins of ARPE-19, in addition to changes in cell growth behavior and gene expression patterns involved in cell structure, growth, shape, migration, adhesion and angiogenesis.

Plasma Neutrophil Gelatinase-Associated Lipocalin Reflects Both Inflammation and Kidney Function in Patients with Myocardial Infarction.

BACKGROUND/AIMS: Neutrophil gelatinase-associated lipocalin (NGAL) has emerged as a marker for acute kidney injury and cardiovascular outcome. However, the relative importance of inflammation versus kidney function on plasma NGAL levels is uncertain, making the interpretation of plasma NGAL unclear. Accordingly, we investigated the relationship between plasma NGAL, inflammation and kidney function in patients with myocardial infarction (MI). METHODS: We prospectively included 584 patients with acute ST-segment elevation MI (STEMI) treated with primary percutaneous coronary intervention (PCI) from 2006 to 2008. Blood samples were drawn immediately before PCI. Additionally, we included 42 patients who had 4 blood samples drawn before and after PCI. Plasma NGAL was measured using a time-resolved immunofluorometric assay. Cross-sectional analyses were performed in these two single-center, prospective study cohorts. RESULTS: Estimated glomerular filtration rate (eGFR) was associated significantly more strongly with plasma NGAL when eGFR was abnormal compared to normal eGFR: a decrease in eGFR of 10 ml/min was associated with an increase in NGAL of 27% (18-36%) versus 4% (1-7%), respectively (p < 0.001). Leukocyte count and C-reactive protein were the main determinants of plasma NGAL in patients with normal eGFR, whereas eGFR was the main determinant at reduced kidney function. CONCLUSIONS: eGFR determines the association of NGAL with either inflammation or kidney function; in patients with normal eGFR, plasma NGAL reflects inflammation but when eGFR is reduced, plasma NGAL reflects kidney function, highlighting the dual perception of plasma NGAL. From a clinical perspective, eGFR may be used to guide the interpretation of elevated NGAL levels in patients with STEMI.

Anti-CD163-dexamethasone conjugate inhibits the acute phase response to lipopolysaccharide in rats.

AIM: To study the effect of a new anti-CD163-dexamethasone conjugate targeting activated macrophages on the hepatic acute phase response in rats. METHODS: Wistar rats were injected intravenous with either the CD163 targeted dexamethasone-conjugate (0.02 mg/kg) or free dexamethasone (0.02 or 1 mg/kg) 24 h prior to lipopolysaccharide (LPS) (2.5 mg/kg intraperitoneal). We measured plasma concentrations of tumour necrosis factor-α (TNF-α) and interleukin 6 (IL-6) 2 h post-LPS and liver mRNAs and serum concentrations of the rat acute phase protein α-2-macroglobulin (α-2-M) 24 h after LPS. Also, plasma concentrations of alanine aminotransferase and bilirubin were measured at termination of the study. Spleen weight served as an indicator of systemic steroid effects. RESULTS: The conjugate halved the α-2-M liver mRNA (3.3 ± 0.6 vs 6.8 ± 1.1, P < 0.01) and serum protein (201 ± 48 µg/mL vs 389 ± 67 µg/mL, P = 0.04) after LPS compared to low dose dexamethasone treated animals, while none of the free dexamethasone doses had an effect on liver mRNA or serum levels of α-2-M. Also, the conjugate reduced TNF-α (7208 ± 1977 pg/mL vs 21583 ± 7117 pg/mL, P = 0.03) and IL-6 (15685 ± 3779 pg/mL vs 25715 ± 4036 pg/mL, P = 0.03) compared to the low dose dexamethasone. The high dose dexamethasone dose decreased the spleen weight (421 ± 11 mg vs 465 ± 12 mg, P < 0.05) compared to controls, an effect not seen in any other group. CONCLUSION: Low-dose anti-CD163-dexamethasone conjugate effectively decreased the hepatic acute phase response to LPS. This indicates an anti-inflammatory potential of the conjugate in vivo.

Identifications of novel mechanisms in breast cancer cells involving duct-like multicellular spheroid formation after exposure to the Random Positioning Machine.

Many cell types form three-dimensional aggregates (MCS; multicellular spheroids), when they are cultured under microgravity. MCS often resemble the organ, from which the cells have been derived. In this study we investigated human MCF-7 breast cancer cells after a 2 h-, 4 h-, 16 h-, 24 h- and 5d-exposure to a Random Positioning Machine (RPM) simulating microgravity. At 24 h few small compact MCS were detectable, whereas after 5d many MCS were floating in the supernatant above the cells, remaining adherently (AD). The MCS resembled the ducts formed in vivo by human epithelial breast cells. In order to clarify the underlying mechanisms, we harvested MCS and AD cells separately from each RPM-culture and measured the expression of 29 selected genes with a known involvement in MCS formation. qPCR analyses indicated that cytoskeletal genes were unaltered in short-term samples. IL8, VEGFA, and FLT1 were upregulated in 2 h/4 h AD-cultures. The ACTB, TUBB, EZR, RDX, FN1, VEGFA, FLK1 Casp9, Casp3, PRKCA mRNAs were downregulated in 5d-MCS-samples. ESR1 was upregulated in AD, and PGR1 in both phenotypes after 5d. A pathway analysis revealed that the corresponding gene products are involved in organization and regulation of the cell shape, in cell tip formation and membrane to membrane docking.

Pathways Regulating Spheroid Formation of Human Follicular Thyroid Cancer Cells under Simulated Microgravity Conditions: A Genetic Approach.

Microgravity induces three-dimensional (3D) growth in numerous cell types. Despite substantial efforts to clarify the underlying mechanisms for spheroid formation, the precise molecular pathways are still not known. The principal aim of this paper is to compare static 1g-control cells with spheroid forming (MCS) and spheroid non-forming (AD) thyroid cancer cells cultured in the same flask under simulated microgravity conditions. We investigated the morphology and gene expression patterns in human follicular thyroid cancer cells (UCLA RO82-W-1 cell line) after a 24 h-exposure on the Random Positioning Machine (RPM) and focused on 3D growth signaling processes. After 24 h, spheroid formation was observed in RPM-cultures together with alterations in the F-actin cytoskeleton. qPCR indicated more changes in gene expression in MCS than in AD cells. Of the 24 genes analyzed VEGFA, VEGFD, MSN, and MMP3 were upregulated in MCS compared to 1g-controls, whereas ACTB, ACTA2, KRT8, TUBB, EZR, RDX, PRKCA, CAV1, MMP9, PAI1, CTGF, MCP1 were downregulated. A pathway analysis revealed that the upregulated genes code for proteins, which promote 3D growth (angiogenesis) and prevent excessive accumulation of extracellular proteins, while genes coding for structural proteins are downregulated. Pathways regulating the strength/rigidity of cytoskeletal proteins, the amount of extracellular proteins, and 3D growth may be involved in MCS formation.

Mechanisms of three-dimensional growth of thyroid cells during long-term simulated microgravity.

Three-dimensional multicellular spheroids (MCS) of human cells are important in cancer research. We investigated possible mechanisms of MCS formation of thyroid cells. Both, normal Nthy-ori 3-1 thyroid cells and the poorly differentiated follicular thyroid cancer cells FTC-133 formed MCS within 7 and 14 days of culturing on a Random Positioning Machine (RPM), while a part of the cells continued to grow adherently in each culture. The FTC-133 cancer cells formed larger and numerous MCS than the normal cells. In order to explain the different behaviour, we analyzed the gene expression of IL6, IL7, IL8, IL17, OPN, NGAL, VEGFA and enzymes associated cytoskeletal or membrane proteins (ACTB, TUBB, PFN1, CPNE1, TGM2, CD44, FLT1, FLK1, PKB, PKC, ERK1/2, Casp9, Col1A1) as well as the amount of secreted proteins (IL-6, IL-7, IL-8, IL-17, OPN, NGAL, VEGFA). Several of these components changed during RPM-exposure in each cell line. Striking differences between normal and malignant cells were observed in regards to the expression of genes of NGAL, VEGFA, OPN, IL6 and IL17 and to the secretion of VEGFA, IL-17, and IL-6. These results suggest several gravi-sensitive growth or angiogenesis factors being involved in 3D formation of thyroid cells cultured under simulated microgravity.

PAPP-A proteolytic activity enhances IGF bioactivity in ascites from women with ovarian carcinoma.

Pregnancy-associated plasma protein-A (PAPP-A) stimulates insulin-like growth factor (IGF) action through proteolysis of IGF-binding protein (IGFBP)-4. In experimental animals, PAPP-A accelerates ovarian tumor growth by this mechanism. To investigate the effect of PAPP-A in humans, we compared serum and ascites from 22 women with ovarian carcinoma. We found that ascites contained 46-fold higher PAPP-A levels as compared to serum (P < 0.001). The majority (80%) of PAPP-A was enzymatically active. This is supported by the finding that ascites contained more cleaved than intact IGFBP-4 (P < 0.03). Ascites was more potent than serum in activating the IGF-I receptor (IGF-IR) in vitro (+31%, P < 0.05); in 8 of 22 patients by more than two-fold. In contrast, ascites contained similar levels of immunoreactive IGF-I, and lower levels of IGF-II (P < 0.001). Immunohistochemistry demonstrated the presence of IGF-IR in all but one tumor, whereas all tumors expressed PAPP-A, IGFBP-4, IGF-I and IGF-II. Addition of recombinant PAPP-A to ascites increased the cleavage of IGFBP-4 and enhanced IGF-IR activation (P < 0.05). In conclusion, human ovarian tumors express PAPP-A, IGFBP-4 and IGFs and these proteins are also present in ascites. We suggest that both soluble PAPP-A in ascites and tissue-associated PAPP-A serve to increase IGF bioactivity and, thereby, to stimulate IGF-IR-mediated tumor growth.

Reduced CD300LG mRNA tissue expression, increased intramyocellular lipid content and impaired glucose metabolism in healthy male carriers of Arg82Cys in CD300LG: a novel genometabolic cross-link between CD300LG and common metabolic phenotypes.

BACKGROUND: CD300LG rs72836561 (c.313C>T, p.Arg82Cys) has in genetic-epidemiological studies been associated with the lipoprotein abnormalities of the metabolic syndrome. CD300LG belongs to the CD300-family of membrane-bound molecules which have the ability to recognize and interact with extracellular lipids. We tested whether this specific polymorphism results in abnormal lipid accumulation in skeletal muscle and liver and other indices of metabolic dysfunction. METHODS: 40 healthy men with a mean age of 55 years were characterized metabolically including assessment of insulin sensitivity by the hyperinsulinemic euglycemic clamp, intrahepatic lipid content (IHLC) and intramyocellular lipid content (IMCL) by MR spectroscopy, and ß-cell function by an intravenous glucose tolerance test. Changes in insulin signaling and CD300LG mRNA expression were determined by western blotting and quantitative PCR in muscle and adipose tissue. RESULTS: Compared with the 20 controls (CC carriers), the 20 CT carriers (polymorphism carriers) had higher IMCL (p=0.045), a reduced fasting forearm glucose uptake (p=0.011), a trend toward lower M-values during the clamp; 6.0 mg/kg/min vs 7.1 (p=0.10), and higher IHLC (p=0.10). CT carriers had lower CD300LG mRNA expression and CD300LG expression in muscle correlated with IMCL (ß=-0.35, p=0.046), forearm glucose uptake (ß=0.37, p=0.03), and tended to correlate with the M-value (ß=0.33, p=0.06), independently of CD300LG genotype. ß-cell function was unaffected. CONCLUSIONS: The CD300LG polymorphism was associated with decreased CD300LG mRNA expression in muscle and adipose tissue, increased IMCL, and abnormalities of glucose metabolism. CD300LG mRNA levels correlated with IMCL and forearm glucose uptake. These findings link a specific CD300LG polymorphism with features of the metabolic syndrome suggesting a role for CD300LG in the regulation of common metabolic traits. TRIAL REGISTRATION NUMBER: NCT01571609.

Anti-Angiogenic Drugs in the Treatment of Metastatic Renal Cell Carcinoma: Advances in Clinical Application.

The current paradigm in attempting to treat metastatic renal cell carcinoma (mRCC) is a first line treatment with a vascular endothelial growth factor (VEGF) antagonist and second and subsequent treatments with either a vascular endothelial growth factor receptor (VEGFR) or an mTOR (mammalian Target of Rapamycin) inhibitor, while conventional chemotherapeutic and hormonal treatments do not play a role in the management of mRCC. Several drugs directed against VEGF and VEGFR have been developed in recent times. Phase III data validates sunitinib, pazopanib and sorafenib as the best-supported drugs in firstline therapy. Second-line treatment possibilities include axitinib, everolimus and sorafenib. Choosing the right combination of first and second line treatments, however, is difficult, because the success of treatment depends on the precondition of the patient. Hence, biomarkers indicating the best choice of therapy in individual patients are searched and newer trials are set to determine the role of surgery and vaccination together with anti-angiogenic drugs in the treatment of mRCC. In this review, current guidelines in mRCC management are summarized and possibilities of future personalized therapies are pointed out.

Anti-vascular endothelial growth factor therapy in breast cancer.

Neo-angiogenesis is a critical process for tumor growth and invasion and has become a promising target in cancer therapy. This manuscript reviews three currently relevant anti-angiogenic agents targeting the vascular endothelial growth factor system: bevacizumab, ramucirumab and sorafenib. The efficacy of anti-angiogenic drugs in adjuvant therapy or as neo-adjuvant treatment has been estimated in clinical trials of advanced breast cancer. To date, the overall observed clinical improvements are unconvincing, and further research is required to demonstrate the efficacy of anti-angiogenic drugs in breast cancer treatments. The outcomes of anti-angiogenic therapy have been highly variable in terms of tumor response. New methods are needed to identify patients who will benefit from this regimen. The development of biomarkers and molecular profiling are relevant research areas that may strengthen the ability to focus anti-angiogenic therapy towards suitable patients, thereby increase the cost-effectiveness, currently estimated to be inadequate.

Plasma neutrophil gelatinase-associated lipocalinin in the general population: association with inflammation and prognosis.

OBJECTIVE: Neutrophil gelatinase-associated lipocalin (NGAL) is a glycoprotein stored in granules of neutrophil leukocytes participating in inflammatory and atherosclerotic processes and possibly plaque rupture. Despite the putative role of NGAL in atherosclerosis and acute myocardial infarction, human studies of plasma NGAL are still limited. APPROACH AND RESULTS: We prospectively followed 5599 randomly selected men and women from the community in the fourth Copenhagen Heart Study. Plasma NGAL was measured at study entry. Participants were followed for 10 years. During follow-up, 20% died (n=1120) and 15% (n=884) developed a major adverse cardiovascular event. Plasma NGAL associated strongly with all inflammatory markers (high-sensitivity C-reactive protein, total leukocyte count, neutrophil count) and inversely with estimated glomerular filtration rate (all, P<0.001). Multivariate analysis identified neutrophil leukocyte count as the main determinant of plasma NGAL. During follow-up, participants with increasing NGAL had increased risk of all-cause mortality and major adverse cardiovascular event (both, P<0.001). Even after adjustment for confounding risk factors by Cox regression analysis, NGAL remained an independent predictor of both all-cause mortality and major adverse cardiovascular event. When added to the Framingham risk score, NGAL improved c-statistics and correctly reclassified ≈15% into more appropriate risk groups. In comparison with high-sensitivity C-reactive protein, when both markers were added to the Framingham risk score, NGAL conferred 3× to 4× the risk. CONCLUSIONS: Plasma NGAL is strongly associated with inflammation in the general population. NGAL independently associated with 10-year outcome, and when added to the Framingham risk score, NGAL both improves c-statistics and correctly reclassifies participants into more accurate risk categories.

Growing tissues in real and simulated microgravity: new methods for tissue engineering.

Tissue engineering in simulated (s-) and real microgravity (r-µg) is currently a topic in Space medicine contributing to biomedical sciences and their applications on Earth. The principal aim of this review is to highlight the advances and accomplishments in the field of tissue engineering that could be achieved by culturing cells in Space or by devices created to simulate microgravity on Earth. Understanding the biology of three-dimensional (3D) multicellular structures is very important for a more complete appreciation of in vivo tissue function and advancing in vitro tissue engineering efforts. Various cells exposed to r-µg in Space or to s-µg created by a random positioning machine, a 2D-clinostat, or a rotating wall vessel bioreactor grew in the form of 3D tissues. Hence, these methods represent a new strategy for tissue engineering of a variety of tissues, such as regenerated cartilage, artificial vessel constructs, and other organ tissues as well as multicellular cancer spheroids. These aggregates are used to study molecular mechanisms involved in angiogenesis, cancer development, and biology and for pharmacological testing of, for example, chemotherapeutic drugs or inhibitors of neoangiogenesis. Moreover, they are useful for studying multicellular responses in toxicology and radiation biology, or for performing coculture experiments. The future will show whether these tissue-engineered constructs can be used for medical transplantations. Unveiling the mechanisms of microgravity-dependent molecular and cellular changes is an up-to-date requirement for improving Space medicine and developing new treatment strategies that can be translated to in vivo models while reducing the use of laboratory animals.

Mechanisms of apoptosis in irradiated and sunitinib-treated follicular thyroid cancer cells.

The multikinase inhibitor sunitinib (S) seems to have promising potential in the treatment of thyroid cancer. We focused on the impact of S and/or irradiation (R) on mechanisms of apoptosis in follicular thyroid cancer cells. The effects of R, S and their combination were evaluated 2 and 4 days after treatment, using the human thyroid cancer cell line CGTH W-1. The transcription of genes involved in the regulation of apoptosis was investigated using quantitative real-time PCR. Western blot analyses of caspases and survivin were also performed. S elevated BAX (day 4), CASP9, CASP3, BIRC5 (day 4) and PRKACA (day 4) gene expression, whereas the mRNAs of BCL2, CASP8, PRKCA, ERK1, and ERK2 were not significantly changed. S, R and R+S clearly induced caspase-9 protein and elevated caspase-3 activity. Survivin was down-regulated at day 4 in control cells and the expression was blunted by S treatment. R+S induced survivin expression at day 2 followed by a reduction at day 4 of treatment. Sunitinib and the combined application with radiation induced apoptosis in follicular thyroid cancer cells via the intrinsic pathway of apoptosis. In addition, sunitinib might induce apoptosis via decreased expression of the anti-apoptotic protein survivin. These findings suggest the potential use of sunitinib for the treatment of poorly differentiated follicular thyroid carcinomas.