Exploratory investigation of eight circulating plasma markers in brain tumor patients.

Several blood biomarkers have been established for the early diagnosis, screening and follow-up of non central nervous system cancers. However, there is lack of knowledge on biochemical blood alterations in brain tumor patients. In this study, we prospectively collected blood plasma samples of 105 adult brain tumor patients with diffuse low-grade glioma (World Health Organization (WHO) II, n = 7), anaplastic glioma (WHO III, n = 10), glioblastoma multiforme (WHO IV, glioblastoma multiforme (GBM)) (n = 34), meningioma (WHO I, n = 8), atypical meningioma (WHO II, n = 5), and intracerebral metastasis (ICM; n = 41). In each case, we measured plasma concentrations of neuropeptide Y, brain-derived neurotrophic factor, glial cell line-derived neurotrophic factor, placental growth factor (PlGF), S100B, secretagogin, interleukin 8, and glial fibrillary acidic protein (GFAP) using enzyme-linked immunosorbent assay. Plasma marker concentrations were correlated to patient parameters including neuropathological diagnosis and neuroradiological features. Most of the markers were detectable in all diagnostic categories in variable concentrations. GFAP plasma detectability was strongly associated with a diagnosis of GBM (p < 0.001). Plasma GFAP and plasma placental growth factor showed promising moderate potential in the differential diagnosis of unifocal GBM versus unifocal supratentorial ICM (area under the curve = 0.73, p < 0.05). To summarize, our data show that none of the investigated markers is suitable to substitute histological diagnosis. However, measurement of circulating GFAP and PlGF may support neuroradiological differential diagnosis of GBM versus ICM.

CXCL12/SDF-1 over-expression in human insulinomas and its biological relevance.

This study was performed on the basis of previously obtained investigative gene array data concerning the over-expression of CXCL12/SDF-1 in human insulinomas versus human pancreatic islet preparations. The presence of CXCL12/SDF-1 was studied by RT-qPCR in human insulinomas (n=8) versus pancreatic islets (n=3), and was found to be significantly up-regulated in the former (p<0.012). The mRNA data were confirmed by immunostaining and confocal microscopy of human normal pancreatic islets, which showed the absence of CXCL12 protein and high expression in insulinoma tissue. Individual human insulinoma cells at cytospins stained positive for CXCL12 in the paranuclear region. These morphological data were extended by consecutive immunoblotting for cell-compartment-specific marker proteins of fractions obtained by sucrose gradient fractionation using Rin-5F insulinoma cells. CXCL12-containing fractions were positive for the membrane marker NSF but negative for SNAP-25 and appeared at a lighter density in the gradient than heavy insulin granules, suggesting packaging in specific granules different from insulin. In order to determine the biological relevance of the protein in insulinomas, we investigated the colony-forming potential of human CXCL12 stable-transfected rat Rin-5F insulinoma cells. These clones secreted human CXCL12 and contained 50-1000-fold higher copy numbers compared to its endogenous rat homologue. In colony-forming assays, these transfectant clones developed greater colony numbers, which were larger than wild-type and sham transfectants. To elucidate the mechanism of action, we identified a CXCL12 transfectant-specific increase in the pro-survival factor Mn-SOD, which is considered important for the inactivation of reactive oxygen species, thereby prolonging cell survival. These data demonstrate the importance of CXCL12 in the tumor biology of insulinoma.

Angiogenic factors in plasma of brain tumour patients.

BACKGROUND: Angiopoiesis and angiopoietic growth factors are of considerable importance in the development and progression of intracranial tumours. However, knowledge of the plasma detectability of distinct angiogenic factors in patients with brain tumour is very limited. This study evaluates the plasma concentrations of the angiogenic factors angiopoietin-2 (Ang-2), vascular endothelial growth factor (VEGF) and platelet-derived growth factor BB (PDGF-BB) in patients with brain tumour. PATIENTS AND METHODS: Plasma samples of 78 patients suffering from various types of intracranial tumours (glioblastoma multiforme, GBM, n = 22; astrocytoma, n = 12; meningioma, n = 16; and intracranial metastasis, n = 28) were analysed. For determination of plasma concentrations of angiogenic factor, highly specific enzyme-linked immuno sorbent assays (ELISAs) were used. RESULTS: Ang-2 plasma concentration in GBM patients was significantly lower when compared with that in patients with meningioma and intracranial metastasis. Highest levels of VEGF concentrations were detected in plasma derived from patients suffering from meningioma. Interestingly, VEGF plasma levels depended on the number of intracranial lesions, with significantly higher concentrations in patients with 3 or more lesions when compared with those with 2 or fewer lesions. However, no correlation between the survival time of the patients and the plasma levels of the tested growth factors was obtained. Plasma levels of PDGF-BB did not differ between the individual tumour groups. CONCLUSION: The detectability of the angiogenic factors Ang-2 and VEGF, as well as of PDGF-BB, in the plasma of patients suffering from various types of brain tumours is described. The plasma detectability of the individual angiopoetic factors seems to depend at least partly on the tumour type as well as on tumour progression. This might be of prognostic and therapeutic relevance.

Localization and characterization of the novel protein encoded by C20orf3.

In the present study, we characterized the gene product of open reading frame 3 encoded at human chromosome 20 (C20orf3), which represents a member of the lactonohydrolase super family. Multiple-tissue Northern blot analysis showed ubiquitous expression of the 2.4 kb transcript coding for 416 amino acids, with highest levels in human liver, placenta and kidney. After recombinant production of protein variants in Escherichia coli and insect cells, antibodies directed against different epitopes within the C20orf3 gene product were generated. Using these immunoreagents, protein expression was demonstrated in the liver, and glomerular and tubular structures of the kidney, as well as in endothelial cells and arterial wall. Positive staining was also observed at the pancreatic islets of Langerhans. Using immunoblotting, we identified three size variants. In line with the results of in silico analysis demonstrating a single transmembrane sequence (amino acids 40-61) at the N-terminus of the full-length protein, FACS cell-surface staining confirmed a mainly extracellular localization of the full-length protein. Sucrose density gradient cell fractionation revealed membrane association of the dominant 50 kDa variant in HepG2 and Rin-5F cells. The finding of a strong arylesterase activity with beta-naphthyl acetate and phenyl acetate of the C20orf3 protein-containing fractions suggests potential involvement of this protein in enzymatic processes. C20orf3 promoter-driven reporter assays, which were verified by gene-specific RT-qPCR (real-time quantitative PCR) showed a strong inhibitory effect of human serum on transcription using the HEK-293 human embryonic kidney cell line. In conclusion, we characterized the structure and expression pattern of the C20orf3 gene product. According to a series of analogies with PON (paraoxonase) family members, we speculate that the C20orf3 gene product represents a new member of this important protein family present at the cellular level.

The Microtubule-Associated Protein Tau and Its Relevance for Pancreatic Beta Cells.

Structural and biochemical alterations of the microtubule-associated protein tau (MAPT) are associated with degenerative disorders referred to as tauopathies. We have previously shown that MAPT is present in human islets of Langerhans, human insulinomas, and pancreatic beta-cell line models, with biophysical similarities to the pathological MAPT in the brain. Here, we further studied MAPT in pancreatic endocrine tissue to better understand the mechanisms that lead to functional dysregulation of pancreatic beta cells. We found upregulation of MAPT protein expression in human insulinomas when compared to human pancreatic islets of Langerhans and an imbalance between MAPT isoforms in insulinomas tissue. We cloned one 3-repeat domain MAPT and transduced this into a beta-cell derived rodent cell line Rin-5F. Proliferation experiments showed higher growth rates and metabolic activities of cells overexpressing MAPT protein. We observed that a MAPT overexpressing cell line demonstrates altered insulin transcription, translation, and insulin secretion rates. We found the relative insulin secretion rates were significantly decreased in a MAPT overexpressing cell line and these findings could be confirmed using partial MAPT knock-down cell lines. Our findings support that MAPT may play an important role in insulin granule trafficking and indicate the importance of balanced MAPT phosphorylation and dephosphorylation for adequate insulin release.

Pneumocystis carinii in a patient with hypercalcemia and renal failure secondary to sarcoidosis.

A case of severe dyspnea, hypercalcemia and renal failure secondary to sarcoidosis is reported. The clinical diagnosis of sarcoidosis in a 48-year-old man was confirmed by histology and cytology. Transiently decreased numbers of CD4+ T cells (282/microliter) indicated impaired immunity in the absence of HIV-infection during the acute phase of the disease. Surprisingly, numerous "trophozoites" of Pneumocystis carinii were detected by immunofluorescence staining and PCR in the bronchoalveolar fluid indicating infection or colonization of the lungs. Corticosteroid therapy was administered together with trimethoprim-sulfamethoxazole and rapidly reduced elevated serum calcium and creatinine concentrations. Since airborne person-to-person transmission of P. carinii to susceptible individuals might be possible, patients with sarcoidosis could be a previously unrecognized reservoir for P. carinii distribution in hospitals and in the community at large.

Expression of secretagogin in clear-cell renal cell carcinomas is associated with a high metastasis rate.

Renal cell carcinomas are divided into several subgroups according to their histopathologic characteristics. The outcome, therapy responses, and the applicability of molecular-targeted therapies depend on the tumor classification and on the tumor stage. Recent advances within the biomarker research facilitated the exact classification of the molecular character of the renal tumor. For example, the calcium-binding proteins parvalbumin and S-100A1 are characteristically expressed in renal cell carcinoma subgroups. This led us to investigate the expression of the novel calcium-binding protein secretagogin in renal cell carcinomas. Tissue microarray cylinders including 94 clear-cell renal cell carcinomas, 61 non-clear-cell renal cell carcinomas (37 papillary renal cell and 24 chromophobe carcinomas), and 30 oncocytomas were analyzed by immunohistochemistry. This showed remarkable secretagogin expression in 37% of the clear-cell renal cell carcinomas. Non-clear-cell renal cell carcinomas and oncocytomas were completely negative. Consequently performed immunoblotting analyses confirmed this expression profile. Because publicly available data direct toward a formation of a hierarchical cluster of secretagogin overexpressing clear-cell renal cell carcinomas, we conducted a clinical follow-up of the patients with clear-cell renal cell carcinoma. This revealed significantly more metastasis within the secretagogin-positive clear-cell renal cell carcinoma subgroup (49% versus 28%; P < .05). In conclusion, we report on detection of the novel calcium-binding protein secretagogin within a subgroup of clear-cell renal cell carcinomas. The increased metastasis rates within the secretagogin-positive subgroup of clear-cell renal cell carcinomas direct toward a clinical impact of our findings.

Age related changes in pancreatic beta cells: A putative extra-cerebral site of Alzheimer's pathology.

Frequent concomitant manifestation of type 2 diabetes mellitus (T2DM) and Alzheimer's disease (AD) has been recently demonstrated by epidemiological studies. This might be due to functional similarities between ß-cells and neurons, such as secretion on demand of highly specific molecules in a tightly controlled fashion. An additional similarity represents the age-related alteration of hyperphosphorylated tau in AD patients. Similarly, alterations have been identified in ß-cells of T2DM patients. The islet amyloid polypeptide has been associated with ß-cell apoptosis. As a consequence of increasing age, the accumulation of highly modified proteins together with decreased regenerative potential might lead to increasing rates of apoptosis. Moreover, reduction of ß-cell replication capabilities results in reduction of ß-cell mass in mammals, simultaneously with impaired glucose tolerance. The new challenge is to learn much more about age-related protein modifications. This can lead to new treatment strategies for reducing the incidence of T2DM and AD.

Plasma neuropeptide Y levels differ in distinct diabetic conditions.

Neuropeptide Y (NPY) is an important hormone in appetite regulation. Although the contribution of NPY to metabolic disease has been previously demonstrated, there are only a few reports addressing NPY plasma levels under distinct diabetic conditions. In this study we evaluated NPY plasma levels in diabetes mellitus type 2 (DM2) patients with (n=34) and without (n=34) diabetic polyneuropathy (PNP) and compared these with age and gender matched healthy controls (n=34). We also analyzed NPY plasma levels in gestational diabetes mellitus (GDM) patients with age and pregnancy-week matched controls with normal glucose tolerance (NGT). NPY concentration was determined using a commercially available radioimmunoassay kit. In addition, metabolic parameters of DM2 and GDM patients were recorded. One-way ANOVA tests with appropriate post hoc corrections showed elevated levels of NPY in DM2 patients with and without PNP when compared with those of healthy controls (122.32±40.86 and 117.33±29.92 vs. 84.65±52.17 pmol/L; p<0.001, p<0.005, respectively). No significant difference was observed between diabetic patients with and without PNP. The NPY levels were similar in the GDM group and in pregnant women with NGT (74.87±14.36 vs. 84.82±51.13 pmol/L, respectively). Notably, the NPY concentration correlated positively with insulin levels in DM2 patients (R=0.35, p<0.01). Our data suggest a potential involvement of circulating NPY in DM2 pathology.

Elevated blood markers 1 year before manifestation of malignant glioma.

We detected distinct plasma concentration profiles of S100B, neuropeptide Y, and secretagogin in 3 of 191 patients enrolled in a previous study investigating brain-tissue-related markers in the blood of patients with atrial fibrillation. Intriguingly, 2 of these 3 patients, both of whom were without neurological symptoms at the time of blood sampling, were diagnosed with malignant glioma (MG) approximately 1 year later. To our knowledge, this is the first report indicating that distinct blood biomarker profiles may be detected long before clinical manifestation of MG.

Brain natriuretic peptide correlates with the extent of atrial fibrillation-associated silent brain lesions.

OBJECTIVE: Identification of plasma markers indicative for atrial fibrillation-associated silent brain lesions. DESIGN AND METHODS: 1. Comparative determination of the plasma concentrations of secretagogin, S100B, neuropeptide Y, brain fatty acid binding protein, matrix metalloprotease 9, brain natriuretic peptide, and of D-Dimer in 222 patients with atrial fibrillation and 28 controls by immunoassays. 2. Correlation of the biochemical marker plasma concentration with the extent of silent white matter brain lesions, as determined by the Fazekas score and N-acetylaspartate-spectroscopy. RESULTS: 1. Plasma concentrations of brain natriuretic peptide, of neuropeptide Y, and of matrix metalloprotease 9 were significantly higher (all with a p<0.05) in patients suffering from atrial fibrillation than in control subjects. 2. Brain natriuretic peptide correlated significantly with the Fazekas score (R=0.41; p<0.005). 3. Brain natriuretic peptide plasma concentrations were significantly higher in patients with a pathological N-acetylaspartate magnetic resonance-spectrometry (p<0.05). CONCLUSION: Brain natriuretic peptide plasma concentrations correlate with the extent of atrial fibrillation-associated silent brain lesions.

The ATP-dependent helicase RUVBL1/TIP49a associates with tubulin during mitosis.

RUVBL1/TIP49a/Pontin52 is a recently identified multi-functional protein with 2 ATP binding (WALKER) sites, which is essential for cell proliferation. We recovered and identified RUVBL1/TIP49a as a tubulin-binding protein from Triton X-100 lysates of U937 promonocytic cells by protein affinity chromatography and tryptic peptide microsequencing. Performing co-immunoprecipitation using newly generated RUVBL1/TIP49a-specific antibodies (mAb and rabbit polyclonal Ab) and RUVBL1/TIP49a-GST fusion protein-pull down assays we demonstrate co-precipitation of alpha- and gamma tubulin with RUVBL1/TIP49a. Confocal immunoflourescence microscopy reveals that RUVBL1/TIP49a was present not only in the nucleus, as expected, but was also concentrated at the centrosome and at the mitotic spindle in colocalization with tubulin. The topology of RUVBL1/TIP49a at the mitotic spindle varied, depending on the mitotic stage. The protein was localized at the centrosome and at the polar and astral microtubules in metaphase, and was detectable at the zone of polar tubule interdigitation in anaphase B and telophase. During cytokinesis the protein reappeared at the area of decondensing chromosomes. Whereas preincubation of U937 cells with colcemid resulted in inhibition of mitotic spindle formation with subsequent loss of RUVBL1/TIP49a mitotic spindle staining, no relevant influence of colcemid on RUVBL1/TIP49a-tubulin binding was observed. An agonistic effect of RUVBL1/TIP49a on in vitro tubulin assembly is demonstrated. Our results reveal a new functional aspect of RUVBL1/TIP49a.