Human epididymis protein 4 reference limits and natural variation in a Nordic reference population

The objectives of this study are to establish reference limits for human epididymis protein 4, HE4, and investigate factors influencing HE4 levels in healthy subjects. HE4 was measured in 1,591 samples from the Nordic Reference Interval Project Bio-bank and Database biobank, using the manual HE4 EIA (Fujirebio) for 802 samples and the Architect HE4 (Abbott) for 792 samples. Reference limits were calculated using the statistical software R. The influence of donor characteristics such as age, sex, body mass index, smoking habits, and creatinine on HE4 levels was investigated using a multivariate model. The study showed that age is the main determinant of HE4 in healthy subjects, corresponding to 2% higher HE4 levels at 30 years (compared to 20 years), 9% at 40 years, 20% at 50 years, 37% at 60 years, 63% at 70 years, and 101% at 80 years. HE4 levels are 29% higher in smokers than in nonsmokers. In conclusion, HE4 levels in healthy subjects are associated with age and smoking status. Age-dependent reference limits are suggested

Effects of hypoxia on angiogenesis and angiogenic factors in crucian carp brain

Most vertebrates depend on an uninterrupted supply of oxygen to maintain the energy production. Some species, like the crucian carp (Carassius carassius) can survive hypoxia for an extended period. This fish is an expert in acquiring the little oxygen that exists in hypoxic water, and can boost its anaerobic ATP production by up-regulating the glycolysis and convert lactate to ethanol. Angiogenesis, the formation of new blood vessels, is induced by hypoxia and controlled through several factors. One of the most important hypoxia driven transcription factors, the hypoxia-inducible factor (HIF)-a, has been found to play a major role in coordinating many adaptive responses to hypoxia. It is continuously synthesized and degraded in normoxic conditions, but accumulates at low oxygen tension. Several factors control the expression of HIF-a, among them the factor-inhibiting HIF-1 (FIH-1). One mechanism by which HIF-a mediates increased oxygen delivery is through inducing the expression of vascular endothelial growth factor (VEGF), which in turn stimulates the formation of blood vessels. In this thesis mRNA levels of HIF-1a, HIF-2a, FIH, VEGFA, VEGFC and VEGFD were quantified by real-time RT-PCR in brain tissue from the crucian carp exposed to hypoxia. The effect of hypoxia on vascular density in crucian carp brain was also studied. A significant increase in mRNA levels was seen in both VEGFA and VEGFC, while no change was seen in VEGFD. VEGFA increased 6-fold and constituted the largest part of the quantified VEGF expression. Different splice isoforms of VEGFA have previously been characterized in mammals and fish, and two of these splice isoforms were cloned in the crucian carp, VEGFA121 and VEGFA165. A trend towards a reduction in VEGFD expression was observed in hypoxia, which may indicate that it does not have the same function in angiogenesis in crucian carp as VEGFA and VEGFC. The expression level of FIH increased 4-fold in hypoxia. No changes in mRNA levels were seen in either HIF-1a or HIF-2a, but a trend towards reduction was seen in HIF-1a. The increase seen in VEGFA and FIH indicates that the fishes have been hypoxic. This might imply that the expression of HIF-a also increases, but in order to verify this protein analyses would have to be performed to verify this. Regarding blood vessel density two parameteres were measured, blood vessel surface area per unit volume and blood vessel length per unit volume. A significant decrease was seen in both of these parameters. The increase in VEGFA expression along with a reduction in blood vessel density can indicate that the hypoxic crucian carp does not require additional blood vessels during hypoxia, but instead suppresses global HIF-a effects, which may work well for local regulation of vascularization, but not for adapting the whole organism to hypoxia

Specific antibodies and sensitive immunoassays for the human epidermal growth factor receptors (HER2, HER3, and HER4)

The use of trastuzumab in patients with breast cancer that overexpresses human epidermal growth factor receptor 2 has significantly improved treatment outcomes. However, a substantial proportion of this patient group still experiences progression of the disease after receiving the drug. Evaluation of the changes in expression of the human epidermal growth factor receptors could be of interest. Monoclonal antibodies against the extracellular domain of the human growth factor receptors, 2, 3, and 4, have been raised, and specific and sensitive immunoassays have been established. Sera from healthy individuals (Nordic Reference Interval Project and Database) were analyzed in the human epidermal growth factor receptor 2 assay (N = 805) and the human epidermal growth factor receptor 3 and 4 assays (N = 114), and reference limits were calculated. In addition, sera from 208 individual patients with breast cancer were tested in all three assays. Finally, the human epidermal growth factor receptor 2 assay was compared with a chemiluminescent immunoassay for serum human epidermal growth factor receptor 2/neu. Reference values were as follows: human epidermal growth factor receptor 2, <2.5 µg/L; human epidermal growth factor receptor 3, <2.8 µg/L; and human epidermal growth factor receptor 4, <1.8 µg/L. There were significant differences in human epidermal growth factor receptor 2 and human epidermal growth factor receptor 3 serum levels between the patients with tissue human epidermal growth factor receptor 2–positive and tissue human epidermal growth factor receptor 2–negative (p = 0.0026, p = 0.000011) tumors, but not in the serum levels of human epidermal growth factor receptor 4 (p = 0.054). There was good agreement between the in-house human epidermal growth factor receptor 2 assay and the chemiluminescent immunoassay. Our new specific antibodies for all the three human epidermal growth factor receptors may prove valuable in the development of novel anti-human epidermal growth factor receptor targeted therapies with sensitive immunoassays for measuring serum levels of the respective targets and in monitoring established treatment

Osteopontin is a prognostic biomarker in non-small cell lung cancer

Background: In a previously published report we characterized the expression of the metastasis-associated proteins S100A4, osteopontin (OPN) and ephrin-A1 in a prospectively collected panel of non-small cell lung cancer (NSCLC) tumors. The aim of the present follow-up study was to investigate the prognostic impact of these potential biomarkers in the same patient cohort. In addition, circulating serum levels of OPN were measured and single nucleotide polymorphisms (SNP) in the −443 position of the OPN promoter were analyzed. Methods: Associations between immunohistochemical expression of S100A4, OPN and ephrin-A1 and relapse free and overall survival were examined using univariate and multivariate analyses. Serum OPN was measured by ELISA, polymorphisms in the −443 position of the tumor OPN promoter were analyzed by PCR, and associations between OPN levels and promoter polymorphisms and clinicopathological parameters and patient outcome were investigated. Results: High expression of OPN in NSCLC tumors was associated with poor patient outcome, and OPN was a strong, independent prognostic factor for both relapse free and overall survival. Serum OPN levels increased according to tumor pT classification and tumor size, and patients with OPN-expressing tumors had higher serum levels than patients with OPN-negative tumors. S100A4 was a negative prognostic factor in several subgroups of adenocarcinoma patients, but not in the overall patient cohort. There was no association between ephrin-A1 expression and patient outcome. Conclusions: OPN is a promising prognostic biomarker in NSCLC, and should be further explored in the selection of patients for adjuvant treatment following surgical resection