Inflammatory profile discriminates clinical subtypes in LRRK2-associated Parkinson's disease.

BACKGROUND AND PURPOSE: The presentation of Parkinson's disease patients with mutations in the LRRK2 gene (PDLRRK2 ) is highly variable, suggesting a strong influence of modifying factors. In this context, inflammation is a potential candidate inducing clinical subtypes. METHODS: An extensive battery of peripheral inflammatory markers was measured in human serum in a multicentre cohort of 142 PDLRRK2 patients from the MJFF LRRK2 Consortium, stratified by three different subtypes as recently proposed for idiopathic Parkinson's disease: diffuse/malignant, intermediate and mainly pure motor. RESULTS: Patients classified as diffuse/malignant presented with the highest levels of the pro-inflammatory proteins interleukin 8 (IL-8), monocyte chemotactic protein 1 (MCP-1) and macrophage inflammatory protein 1-ß (MIP-1-ß) paralleled by high levels of the neurotrophic protein brain-derived neurotrophic factor (BDNF). It was also possible to distinguish the clinical subtypes based on their inflammatory profile by using discriminant and area under the receiver operating characteristic curve analysis. CONCLUSIONS: Inflammation seems to be associated with the presence of a specific clinical subtype in PDLRRK2 that is characterized by a broad and more severely affected spectrum of motor and non-motor symptoms. The pro-inflammatory metabolites IL-8, MCP-1 and MIP-1-ß as well as BDNF are interesting candidates to be included in biomarker panels that aim to differentiate subtypes in PDLRRK2 and predict progression.

Screening for anorexia nervosa via measurement of serum leptin levels.

Due to their sub-normally low fat mass, leptin levels in patients with acute anorexia nervosa (AN) are well below reference levels for age and sex-matched controls. This hypoleptinemia entails endocrinological and behavioral characteristics observed in AN patients during starvation. We aimed to study the appropriateness of hypoleptinemia as a diagnostic marker for AN by assessing sensitivity, specificity and likelihood ratios for different referral serum leptin levels for predicting anorexia nervosa and healthy leanness. For prediction, we additionally generated a score based on a multivariate logistic model including body mass index (BMI; kg/m²) and leptin level. For this purpose, we measured leptin levels in 74 female patients with acute AN upon admission for inpatient or outpatient treatment. Adolescent and adult patients were recruited according to DSM-IV criteria from two multi-center studies. Additionally, leptin levels were measured in 65 female healthy, lean students. Mean serum leptin level was significantly decreased in patients with AN compared to underweight controls (0.87 ± 0.90 vs. 6.43 ± 3.55 µg/L, p < 0.001). Leptin predicted AN independently of BMI; we confirmed a cutoff value in the range of 2 µg/L as having both high specificity and sensitivity. Hypoleptinemia represents a state marker of acute AN and is useful for a laboratory-based diagnostic screening.

Validation of functional insulin-like growth factor binding protein-3 measurement by a ligand immunoassay.

BACKGROUND: Proteolysis of Insulin-like Growth Factor Binding Protein (IGFBP)--3 is a well known mechanism regulating IGF-I bioavailability and IGF-independent actions of IGFBP-3 fragments. Measurement of functional IGFBP-3 can be of use in diagnostics of growth failure or renal impairment. We herein characterize the properties of a commercially available immunoassay for the measurement of functional (IGF-I binding) IGFBP-3. METHOD: Fragmentation of IGFBP-3 is analyzed by gel filtration, SDS-PAGE, and western ligand and immunoblotting and compared with subsequent measurement of total and functional IGFBP-3 by ELISA/IFA. Furthermore, assay characteristics such as reproducibility, linearity, and sensitivity are surveyed. RESULTS: Functional IGFBP-3 was reproducibly measured (6.8/5.6% Inter-/Intra assay variance). A broad range of linearity (1:50-1:300) and a high sensitivity (0.18 microg/L) allowed reliable measurement of IGF-binding IGFBP-3. Analysis of IGFBP-3 fragments reveals that the assay described only detects intact IGFBP-3. Analysis of 189 serum samples from healthy blood donors showed that on average 84% and 69% of total IGFBP-3 was functional in men and women, respectively (p < 0.01). CONCLUSIONS: Functional IGFBP-3 can be measured reliably by the assay system used. Thus, this assay system is suited for the investigation of the diagnostic value of functional IGFBP-3 in human body fluids.

Integrin-mediated action of insulin-like growth factor binding protein-2 in tumor cells.

The neoplastic production of the insulin-like growth factor binding protein (IGFBP)-2 often correlates with tumor malignancy and aggressiveness. Since IGFBP-2 contains an RGD motif in its C-terminus, it was hypothesized that this protein may act independently of IGF on tumor cells through integrins. To investigate this, integrin binding, intracellular signaling and the impact of IGFBP-2 on cell adhesion and proliferation were examined in two tumor cell lines. In tracer displacement studies, up to 30% of the added (125)I-hIGFBP-2 specifically bound to the cells. Bound (125)I-hIGFBP-2 was reversibly displaced by IGFBP-2, IGFBP-1 and RGD-(Gly-Arg-Asp)-containing peptides, but not by IGFBP-3, -4, -5, -6 and RGE-(Gly-Arg-Glu)-containing peptides. Blocking with antibodies directed against different integrins and with fibronectin demonstrated that IGFBP-2 cell surface binding is specific for alpha5beta1-integrin. Incubation of IGFBP-2 with equimolar quantities of IGF-I and IGF-II annihilated RGD-specific binding. IGFBP-2 binding at the cell surface led to dephosphorylation of the focal adhesion-kinase (FAK) of up to 37% (P<0.01), and of the p42/44 MAP-kinases of up to 40% (P<0.01). In addition, IGFBP-2 promoted de-adhesion of the cells dose-dependently by up to 30% (P<0.05), and reduced proliferation by 24% (P<0.01). Since one of the cell lines used does not express a functional IGF-I receptor, these data demonstrate that IGFBP-2 can act in an IGF-independent manner, at least in part by an interaction with alpha5beta1-integrin.

Transactivation of the IGFBP-2 promoter in human tumor cell lines.

Many cancers produce high amounts of the insulin-like growth factor binding protein (IGFBP)-2, which can influence the tumorigenicity and growth of tumor cells. In order to study the possible cause of elevated expression of IGFBP-2 in tumors, we investigated the transcriptional regulation by IGF of a 633-bp fragment of the human IGFBP-2 promoter in a transiently transfected choriocarcinoma (JAR) and a leukemic T-cell line (Molt-4) that express IGFBP-2 highly, and in a leukemic B-cell line (Raji) that expresses little IGFBP-2. Strong basal promoter activity, i.e. luciferase activity was measurable in all of the tumor cell lines. The introduction of equal amounts of normal IGF-I and IGF-II stimulated the transcription of IGFBP-2 only slightly. Synthetic IGF analogues with increased biological activity, however, caused a specific 2.0-3.3-fo1d transactivation of the promoter, as well as a 25% increase in IGFBP-2 mRNA. Synchronously, IGF analogues caused a decrease in the level of IGFBP-3 mRNA of about 45%, while the production of IGFBP-2 as measured by RIA increased in relation to IGFBP-3 by up to 15 times. Blocking with the IGF antagonist JB1 revealed partial involvement of the IGF-I receptor in the regulation of IGFBP-2 expression by locally produced IGF. We conclude, that the reduced ability of IGF analogues to form complexes with locally produced IGFBP may account for their increased biological activity in the stimulation of expression of IGFBP-2 and of cell growth. Since increased biological activity had also been demonstrated for natural pro-IGF forms often produced by tumors, pro-IGFs may be involved in the mechanism leading to elevated IGFBP-2 expression of tumors in vivo.

Functional and total IGFBP3 for the assessment of disorders of the GH/IGF1 axis in children with chronic kidney disease, GH deficiency, or short stature after SGA status at birth.

OBJECTIVE: IGFBP3 immunoreactivity may appear elevated in patients with chronic kidney disease (CKD), in part due to accumulation of low molecular fragments. The importance of these IGFBP3 variants for binding and inactivation of IGF1 and their relevance for the impaired growth of uremic children are unclear. Nevertheless, IGFBP3, measured as total (t-)IGFBP3, is frequently used as a diagnostic parameter in pediatric CKD patients. A new assay for functional (f-)IGFBP3 exclusively detects IGFBP3 capable of IGF binding. The aim of the study was to evaluate the significance of f-IGFBP3 measurements for the assessment of uremic abnormalities of the GH/IGF1 axis. DESIGN: Prospective cross-sectional study. METHODS: t-IGFBP3, f-IGFBP3, and IGF1 were measured in pediatric CKD patients, including patients with CKD stage 3-4 not on dialysis (CKD, n=33), on dialysis treatment (DT, n=26), patients after renal transplantation (RTx, n=89), healthy children (n=29), children with GH deficiency (GHD, n=42), and small for gestational age (SGA) children (SGA, n=34). RESULTS: Mean t-IGFBP3 SDS was elevated in CKD, DT, and RTx children compared with controls and GHD patients (P≤0.0004). Highest values were reached in DT (P<0.0001 vs all groups). In contrast, mean f-IGFBP3 was similar in all groups (P=0.30). CONCLUSIONS: Pediatric CKD patients displayed elevated serum concentrations of t-IGFBP3 but not f-IGFBP3, supporting the hypothesis that IGFBP3 fragments not binding IGF1 accumulate during uremia. f-IGFBP3 is an indicator of IGFBP3 fragmentation and seems to reflect IGF1 binding in CKD better than t-IGFBP3. However, the role of f-IGFBP3 for the diagnosis of disturbances of the GH/IGF hormonal axis appears to be limited.

Rational approach to the diagnosis of severe growth hormone deficiency in the newborn.

CONTEXT: Severe congenital GH deficiency (GHD) of the newborn is a rare disease, which can cause life-threatening hypoglycemias beginning in the first week of life. Reviews and consensus papers on the diagnosis of GHD repeatedly state the lack of a practical evidence-based approach to the diagnosis of GHD in the newborn. OBJECTIVE: Here we provide for the first time sound reference values and a diagnostic cutoff for the GH levels in newborns at the age between d 3 and 5. DESIGN, SETTING, AND PATIENTS: GH was measured in the eluate from 314 filter papers of the newborn screening test performed in our university hospital by using a highly sensitive human GH-ELISA. Reference data are compared with measurements from nine newborns with very high likelihood of having severe GHD, and cutoffs for the diagnostic work-up are defined. RESULTS: In the presence of clinical evidence, the diagnosis of neonatal GHD can be confirmed during the first week of life by a single randomly taken GH level less than 7 microg/liter with 100% sensitivity and 98% specificity on the basis of our assay method. GH content in newborn screening cards stored for almost 3 yr were not different from the content found in recently used screening cards indicating high immunological stability of GH over time. Therefore, the diagnostic approach can use stored screening cards. In addition, we observed a clear gender dichotomy in respect to GH, with healthy female newborns having significantly higher GH levels than males. Cigarette smoking during pregnancy was associated with higher, transient tachypnea of the newborn with lower GH levels. CONCLUSIONS: We provide the first rational approach to the diagnosis of severe GHD in the newborn and evidence for gender dichotomy of the neonatal GH axis.

Human growth hormone measurement by means of a sensitive ELISA of whole blood spots on filter paper.

BACKGROUND: Measurements of human growth hormone (hGH) are a prerequisite for identifying a deficiency or excess. Our study is the first to investigate the reliability of a very sensitive assay for the quantification of GH in dried blood spots on filter paper. OBJECTIVE: Validation of a commercially-available enzyme-linked immunoassay (ELISA) for measuring hGH from filter paper samples of dried blood. METHODS: We used an assay system (ELISA, E022, Mediagnost) based on polyclonal rabbit antibodies. Its suitability is ascribable to its very high sensitivity (1.6 ng/L) and virtual absence of interfering factors, excepting for a cross-reactivity with high pegvisomant concentrations. RESULTS: hGH was found to be stable in dried blood spots on filter paper (No. 903, Whatman) over eight days at 37 degrees C. Extraction of hGH from filter paper, in comparison to EDTA plasma, was 107% (SD 8.1%; n=6) over a range from 2.4 to 34.5 microg/L. Linear regression analysis (n=119) showed a correlation of R(2)=0.97 for the hGH concentration in serum and on filter paper samples. CONCLUSION: Our findings demonstrate the reliability of measurements of hGH in dried blood spots on filter paper. The advantages of this method are the low sample volume and the easy transport, storage, and handling of samples. This method contributes to the standardisation of diagnostics pertaining to abnormal hGH secretion as it facilitates the comparison of decisive measurements.