Circulating noggin levels following treatment with denosumab or teriparatide in postmenopausal women with low bone mass.

OBJECTIVES: Noggin inactivates bone morphogenetic proteins (BMPs), possibly exerting negative effects on the skeleton.We aimed to compare the effect of agents with opposite impact on bone turnover on noggin circulating levels. METHODS: In this observational, open label, non-randomized clinical study postmenopausal women with low bone mass were treated with either denosumab (n=30) or teriparatide (n=30). Serum samples were obtained at baseline, three and twelve months after treatment initiation. Prevalent fractures were recorded at baseline and lumbar spine bone mineral density (LS BMD) was measured at baseline and twelve months. Measured parameters included noggin, BMP-2, BMP-4, procollagen type 1 N-terminal propeptide (P1NP) and C-terminal cross-linking telopeptide of type 1 collagen (CTx). RESULTS: Noggin levels remained unchanged after either denosumab or teriparatide treatment. Baseline noggin levels were not different between women with vs. without previous anti-osteoporotic treatment, or between those with vs. without vertebral or non-vertebral fractures and were not correlated with age or LS BMD. At twelve months, noggin levels were positively correlated with P1NP within the denosumab (rs= 0.47; p=0.014), whereas negatively within the teriparatide group (rs= -0.43; p=0.019). CONCLUSIONS: In postmenopausal women with low bone mass noggin levels were not correlated with bone parameters at any time point, except with P1NP at 12 months, and remained stable with both denosumab and teriparatide treatment.

Rational design of a hypoallergenic Phl p 7 variant for immunotherapy of polcalcin-sensitized patients.

Polcalcins are important respiratory panallergens, whose IgE-binding capacity depends on the presence of calcium. Since specific immunotherapy is not yet available for the treatment of polcalcin-sensitized patients, we aimed to develop a molecule for efficient and safe immunotherapy. We generated a hypoallergenic variant of the grass pollen polcalcin Phl p 7 by introducing specific point mutations into the allergen's calcium-binding regions. We thereby followed a mutation strategy that had previously resulted in a hypoallergenic mutant of a calcium-binding food allergen, the major fish allergen parvalbumin. Dot blot assays performed with sera from Phl p 7-sensitized patients showed a drastically reduced IgE reactivity of the Phl p 7 mutant in comparison to wildtype Phl p 7, and basophil activation assays indicated a significantly reduced allergenic activity. Rabbit IgG directed against mutant rPhl p 7 blocked patients' IgE binding to wildtype Phl p 7, indicating the mutant's potential applicability for immunotherapy. Mass spectrometry and circular dichroism experiments showed that the mutant had lost the calcium-binding capacity, but still represented a folded protein. In silico analyses revealed that the hypoallergenicity might be due to fewer negative charges on the molecule's surface and an increased molecular flexibility. We thus generated a hypoallergenic Phl p 7 variant that could be used for immunotherapy of polcalcin-sensitized individuals.

Denosumab effects on serum levels of the bone morphogenetic proteins antagonist noggin in patients with transfusion-dependent thalassemia and osteoporosis.

INTRODUCTION: Noggin is an antagonist of bone morphogenetic proteins (BMPs) and has a strong effect on osteogenesis. Osteoporosis is a common complication of transfusion dependent beta-thalassemia (TDT) and denosumab has been recently emerged as a promising therapeutic option. This was a post hoc investigation of serum noggin levels among TDT patients with osteoporosis who participated in a randomized, placebo-control, phase 2b study. METHODS: Patients received either 60 mg denosumab (n = 32) or placebo (n = 31) every 6 months for 12 months. Noggin was measured, for the first time in thalassemia patients, at baseline and at 12 months, using a recently developed high sensitivity fluorescent immunoassay. RESULTS: Both groups showed a significant increase in noggin serum levels (denosumab p < 0.001; placebo p < 0.0001). Interestingly, the increase was higher in the placebo group. Furthermore, we observed a strong correlation between noggin and wrist bone mineral density (r = -0.641, p = 0.002) only in the denosumab group. CONCLUSION: In conclusion, higher noggin levels reflected more BMP inhibition, since our assay detects free bioactive noggin, which in turn impaired bone formation in placebo group. Therefore, denosumab possibly regulates noggin and favours bone turnover in TDT patients with osteoporosis through a novel mechanism of action.

Noggin levels in nonalcoholic fatty liver disease: the effect of vitamin E treatment.

AIM: The evaluation of (a) noggin levels in patients with simple steatosis (SS) vs. nonalcoholic steatohepatitis (NASH) vs. controls, and (b) the effect of combined spironolactone plus vitamin E vs. vitamin E monotherapy on noggin levels in biopsy-proven patients with nonalcoholic fatty liver disease (NAFLD). METHODS: In the case-control study, 15 patients with SS, 16 with NASH, and 24 controls were included. In the randomized controlled trial, NAFLD patients were assigned to vitamin E (400 IU/d) or spironolactone (25 mg/d) plus vitamin E for 52 weeks. RESULTS: Noggin levels were lower in SS (5.8 ± 1.5 pmol/l) and NASH (8.7 ± 2.4 pmol/l) patients than in controls (13.7 ± 2.7 pmol/l; p for trend = 0.040), but were similar in SS and NASH patients. After adjustment for potential cofounders, log(noggin) remained different between groups. Log(noggin) levels similarly increased post-treatment in both groups: log(noggin) was not different between groups (p = 0.20), but increased within groups over time (p < 0.001), without a significant group × time interaction (p = 0.62). Log(noggin) significantly increased at month 2 post-treatment (p = 0.008 vs. baseline) and remained stable thereafter. CONCLUSIONS: Lower noggin levels were observed in NAFLD patients than in controls. Noggin levels increased similarly by either combined low-dose spironolactone plus vitamin E or vitamin E monotherapy. TRIAL REGISTRATION: NCT01147523.

Single step, direct fluorescence immunoassays based on metal enhanced fluorescence (MEF-FIA) applicable as micro plate-, array-, multiplexing- or point of care-format.

Although Enzyme Linked Immuno Sorbent Assay (ELISA) technology is approaching it's 45th year of existence since first described in 1971, it is still the main diagnostic tool in clinical research and routine diagnostics. However, despite its broad usage it suffers from some drawbacks, limiting its use especially in more advanced assay formats like multiplexing platforms, point of care devices or protein arrays. Those limitations result from the need for an enzyme label, a soluble enzyme substrate, washing steps (multiplexing, point care, arrays) and in some cases also insufficient sensitivity, because the majority of circulating proteins and thus potential biomarkers may be found in lower sub-picomolar concentrations. We hereby present a new assay platform based on metal enhanced fluorescence (MEF), that remedies these problems since it eliminates the need for washing steps, for using enzyme labels and allows detection of analytes down to sub-picomolar concentrations. In addition this technology is fully compatible to standard fluorescence reader equipment as it is found in many laboratories nowadays. Since our present work is focused on single biomarker evaluation, we chose a 96 well plate format for convenience, but any other formate like antibody arrays, strip-like point of care devices etc. is feasible too.