Multivalent Fcγ-receptor engagement by a hexameric Fc-fusion protein triggers Fcγ-receptor internalisation and modulation of Fcγ-receptor functions.

Engagement of Fcγ-receptors triggers a range of downstream signalling events resulting in a diverse array of immune functions. As a result, blockade of Fc-mediated function is an important strategy for the control of several autoimmune and inflammatory conditions. We have generated a hexameric-Fc fusion protein (hexameric-Fc) and tested the consequences of multi-valent Fcγ-receptor engagement in in vitro and in vivo systems. In vitro engagement of hexameric-Fc with FcγRs showed complex binding interactions that altered with receptor density and triggered the internalisation and degradation of Fcγ-receptors. This caused a disruption of Fc-binding and phagocytosis. In vivo, in a mouse ITP model we observed a short half-life of hexameric-Fc but were nevertheless able to observe inhibition of platelet phagocytosis several days after hexameric-Fc dosing. In cynomolgus monkeys, we again observed a short half-life, but were able to demonstrate effective FcγR blockade. These findings demonstrate the ability of multi-valent Fc-based therapeutics to interfere with FcγR function and a potential mechanism through which they could have a sustained effect; the internalisation and degradation of FcγRs.

Alpha synuclein dimers and oligomers are increased in overexpressing conditions in vitro and in vivo.

Parkinson's disease is characterized by degeneration of dopaminergic neurons in the substantia nigra pars compacta along with the formation of intracellular fibrillar inclusions (Lewy bodies and Lewy neuritis), which are mainly composed of aggregated α-synuclein (ASYN). This latter is a 14 kDa protein that localizes to synaptic vesicles in nerve terminals and promotes soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex assembly. We explored the monomeric and oligomeric state of ASYN in vitro in HEK293s and SH-SY5Y cell lines. In addition rats were injected in the substantia nigra with an Adeno associated virus carrying the human A53T mutation of ASYN (in vivo experiments). We show that human wild type ASYN as well as PD-linked mutations (A30P, E46K and A53T) in overexpressing conditions mostly exists in a monomeric state in equilibrium with dimeric forms. The monomer/dimer ratio is unaffected by PD-linked mutation. Furthermore, the A30P, E46K and A53T mutations overexpression strongly increased cell death compared to wild type ASYN. Taken together, our data suggest that ASYN dimers amount do not directly correlate to reduced cellular viability, suggesting a different role in protein function and induced pathology. Our data suggest that early ASYN neuro-pathogenic effects are probably mediated by other molecular processes than increased oligomerization alone.

A simple HPLC-UV method for the determination of lacosamide in human plasma.

Lacosamide (Vimpat®) is an antiepileptic drug approved in the USA, Europe and several other countries as adjunctive therapy for partial-onset seizures. We report a simple HPLC method with UV detection for the quantification of lacosamide in human plasma. The method involves protein precipitation with methanol followed by chromatographic separation using an ACE® C18-AR column (2.1 mm × 150 mm, 3.0 µm) and mobile phases consisting of mixtures of ammonium formate buffer at pH 9 and acetonitrile. Briefly, 25 µl of internal standard and 300 µl of methanol are added to 100 µl of plasma. After vortexing and centrifugation, 70 µl of supernatant is transferred to an autosampler vial and 5.0 µl is injected. Calibration curves are linear in the range of 0.5 to 12.5 µg/ml. A validation was performed that consisted of the evaluation of accuracy and precision, specificity, limit of detection and carryover. Moreover, the possibility of using single-point calibration was evaluated and a crossvalidation between this method and an established LC-MS/MS method using pooled clinical study samples was performed. The method's sensitivity, simplicity and reliance on simpler HPLC equipment should allow for straightforward application in drug monitoring.

Iodine-123-interleukin-2 scintigraphy in metastatic hypernephroma: a pilot study.

AIM: This pilot study reports on the uptake of (123)I-interleukin 2 (IL-2) in metastatic renal cell carcinoma (MRCC) patients and its relationship to prognostic factors of response or failure of MRCC to cytokines treatment. METHODS: Nine consecutive patients with MRCC underwent an (123)I-IL-2 scan (6 male and 3 female; mean age 64 years; range 51-78). Uptake in metastases was related to a summed score of 4 independent factors, predictive of rapid progression under cytokine treatment as defined by Negrier et al. RESULTS: Four patients presented with metastases at one site, 4 at 2 sites and one patient at 3 different sites. Summed scores were: 5 patients had a summed score of 1; 3 a summed score of 2 and 1 patient a summed score of 3. Uptake of (123)I-IL-2 by tumor tissue was found in only 2 patients. Uptake occurred in 1 patient with a summed score of 3 and in 1 with a summed score of 2. CONCLUSION: In this small series of patients with MRCC, (123)I-IL-2 uptake was found in tumors of 2 patients who less likely will benefit from cytokine treatment. Additional studies are needed to assess the relationship between the pretreatment uptake of (123)/I-IL2 in MRCC and the response to IL-2 therapy.

Comparison between 1 T MRI and non-MRI based volumetry in inoculated tumours in mice.

Tumour volume is an important therapeutic endpoint for mouse tumour models in the evaluation of new chemotherapeutic drugs and in pre-clinical evaluation of new radioimmunotherapy pharmaceuticals. In this study, two 1 T MRI-based methods both using T1-T2 hybrid weighting, a manual method (determination of the area per slice) and a semi-automated method (using thresholding), are compared with two classical methods, the abovementioned calliper method and volumetry by water displacement after dissection of the tumour. Interoperator and intraoperator differences for both MRI-based methods were good (no differences p<0.05 using a repeated measures analysis of variance (ANOVA) test). Correlation between the different methods was excellent. No significant differences were obtained (p<0.05), except for the semi-automated method, because it automatically excludes necrotic regions from the tumour. Therefore, we conclude that both manual and semi-automated tumour volumetry in subcutaneous tumour bearing athymic mice by low-field MRI are accurate and reliable methods. The semi-automated method is especially useful for larger tumour volumes, since it accounts for necrotic areas within the tumour.

Synthesis, radiosynthesis and in vivo evaluation in mice of [123I]-(4-fluorophenyl) {1-[2-(4-iodophenyl)ethyl]piperidin-4-yl}methanone for visualization of the 5-HT2A receptor with SPECT.

This work reports the synthesis, radiolabelling and preliminary in vivo evaluation of [(123)I]-(4-fluorophenyl){1-[2-(4-iodophenyl)ethyl]piperidin-4-yl}methanone. The tributylstannylprecursor was synthesized with a yield of 30%. Radiolabelling was performed using an electrophilic iododestannylation. Tracer yield was 80%, radiochemical purity was >95% and specific activity was at least 55 Ci/micromol. Log P was 1.5. The tracer showed uptake in mice brain (2.72% ID/g tissue at 5 min p.i.) and therefore will be evaluated further by regional brain biodistribution and displacement studies in rabbits.

Optimization of the chiral separation of some 2-arylpropionic acids on an avidin column by modeling a combined response.

The enantiomeric separation of some nonsteroidal antiinflammatory drugs was investigated on an avidin column. An experimental design approach (central composite design) was used to evaluate the effects of three method parameters (pH, concentration of organic modifier, and buffer concentration) on the analysis time and the resolution, as well as to model these responses. This revealed that the organic modifier concentration and sometimes the pH are significant parameters to control because of their influence on both analysis time and resolution. Furthermore, the central composite design results were combined in a multicriteria decision-making approach in order to obtain a set of optimal experimental conditions leading to the most desirable compromise between resolution and analysis time.

The relation of CT-determined tumor parameters and local and regional outcome of tonsillar cancer after definitive radiation treatment.

PURPOSE: To investigate the value of CT-derived tumor parameters as predictor of local and regional outcome of tonsillar squamous cell carcinoma treated by definitive radiation therapy. METHODS AND MATERIALS: The pretreatment CT studies of 112 patients with tonsillar squamous cell carcinoma were reviewed. After redigitizing the films, primary and nodal tumor volume was calculated with the summation-of-areas technique. The nodal CT aspect was graded using a 3-point scale (homogenous, inhomogeneous, and necrotic). Mean follow-up time was 33 months. Actuarial statistical analysis of local and regional outcome was done for each of the covariates; multivariate analysis was performed using Cox's proportional hazards model. RESULTS: In the actuarial analysis, CT-determined primary tumor volume was significantly correlated with local recurrence rate (p < 0.05) when all patients were considered, but primary tumor volume did not predict local control within the T2, T3, and T4 category. CT-determined nodal volume was significantly related to regional outcome (p < 0.01), but nodal density was not. Total tumor volume was not significantly related to locoregional outcome (p = 0.1). In the multivariate analysis, the T and N categories were the independent predictors of local and regional outcomes, respectively. CONCLUSION: Compared to other head-and-neck sites, primary and nodal tumor volume have only marginal predictive value regarding local and regional outcome after radiation therapy in tonsillar cancer.

Sarcomatoid renal cell carcinoma: case report and review of the literature.

Sarcomatoid renal cell carcinoma (SRCC) is an uncommon tumor of the renal parenchyma, representing one to five percent of all primary kidney tumors. The major symptoms are the same as in the classic renal cell carcinoma: haematuria and flank pain. The tumor consists of a bimorph feature of clear cells with areas of spindle cells and giant cells, resembling a sarcoma. Immunohistochemistry and electronmicroscopy are often necessary to prove the epithelial origin of these spindle cells. Therapy is essentially surgical but in some cases there can be a benefit of chemotherapy.