Potency determination of factor VIII and factor IX for new product labelling and postinfusion testing: challenges for caregivers and regulators.

A workshop organized by the European Medicines Agency and the European Directorate for the Quality of Medicines and HealthCare was held in London, UK on November 28-29, 2013, to provide an overview of the current knowledge of the characterization of new factor VIII (FVIII) and factor IX (FIX) concentrates with respect to potency assays and testing of postinfusion material. The objective was to set the basis for regulatory authorities' discussion on the most appropriate potency assay for the individual products, and European Pharmacopoeia (Ph. Eur.) discussion on whether to propose revision of the Ph. Eur. monographs with respect to potency assays in the light of information on new FVIII and FIX concentrates. The workshop showed that for all products valid assays vs. the international concentrate standards were obtained and potency could be expressed in International Units. The Ph. Eur. chromogenic potency assay gave valid assay results which correlate with in vivo functionality of rFVIII products. For some modified rFVIII products and all modified rFIX products, one-stage clotting assay methods result in different potencies depending on the activated partial thromboplastin time reagent. As a consequence, monitoring of patients' postinfusion levels is challenging but it was pointed out that manufacturers are responsible for providing the users with appropriate information for use and laboratory testing of their product. Strategies to avoid misleading determination of patents' plasma levels, e.g. information on suitable assays, laboratory standards or correction factors were discussed.

Central adjudication of venous ultrasound in VTE screening trials: reasons for failure.

BACKGROUND: The accuracy of screening ultrasound for venous thrombosis in asymptomatic patients is still a matter of debate. The VENUS study evaluated the accuracy of centrally adjudicated venous ultrasound against venography in patients after major orthopedic surgery and found the sensitivity of ultrasound to be poor for both proximal and distal deep vein thrombus (DVT). OBJECTIVES: To evaluate whether thrombus characteristics such as location or size influence the diagnostic performance of centrally adjudicated venous ultrasound. METHODS: All false negative sonograms of the VENUS study were re-evaluated against the corresponding venograms. Discrepancies were categorized into types of diagnostic failures. Within these categories, thrombus characteristics such as location, length or size of thrombus were evaluated. RESULTS: One hundred and twelve pairs of discrepant ultrasound and venography documents were compared with 28 pairs with concordant results. Discrepancies were caused by local documentation failure (37.5%), failure of the ultrasound method (43.7%) and failure of the central adjudication process (18.7%). The overall size of thrombi was small, which caused about 40% of all sonographic failures with a detection threshold of five Marder points, a thrombus length of 9.5 cm and a number of 3.5 pathological compression manoeuvres. Proximal or distal location of DVT did not affect thrombus detection. CONCLUSION: If centrally adjudicated ultrasound is to be used in future VTE screening trials, training of local sonographers and central adjudicators needs to be intensified, because asymptomatic DVTs seem to be small and ultrasound sensitivity depends on the number of pathological compression manoeuvres documented in the ultrasound document. In contrast, distal or proximal thrombus location itself does not influence sensitivity.

Angiogenesis and fibroblast proliferation precede formation of recurrent tumors after radiation therapy in nude mice.

Recently, the combination of ionizing radiation with inhibitors of angiogenesis has been reported to improve tumor eradication compared to treatment with irradiation alone. However, the mechanisms of this effect have not been defined. For this purpose [corrected] we established a non-small cell lung cancer model in nude mice. Tumor vascularization was visualized in vivo by MRI using gadolinium-DTPA as contrast agent. Further, cryosections were produced as close as possible to the MRI slice positions. Since we were interested in examining the formation of a recurrent tumor, irradiation was performed with a single fraction of 4 Gy. This dose caused a partial remission followed by recurrent tumor growth 25 to 35 days after therapy. The process of partial remission as well as formation of the recurrent tumor was examined in 28 nude mice analysing the following parameters: (i) contrast agent enhancement using high-resolution MRI, (ii) proliferation of tumor cells and fibroblasts using Ki-67 immunohistochemistry and (iii) formation of microvessels using CD31 immunohistochemistry. The latter analyses led to differentiation of three stages. Stage 1 (day 1 to day 15 after irradiation) was characterized by increasing areas of dead cell mass in hematoxylin-eosin-stained slides that corresponded to a decrease in tumor cell proliferation as well as contrast agent enhancement in MRI. The percentage of Ki-67-positive tumor cells decreased from initially 45.1% +/- 6.0% (mean +/- standard deviation) to 1.4% +/- 1.2% (mean +/- standard deviation) on day 15. Stage 2 (day 6 to day 20 after irradiation; overlapping with stage 1) was characterized by proliferation of fibroblasts leading to formation of fibrotic septae with abundant microvessels. Already during late stage 2, MRI identified new contrast agent enhancing areas. Stage 3 (day 20 to day 40 after irradiation) was characterized by new tumor cell proliferation. Interestingly, tumor cells almost exclusively proliferated in the direct neighbourhood of the fibrotic septae that had been formed in stage 2. Obviously, proliferation of fibroblasts and blood vessels was a condition prior to formation of recurrent tumor tissue. Thus, our results are in contrast with the view that tumors or recurrent tumors begin as avascular masses that later induce neovascularization. With respect to clinical practice, our results suggest that: (i) adjuvant anti-angiogenic therapy should not be limited to the day of irradiation but should cover a critical period until day 5 to day 20 after radiotherapy, (ii) adjuvant therapy should also include inhibition of fibroblast proliferation and (iii) MRI can identify a recurrent tumor 10 to 15 days before occurrence of new tumor growth.

[Cryotherapy of malignant tumors: studies with MRI in an animal experiment and comparison with morphological changes]. / Kryotherapie maligner Tumoren: Untersuchungen mittels MRT im Tierexperiment und Vergleich mit morphologischen Veränderungen.

PURPOSE: Aim of our study was to investigate the efficacy of 7 F cryoprobes for percutaneous use morpho- and histologically, to examine the role of apoptosis after cryotherapy, and to compare contrast-enhanced MRI with histopathological findings at different time intervals in a tumor-mouse model. METHODS: Percutaneous cryotherapy was performed in 15 immunocompromised nude mice with subcutaneously implanted tumors using the non-small-cell lung cancer cell line Lu 1. In group a) 7 mice were sacrificed after definite time intervals and histological examinations were done for evaluation of necrosis and apoptosis (HE; TUNEL assay); 2 mice are in long-term follow-up. In group b) in 6 mice tumor destruction and perfusion before and after freezing were investigated with native and contrast-enhanced MR imaging (T1- and T2-weighted spin-echo) and compared with histopathological findings. Histological control were done in 2 untreated mice. RESULTS: We observed fast tumor-reduction within two weeks (ca. 50%). On long-term follow-up (> 6 months) no recurrence has been noticed so far. Tumors were well vascularized prior to treatment and did not-show contrast enhancement an any time after cryotherapy. A narrow contrast-enhanced zone was seen on the tumor border subcutaneously as a sign of peripheral hyperemia and central vascular stasis after cryotherapy. On histology there was evidence of both apoptosis and necrosis. CONCLUSION: We have established a tumor-mouse model for further investigations. Two minutes freezing of a 2-cm tumor in the mouse model is sufficient for tumor ablation with scarred healing. Apoptosis may play a role in cryotherapy of experimental tumors. Contrast-enhanced MRI is suitable for the estimation of the cryolasion.

A fusion protein designed for noncovalent immobilization: stability, enzymatic activity, and use in an enzyme reactor.

We have designed a new method for enzyme immobilization using a fusion protein of yeast alpha-glucosidase containing at its C-terminus a polycationic hexa-arginine fusion peptide. This fusion protein can be directly adsorbed from crude cell extracts on polyanionic matrices in a specific, oriented fashion. Upon noncovalent immobilization by polyionic interactions, the stability of the fusion protein is not affected by pH-, urea-, or thermal-denaturation. Furthermore, the enzymatic properties (specific activity at increasing enzyme concentration, Michaelis constant, or activation energy of the enzymatic reaction) are not influenced by this noncovalent coupling. The operational stability of the coupled enzyme under conditions of continuous substrate conversion is, however, increased significantly compared to the soluble form. Fusion proteins containing polyionic peptide sequences are proposed as versatile tools for the production of immobilized enzyme catalysts.

Improved refolding of an immobilized fusion protein.

Fusion proteins of monomeric alpha-glucosidase from Saccharomyces cerevisiae containing N- or C-terminal hexa-arginie peptides were expressed in the cytosol of Escherichia coli in soluble form. The polycationic peptide moieties allow noncovalent binding of the denatured fusion proteins to a polyanionic solid support. Upon removal of the denaturant, refolding of the matrix-bound protein can proceed without perturbation by aggregation. However, nonspecific interactions of the denatured polypeptide, or of folding intermediates, with the matrix cause a drastic decrease in renaturation under suboptimal folding conditions. At low salt concentrations, ionic interactions of the refolding polypeptide with the matrix result in lower yields of renaturation. At higher salt concentrations, renaturation is prevented by hydrophobic interactions with the matrix. Apart from ionic strength, renaturation of the denatured matrix-bound fusion protein must be optimized with respect to pH, temperature, cosolvents, and matrix material used. Under optimum conditions, immobilized alpha-glucosidase can be renatured with a high yield at protein concentrations up to 5 mg/ml, whereas folding of the wild-type enzyme in solution is feasible only at an extremely low protein concentration (15 micrograms/ml). Thus, folding of the immobilized alpha-glucosidase allows an extremely high yield of the renaturated model protein. The technology should be applicable to other proteins that tend to aggregate during refolding.

Characterization of structural sequences in the chicken osteocalcin gene: expression of osteocalcin by maturing osteoblasts and by hypertrophic chondrocytes in vitro.

Osteocalcin is one of the major noncollagenous proteins specific to mineralized connective tissues of vertebrates. A cDNA clone encoding the chicken osteocalcin gene was isolated, and the complete coding sequence for the 97-amino-acid pre-pro-osteocalcin was deduced. The 48-amino-acid pre-pro-peptide contains the expected hydrophobic leader sequence and the dibasic Lys-Arg sequence preceding the NH2-terminal His of the mature 49-amino-acid chicken osteocalcin, which is believed to be necessary for pro-peptide cleavage. The pro-peptide sequence also contains the expected motif of polar and hydrophobic residues, including Phe at -16, which targets vitamin K-dependent gamma-carboxylation of the three specific Glu residues at positions 17, 21, and 24 in the mature protein. Northern blots of total RNA were prepared from embryonic and adult chicken tissues (bone, brain, heart, intestine, kidney, muscle) and probed with chicken osteocalcin cDNA. The appearance of a single 0.5 kb mRNA species confirms that bone is the major site of osteocalcin expression in vivo. In primary osteoblasts isolated from 17-day embryonic chicken calvaria, an osteocalcin mRNA of similar size is expressed concurrently with culture mineralization in vitro. Hypertrophic chondrocytes from 12-day ventral vertebrae and from the cephalic half of 17-day caudal sternae also express osteocalcin mRNA, but nonhypertrophic chondrocytes from the caudal half of 17-day sternae do not express osteocalcin mRNA.

Human interleukin-13 activates the interleukin-4-dependent transcription factor NF-IL4 sharing a DNA binding motif with an interferon-gamma-induced nuclear binding factor.

The effects of interleukin-13 (IL-13) and interleukin-4 (IL-4) on cellular functions were shown to be quite similar. We provide evidence that in monocytes as well as in T lymphocytes both IL-4 and IL-13 activate the same recently identified transcription factor NF-IL4 which binds to the specific responsive element IL-4RE. In addition, we show that a nuclear factor activated by interferon-gamma also interacts with the IL-4RE. It differs from NF-IL4 in the electrophoretic mobility of the complex with DNA, in its DNA-binding specificity and in the proteins interacting with the DNA sequence. Sensitivity against various enzyme inhibitors suggests that components of the signal transduction pathway are shared by all three cytokines.

Reconstitution of a heat shock effect in vitro: influence of GroE on the thermal aggregation of alpha-glucosidase from yeast.

alpha-Glucosidase from yeast is inactivated rapidly at temperatures above 42 degrees C. The thermal inactivation is accompanied by aggregation. The molecular chaperone GroEL suppresses the formation of aggregates by binding the thermally inactivated alpha-glucosidase. Spectroscopic studies suggest that GroEL binds alpha-glucosidase in an intermediately folded state. The complex between alpha-glucosidase and GroEL can be dissolved by MgATP. GroES accelerates the MgATP-dependent dissociation of the alpha-glucosidase-GroEL complex. At elevated temperatures this release leads to the formation of aggregates, while at lower temperatures native, enzymatically active molecules are formed.

Localization of acid microclimate along intestinal villi of rat jejunum.

Considering the significance that pH value could have for digestive and absorptive processes, these investigations were aimed at precisely localizing the position of the acid microclimate, i.e., of proton accumulation along the surface of intestinal villi. The determinations were carried out under microscopic control on jejunal segments of rats incubated at 25 degrees C in O2-saturated phosphate buffer (pH 7.4). Specially manufactured antimony microelectrodes (tip diam 50 microns) and calomel reference electrodes were used for pH registration. Highest proton concentration (214-224 nmol/l not equal to pH 6.67-6.65) was found 10-100 microns below the tip of the villus in the zone of digestive and absorptive epithelial cells. Toward the crypt, a steep decrease of proton concentration was registered with alkaline values 200 microns below the villus tip. Toward the bulk phase, the decrease of the proton concentration was moderate due to the existence of the unstirred water layer as an effective diffusion barrier. The pH value of the bulk phase was reached 440 microns over the villus tip, a distance possibly identical to the thickness of the unstirred water layer.