Monitoring of plasma levels of activated protein C using a clinically applicable oligonucleotide-based enzyme capture assay.

BACKGROUND: Human-activated protein C (APC) is a serine protease with anticoagulant, anti-inflammatory and cytoprotective functions. This feature renders APC to be a promising vascular-inflammatory biomarker. OBJECTIVE: The aim of the present study was the development and validation of a technique that allows the measurement of APC plasma levels under practical laboratory conditions. METHODS/PATIENTS: Based on the APC-binding ssDNA aptamer HS02-52G we developed an oligonucleotide-based enzyme capture assay (OECA) that quantifies aptamer-captured APC through hydrolysis rates of a fluorogenic peptide substrate. After optimization of pre-analytical conditions, plasma APC levels were measured in healthy individuals and patients undergoing hip replacement surgery. RESULTS AND CONCLUSION: A combination of APC-OECA with an aprotinin-based quenching strategy allowed APC analysis with a limit of detection as low as 0.022 ± 0.005 ng mL(-1) (0.39 ± 0.10 pmol L(-1)) and a limit of quantification of 0.116 ± 0.055 ng mL(-1) (2.06 ± 0.98 pmol L(-1)). While APC plasma levels in healthy individuals fell below the quantifiable range of the APC-OECA platform, levels substantially increased in patients undergoing hip replacement surgery reaching peak values of up to 12 ng mL(-1) (214 pmol L(-1)). When normalized to the amount of thrombin generated, interindividual variabilities in the APC generating capacity were observed. In general, with a turn-around time from blood sampling to generation of test results of < 7 h, the APC-OECA platform allows sensitive and rapid determination of circulating APC levels under pathological conditions.

Aptamer-based modulation of blood coagulation.

Nucleic acid based aptamers are single-stranded oligonucleotide ligands isolated from random libraries by an in-vitro selection procedure. Through the formation of unique three-dimensional structures, aptamers are able to selectively interact with a variety of target molecules and are therefore also promising candidates for the development of anticoagulant drugs. While thrombin represents the most prominent enzymatic target in this field, also aptamers directed against other coagulation proteins and proteases have been identified with some currently being tested in clinical trials. In this review, we summarize recent developments in the design and evaluation of aptamers for anticoagulant therapy and research.

Induction of effective and antigen-specific antitumour immunity by a liposomal ErbB2/HER2 peptide-based vaccination construct.

Efficient delivery of tumour-associated antigens to appropriate cellular compartments of antigen-presenting cells is of prime importance for the induction of potent, cell-mediated antitumour immune responses. We have designed novel multivalent liposomal constructs that co-deliver the p63-71 cytotoxic T Lymphocyte epitope derived from human ErbB2 (HER2), and HA307-319, a T-helper (Th) epitope derived from influenza haemagglutinin. Both peptides were conjugated to the surface of liposomes via a Pam3CSS anchor, a synthetic lipopeptide with potent adjuvant activity. In a murine model system, vaccination with these constructs completely protected BALB/c mice from subsequent s.c. challenge with ErbB2-expressing, but not ErbB2-negative, murine renal carcinoma (Renca) cells, indicating the induction of potent, antigen-specific immune responses. I.v. re-challenge of tumour-free animals 2 months after the first tumour cell inoculation did not result in the formation of lung tumour nodules, suggesting that long-lasting, systemic immunity had been induced. While still protecting the majority of vaccinated mice, a liposomal construct lacking the Th epitope was less effective than the diepitope construct, also correlating with a lower number of CD8+ IFN-gamma+ T-cells identified upon ex vivo peptide restimulation of splenocytes from vaccinated animals. Importantly, in a therapeutic setting treatment with the liposomal vaccines resulted in cures in the majority of tumour-bearing mice and delayed tumour growth in the remaining ones. Our results demonstrate that liposomal constructs which combine Tc and Th peptide antigens and lipopeptide adjuvants can induce efficient, antigen-specific antitumour immunity, and represent promising synthetic delivery systems for the design of specific antitumour vaccines.

Construction and characterization of bispecific costimulatory molecules containing a minimized CD86 (B7-2) domain and single-chain antibody fragments for tumor targeting.

Efficient T-cell activation requires two signals. The first signal, which confers specificity, is provided by interaction of the T-cell receptor with peptides presented by MHC molecules. One of the second costimulatory signals is induced by binding of B7 proteins on the surface of antigen-presenting cells to CD28 on the T-cell surface. Expression of B7 molecules on tumor cells can result in the activation of tumor specific T lymphocytes and induce protective antitumor immunity. However, at present such gene-therapeutic approaches are limited by the inability to selectively target B7 gene expression to cancer cells. As an alternative approach we exploited recombinant antibody fragments to localize a costimulatory B7 molecule to the surface of tumor cells. We constructed chimeric proteins that contain in a single polypeptide chain a portion of human B7-2 (CD86) genetically fused to single-chain (sc) Fv antibody domains specific for the tumor-associated antigens epidermal growth factor receptor and the closely related ErbB2 receptor tyrosine kinase. A small recombinant fragment of human CD86 was characterized that corresponds to amino acid residues 1-111 (CD86(111)) of the mature protein. CD86(111) produced in the yeast Pichia pastoris and CD86(111) expressed in bacteria was functionally active and displayed specific binding to B7 counter receptors. Bacterially expressed CD86(111)-scFv fusion proteins specifically localized to the respective target antigens on the surface of tumor cells and markedly enhanced the proliferation of primary T cells when bound to immobilized tumor antigen.

[Prevention of retinal detachment in Vorarlberg]. / Ablatioprophylaxe in Vorarlberg.

Since the eye department in Feldkirch is the only surgical eye department in Vorarlberg, it is possible to survey all patients from within a geographically defined area and draw conclusions from the morbidity of certain retinal degenerations concerning the percentage distribution in the population as a whole. In this paper, peripheral retinal degenerations are analyzed with regard to frequency of occurrence, symptoms, age and sex distribution as well as localization on the fundus. The prophylactically treated eyes are compared with all cases of ablatio retinae which occurred during the same period. The need for treatment of peripheral retinal degenerations and the efficacy of such treatment are discussed.

[Schimek's method of correcting senile entropion (author's transl)]. / Zur Korrektur des Entropium senile nach Schimek.

Laxity of skin and tissue over the tarsal plate and spasm of the orbicular muscle along the lid margin lead to senile entropion. The lashes rub against the cornea and this irritation incites further lid spasm. The inferior margin of the tarsus everts easily and in strong cases of entropion the tarsal plate has turned 180 degrees. In 1957 Schimek described a method of correcting senile entropion by a permanently buried horizontal suture which runs from the medial part of the orbicular muscle to the periost of temporal orbital margin. This suture tightens the orbicular muscle and tarsoorbital fascia and provides eversion of the lower border of the tarsus. We have done this operation in 36 patients and were able to examine 24 of them (29 eyes) after a period of one month to 5 years after operation. In 21 patients we noted good results, in 8 cases we found a recurrence of entropion, which means a relatively high recidivity rate with this method.