Reproductive decision-making in the context of hereditary cancer: the effects of an online decision aid on informed decision-making.

Individuals having a genetic predisposition to cancer and their partners face challenging decisions regarding their wish to have children. This study aimed to determine the effects of an online decision aid to support couples in making an informed decision regarding their reproductive options. A nationwide pretest-posttest study was conducted in the Netherlands among 131 participants between November 2016 and May 2018. Couples were eligible for participation if one partner had a pathogenic variant predisposing for an autosomal dominant hereditary cancer syndrome. Participants completed a questionnaire before use (T0), and at 3 months (T3) after use of the decision aid to assess the primary outcome measure informed decision-making, and the secondary outcome measures decisional conflict, knowledge, realistic expectations, level of deliberation, and decision self-efficacy. T0-T3 comparisons show an overall positive effect for all outcome measures (all ps < 0.05; knowledge (ES = - 1.05), decisional conflict (ES = 0.99), participants' decision self-efficacy (ES = -0.55), level of deliberation (ES = - 0.50), and realistic expectations (ES = - 0.44). Informed decision-making increased over time and 58.0% of the participants made an informed reproductive decision at T3. The online decision aid seems to be an appropriate tool to complement standard reproductive counseling to support our target group in making an informed reproductive decision. Use of the decision aid may lessen the negative psychological impact of decision-making on couples' daily life and wellbeing.

Online decision support for persons having a genetic predisposition to cancer and their partners during reproductive decision-making.

A nationwide pretest-posttest study was conducted in all clinical genetic centres in the Netherlands, to evaluate the effects of an online decision aid to support persons who have a genetic predisposition to cancer and their partners in making an informed decision regarding reproductive options. Main outcomes (decisional conflict, knowledge, realistic expectations, level of deliberation, and decision self-efficacy) were measured before use (T0), immediately after use (T1), and at 2 weeks (T2) after use of the decision aid. Paired sample t tests were used to compute differences between the first and subsequent measurements. T0-T1 and T0-T2 comparisons indicate a significant reduction in mean decisional conflict scores with stronger effects for participants with high baseline decisional conflict. Furthermore, use of the decision aid resulted in increased knowledge levels and improved realistic expectations. Level of deliberation only increased for participants with lower baseline levels of deliberation. Decision self-efficacy increased for those with low baseline scores, whereas those with high baseline scores showed a reduction at T2. It can be concluded that use of the decision aid resulted in several positive outcomes indicative of informed decision-making. The decision aid is an appropriate and highly appreciated tool to be used in addition to reproductive counseling.

The development of an online decision aid to support persons having a genetic predisposition to cancer and their partners during reproductive decision-making: a usability and pilot study.

An online decision aid to support persons having a genetic predisposition to cancer and their partners during reproductive decision-making was developed. A two-phase usability test was conducted among 12 couples (N = 22; 2 persons participated without their partner) at risk for hereditary cancer and 15 health care providers. Couples and health care providers expressed similar suggestions for improvements, and evaluated the modified decision aid as acceptable, easy to use, and comprehensible. The final decision aid was pilot tested (N = 16) with paired sample t tests comparing main outcomes (decisional conflict, knowledge, realistic expectations regarding the reproductive options and decision self-efficacy) before (T0), immediately (T1) and 2 weeks after (T2) use of the decision aid. Pilot testing indicated decreased decisional conflict scores, increased knowledge, and improved realistic expectations regarding the reproductive options, at T1 and T2. No effect was found for couples' decision self-efficacy. The positive findings during usability testing were thus reflected in the pilot study. The decision aid will be further evaluated in a nationwide pretest-posttest study to facilitate implementation in the onco-genetic counselling setting. Ultimately, it is expected that the decision aid will enable end-users to make an informed decision.

Anti-ADDL antibodies differentially block oligomer binding to hippocampal neurons.

Abeta-derived diffusible ligands (ADDLs) are abundant in AD brain, bind to hippocampal neurons and induce deficits in rodent cognition. To further investigate ADDL binding to neurons and identify antibodies that block this association, a panel of anti-Abeta and anti-ADDL antibodies was characterized for their ability to immuno-detect neuronally bound ADDLs and attenuate the binding of ADDLs to neurons. The results showed that anti-Abeta and anti-ADDL antibodies were able to abate ADDLs binding to hippocampal neurons, but to different degrees. Quantitative assessment of binding showed that one antibody, ACU-954 was markedly more effective at blocking ADDL binding than other antibodies assessed. ACU-954 was also found to block ADDL binding to hippocampal slice cultures, attenuate the ADDL-induced loss of dendritic spines and detect "natural ADDLs" in human AD tissue. These results demonstrated that antibodies that bind to and block ADDL binding to neurons can be identified, although their efficacy is conformationally specific since it is not readily apparent or predictable based on the core linear epitope or affinity for monomeric Abeta.

HIV-1 vaccine development: constrained peptide immunogens show improved binding to the anti-HIV-1 gp41 MAb.

The human immunodeficiency virus type I (HIV-1) transmembrane glycoprotein gp41 mediates viral entry through fusion of the target cellular and viral membranes. A segment of gp41 containing the sequence Glu-Leu-Asp-Lys-Trp-Ala has previously been identified as the epitope of the HIV-1 neutralizing human monoclonal antibody 2F5 (MAb 2F5). The 2F5 epitope is highly conserved among HIV-1 envelope glycoproteins. Antibodies directed at the 2F5 epitope have neutralizing effects on a broad range of laboratory-adapted HIV-1 variants and primary isolates. Recently, a crystal structure of the epitope bound to the Fab fragment of MAb 2F5 has shown that the 2F5 peptide adopts a beta-turn conformation [Pai, E. F., Klein, M. H., Chong, P., and Pedyczak, A. (2000) World Intellectual Property Organization Patent WO-00/61618]. We have designed cyclic peptides to adopt beta-turn conformations by the incorporation of a side-chain to side-chain lactam bridge between the i and i + 4 residues containing the Asp-Lys-Trp segment. Synthesis of extended, nonconstrained peptides encompassing the 2F5 epitope revealed that the 13 amino acid sequence, Glu-Leu-Leu-Glu-Leu-Asp-Lys-Trp-Ala-Ser-Leu-Trp-Asn, maximized MAb 2F5 binding. Constrained analogues of this sequence were explored to optimize 2F5 binding affinity. The solution conformations of the constrained peptides have been characterized by NMR spectroscopy and molecular modeling techniques. The results presented here demonstrate that both inclusion of the lactam constraint and extension of the 2F5 segment are necessary to elicit optimal antibody binding activity. The ability of these peptide immunogens to stimulate a high titer, peptide-specific immune response incapable of viral neutralization is discussed in regard to developing an HIV-1 vaccine designed to elicit a 2F5-like immune response.

An enhanced and scalable process for the purification of SIV Gag-specific MHC tetramer.

A recently developed method for the identification and quantitation of antigen-specific T lymphocytes involves the use of complexes of biotinylated major histocompatibility complex (MHC) and avidin conjugated to a fluorescent reporter group. This complex, dubbed the "tetramer," binds to antigen-specific T lymphocytes in vitro, which can then be sorted and counted by fluorescence-activated flow cytometry to measure immune response. Our research has focused on developing the purification process for preparing tetramer reagent. Our goal was to reengineer a published lab-scale purification process to reduce the number of processing steps and to make the process scalable. In our reengineered process, recombinant MHC alpha chain is isolated from Escherichia coli as inclusion bodies by tangential flow filtration. The purified MHC alpha chain is refolded with beta-2-microglobulin and the target peptide antigen to form the class I MHC. The resulting MHC is purified by hydrophobic interaction chromatography (HIC) and biotinylated enzymatically, and the biotinylated MHC is purified by a second HIC step. The tetramer is prepared by mixing biotinylated MHC with an avidin-fluorophore conjugate. The tetramer is further purified to remove any excess MHC or avidin components. Analysis by flow cytometry confirmed that the tetramers generated by this new process gave bright staining and specific binding to CD3+/CD8+ cells of vaccinated monkeys and led to results that were equivalent to those generated with tetramer produced by the original process.

Antibody, cytokine and cytotoxic T lymphocyte responses in chimpanzees immunized with human papillomavirus virus-like particles.

We evaluated antibody, cytokine (IFN-gamma, IL-5, TNF-alpha), and cytotoxic T lymphocyte (CTL) responses in chimpanzees immunized with monovalent or quadrivalent (HPV-6, -11, -16, -18) L1 virus-like particle (VLP) vaccines administered i.m. on aluminum hydroxyphosphate (alum) at weeks 0, 8 and 24. Maximum serum antibody titers to type-specific, neutralizing, conformational epitopes on HPV-11 or -16 L1 VLPs were detected by radioimmunoassay (RIA) four weeks after the second and third immunizations. HPV-11 and -16 neutralizing antibodies were also detected at similar time points with an Human papillomaviruses (HPV) neutralization assay using pseudovirions. Depending on the VLP type used for immunization, HPV type-specific cytokine responses were most frequently seen four weeks after the second or third immunizations and between weeks 44-52. Transient HPV-16 L1-specific CTL activity was observed only between weeks 16-24 in 3 of 22 (13.6%) chimpanzees immunized with HPV-16 L1 VLPs. These findings provide evidence that immunization with multivalent L1 VLPs on alum can evoke both neutralizing antibodies and Th1 and Th2 cytokine responses to several HPV types; however, induction of CTLs is infrequent.

Purification of virus-like particles of recombinant human papillomavirus type 11 major capsid protein L1 from Saccharomyces cerevisiae.

Recombinant major capsid protein, L1 (M(r) = 55,000), of human papillomavirus type 11 was expressed intracellularly at high levels in a galactose-inducible Saccharomyces cerevisiae expression system by an HPV6/11 hybrid gene. The capsid protein self-assembled into virus-like particles (VLPs) and accounted for 15% of the total soluble protein. A purification process was developed that consisted of two main steps: microfiltration and cation-exchange chromatography. The purified VLPs were 98% homogeneous, and the overall purification yield was 10%. The final product was characterized by several analytical methods and was highly immunogenic in mice.

The L1 major capsid protein of human papillomavirus type 11 recombinant virus-like particles interacts with heparin and cell-surface glycosaminoglycans on human keratinocytes.

The L1 major capsid protein of human papillomavirus (HPV) type 11, a 55-kDa polypeptide, forms particulate structures resembling native virus with an average particle diameter of 50-60 nm when expressed in the yeast Saccharomyces cerevisiae. We show in this report that these virus-like particles (VLPs) interact with heparin and with cell-surface glycosaminoglycans (GAGs) resembling heparin on keratinocytes and Chinese hamster ovary cells. The binding of VLPs to heparin is shown to exhibit an affinity comparable to that of other identified heparin-binding proteins. Immobilized heparin chromatography and surface plasmon resonance were used to show that this interaction can be specifically inhibited by free heparin and dextran sulfate and that the effectiveness of the inhibitor is related to its molecular weight and charge density. Sequence comparison of nine human L1 types revealed a conserved region of the carboxyl terminus containing clustered basic amino acids that bear resemblance to proposed heparin-binding motifs in unrelated proteins. Specific enzymatic cleavage of this region eliminated binding to both immobilized heparin and human keratinocyte (HaCaT) cells. Removal of heparan sulfate GAGs on keratinocytes by treatment with heparinase or heparitinase resulted in an 80-90% reduction of VLP binding, whereas treatment of cells with laminin, a substrate for alpha6 integrin receptors, provided minimal inhibition. Cells treated with chlorate or substituted beta-D-xylosides, resulting in undersulfation or secretion of GAG chains, also showed a reduced affinity for VLPs. Similarly, binding of VLPs to a Chinese hamster ovary cell mutant deficient in GAG synthesis was shown to be only 10% that observed for wild type cells. This report establishes for the first time that the carboxyl-terminal portion of HPV L1 interacts with heparin, and that this region appears to be crucial for interaction with the cell surface.

Expression and purification of recombinant tick anticoagulant peptide (Y1W/D10R) double mutant secreted by Saccharomyces cerevisiae.

A double mutant of tick anticoagulant peptide (TAP) was cloned as a chimeric fusion with the yeast alpha-mating factor pre-proleader peptide. Expression in yeast (Saccharomyces cerevisiae) resulted in the secretion of the TAP mutein into the culture medium. An HPLC-based assay was used to screen yeast strains to find those giving highest expression levels. Efficiency of cleavage at the junction of the leader-TAP mutein varied from strain to strain, and a rapid purification method followed by N-terminal sequence analysis was used to identify a host strain that minimized undesirable cleavage products. A purification scheme was developed which separated the TAP mutein from improperly processed peptides present in the medium. This scheme employed cation-exchange chromatography and reversed-phase HPLC. Scale-up of the process was successful and produced 100 mg of fully functional TAP mutein of >96% homogeneity from a 50-L yeast culture.

Human papillomavirus type 11 (HPV-11) neutralizing antibodies in the serum and genital mucosal secretions of African green monkeys immunized with HPV-11 virus-like particles expressed in yeast.

It has been shown previously that immunization of animals with recombinant virus-like particles (VLPs) consisting of the viral capsid proteins L1 or L1 plus L2 protected animals against experimental viral challenge. However, none of these experimental models addresses the issue of whether systemic immunization with VLPs elicits a neutralizing antibody response in the genital mucosa. Such a response may be necessary to protect the uterine cervix against infection with genital human papillomavirus (HPV) types. African green monkeys systemically immunized with HPV-11 VLPs expressed in Saccharomyces cerevisiae and formulated on aluminum adjuvant elicited high-titered HPV-11 VLP-specific serum antibody responses. Sera from these immunized monkeys neutralized HPV-11 in the athymic mouse xenograft system. Significant levels of HPV-11-neutralizing antibodies also were observed in cervicovaginal secretions. These findings suggest that protection against HPV infection of the uterine cervix may be possible through systemic immunization with HPV VLPs.

Sequence determination of human papillomavirus type 6a and assembly of virus-like particles in Saccharomyces cerevisiae.

Human papillomavirus 6a (HPV6a), the most abundant HPV6 subtype, was detected in a vulvar condyloma acuminatum. The complete genome of HPV6a was cloned, and its DNA sequence was shown to be over 97% identical to the HPV6b sequence. Of the eight open reading frames (ORFs) of HPV6a, only the imputed amino acid sequence of the major capsid protein L1 was identical to the corresponding HPV6b sequence; all other HPV6a ORFs showed amino acid changes compared to the HPV6b ORFs. The HPV6a L1 or the L1 + L2 ORFs were expressed in the yeast Saccharomyces cerevisiae. Self-assembly of the L1 capsid protein into virus-like particles (VLPs) was demonstrated both in the L1 as well as L1 + L2 coexpressing yeast strains. Copurification of the L1 and L2 proteins showed complex formation of the L1 and L2 proteins in the yeast-derived VLPs of coexpressing strains.

Chromatographic separation of low-molecular-mass recombinant proteins and peptides on Superdex 30 prep grade.

The chromatographic properties of Superdex 30 prep grade medium have been investigated in non-denaturing and denaturing mobile phases using commercially available proteins and peptides as well as low-molecular-mass (M(r)) recombinant polypeptides. The medium is a macroreticular gel composed of crosslinked agarose beads to which dextran has been covalently bound. The mean particle size is approximately 34 microns. Experimental results show a linear relation between the distribution coefficient (KD) and the log10 M(r) in the fractionation range 24,000-3000. The relationships between resolution and flow-rate or load volume were investigated and shown to be comparable with those of Superdex 75 and 200 prep grade media. Minimal loss of resolution occurred in the flow-range from 30-60 cm/h. Load volumes of up to 5% total column volume could be applied while maintaining baseline resolution of polypeptide mixtures. Non-specific interactions between the matrix and certain samples were characterized. The predominant interactions with the resin appear to be hydrophobic in nature rather than ionic. Hydrogen bonding may also play a role in the retardation of certain small molecules. The applicability of the resin for separating dimeric and oligomeric forms of low-molecular-mass recombinant proteins was shown.

Expression, purification and characterization of multigram amounts of a recombinant hybrid HV1-HV2 hirudin variant expressed in Saccharomyces cerevisiae.

Hirudin (HIR), derived from leeches, and tick anticoagulant peptide (TAP) are polypeptide protease inhibitors of thrombin and coagulation factor Xa (fXa), respectively, and they have both shown utility in vitro and in vivo as potent antithrombotic agents. A thorough side-by-side comparison of the in vivo efficacy of factor Xa inhibition compared to thrombin inhibition by TAP and HIR, respectively, required purification and characterization of multigram amounts of hirudin. Therefore, a recombinant Saccharomyces cerevisiae strain was developed using a plasmid containing the gene encoding the MF alpha 1 preproleader, a synthetic hybrid HV1-HV2 HIR gene, and a galactose-inducible promoter which directed the secretion of 44 mg/liter of recombinant HIR (rHIR) after induction. rHIR was purified by a process that consisted of two chromatographic steps and decolorization. Total yield for the purification process was 3.6 g, or 41%. This process gave 59-fold purification of rHIR that was judged to be > 96% pure with regard to polypeptide content by capillary zonal electrophoresis and reversed-phase high-performance liquid chromatography. Single, unique N- and C-termini were obtained by sequencing and were identical to those predicted from the deduced sequence of the cDNA. Determination of the dissociation constant, by thrombin:hirudin inhibition reaction, and anticoagulant activity, by the activated partial thromboplastin time, demonstrated that the hybrid rHIR HV1-HV2 protein discussed in this report was essentially equipotent with rHIR preparations HV1 and HV2 reported by others.

A novel process for the large-scale purification of recombinant tick anticoagulant peptide using perfusion chromatography.

Tick anticoagulant peptide (TAP) is a 60 amino acid peptide (Mr = 6977, pI = 4.9) found in the saliva of the soft tick Ornithodorous moubata that specifically inhibits blood coagulation factor Xa (fXa). A recombinant form of TAP (rTAP) secreted by Saccharomyces cerevisiae was purified from 200 liters of fermentation broth with POROS, strong cation exchange (SCX) and reversed-phase high performance liquid perfusion chromatography (RP-HPLC) media (20 microns nominal particle diameter). The higher linear flow rates and dynamic capacities, as well as low back pressures, obtained with perfusion chromatography media permitted 37.5 g of rTAP to be efficiently captured and significantly enriched from 400 liters of diafiltered fermentation broth (24.3 g yield) with 1.25 liter of SCX media using a low pressure column and peristaltic pump in 4.5 hours. Subsequently, 16.7 g of rTAP obtained from the capture step was purified in a high-resolution mode with the same SCX media, after the media was cleaned and repacked into an 800 ml high-pressure column. By doing multiple rapid cycles at high linear flow rates, preparative-scale high-resolution purification of multi-gram amounts of the peptide was done on this relatively small column in only 10.5 hours. Finally, the peptide was desalted and decolorized on a 200 ml RP-HPLC column of perfusion chromatography media by doing multiple rapid cycles. After lyophilization, 12 g of peptide (46.9% yield) was obtained that was > 96% homogeneous by several analytical criteria and fully active in inhibiting blood coagulation factor Xa (fXa).(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of recombinant antistasin secreted by Saccharomyces cerevisiae.

Secretion from recombinant yeast was used as a potential source of large quantities of the leech protein antistasin (ATS), a potent and highly specific inhibitor of the serine protease coagulation factor Xa. Mature recombinant ATS (r-ATS) is obtained after intracellular cleavage by the yscF protease of the mating factor alpha-1 pre-proleader from the fusion protein at the Lys-Arg sequence junction. Production levels are relatively low (ca. 1 mg/liter). Purification of the secreted product from a complex growth medium involved cell removal by microfiltration and diafiltration, cation-exchange capture and concentration on S-Sepharose Fast Flow, C-4 reverse-phase high-performance liquid chromatography (RP-HPLC), and HPLC cation-exchange chromatography step, and RP-HPLC concentration and desalting. The process was scaled up from the 16- to the 250-liter level with a corresponding increase in amount of r-ATS. From the 250-liter fermentation two major forms, r-ATS-I and r-ATS-II, distributed approximately 60:40, and a minor form, r-ATS-minor (ca. 1% of the purified r-ATS), were characterized. Limited N-terminal sequence analysis by Edman degradation indicated that r-ATS-I has the predicted mature N-terminus starting with Gln, that r-ATS-II is N-terminally blocked with pyroglutamate, and that r-ATS-minor is an incompletely processed form. RP-HPLC, hydrophilic-interaction HPLC, cation-exchange HPLC analysis, and electrophoresis results are consistent with the differences observed by sequencing. Preliminary in vitro characterization by intrinsic Ki determination for factor Xa inhibition indicated that the yeast r-ATS forms are indistinguishable from each other as well as from r-ATS expressed by the insect baculovirus host-vector system. Nevertheless, r-ATS-I and r-ATS-II appear less potent than insect-derived r-ATS in the activated partial thromboplastin time clotting assay. Further characterization indicated that C-terminal cleavage at Pro-116 had occurred in r-ATS-I and r-ATS-II as well as oxidation of methionine residues to methionine sulfoxide. The possible role of the C-terminus in inhibition of the prothrombinase complex is discussed.

Large-scale purification and characterization of recombinant tick anticoagulant peptide.

Recombinant tick anticoagulant peptide (r-TAP), a potent and specific inhibitor of blood coagulation factor Xa, was purified to greater than 99% homogeneity at the multi-gram scale. Genetically engineered yeast secreted 200-250 mg/l of the heterologous protein into the medium. Cells were separated from broth by diafiltration and purification was done by two chromatographic steps, both conducive to operation on a large scale. Analysis of the purified protein by several methods indicated that it was greater than 99% homogeneous and no incompletely processed or truncated proteins were detected. Physico-chemical characterization data of r-TAP show that it exists as a monomer in solution and no evidence of post-translational modification was observed. The purified protein was fully active in inhibiting human coagulation factor Xa.

Venous and aortic porcine endothelial cells cultured under standardized conditions synthesize heparan sulfate chains which differ in charge.

The identification of a specific required carbohydrate structure for the antithrombin III binding site on heparin suggests that there may be specific structures in glycosaminoglycan chains which are necessary for other vascular functions of these carbohydrates. Determining that such differences exist requires a mechanism to isolate heparan sulfates from endothelial cells of specific vascular beds. The present report indicates that cultured venous and aortic endothelial cells synthesize heparan sulfate chains differing in charge density. There are two important conclusions from this work. (i) Endothelial cells from different blood vessels (i.e., vena cava and thoracic aorta) synthesize heparan sulfates which differ in negative charge and sulfation pattern. Specifically, aortic endothelial heparan sulfates have a higher negative charge than venous heparan sulfates. Differences are also observed in the nitrous acid degradation products of the heparan sulfates. (ii) Endothelial cells in culture retain the ability to synthesize different heparan sulfates in vitro after months of subculture under defined conditions. These results indicate that it is feasible to characterize heparan sulfates using cultured endothelial cells from a variety of vascular beds.

Carcinoma of the bladder treated by partial or total cystectomy.

The results obtained in 37 patients treated by partial cystectomy and 51 patients treated by total cystectomy are presented. Partial cystectomy was usually performed for deeply invasive tumours. THere were no operative deaths, but 85% of the patients died in the first two years of follow-up and the five-year survival was 11%. Of the 51 patients having total cystectomy, 22 were operated upon after other treatments had failed. There were six (11.8%) operative deaths, only one following simple cystectomy, and the remainder following radical cystectomy with or without urethrectomy. Almost half the survivors died in the first two years, and the five-year survival was 31%. The better prognosis in total cystectomy is attributable to the presence of a number of patients with multicentric but relatively non-invasive tumours. Thirteen patients, all with invasive bladder tumours, had 4,000 R irradiation to the pelvis before operation. There was no benefit demonstrated in this combined treatment. The loss survival rate despite treatment in deeply invasive bladder tumours indicates the value of early diagnosis and the need for more effective forms of treatment.