Transgenic expression of a CD46 (membrane cofactor protein) minigene: Studies of xenotransplantation and measles virus infection

CD46 (membrane cofactor protein) is a human cell-surface regulator of activated complement and a receptor for the measles virus. A CD46 transgenic mouse line with an expression pattern similar to that of human tissues has been produced, to develop an animal model of (i) the control of complement activation by complement regulators in hyperacute rejection of xenografts, and (ii) measles virus infection. The mouse line was made using a CD46 minigene that includes promoter sequence and the first two introns of genomic CD46, which was coinjected into mouse ova with chicken lysozyme matrix attachment region DNA. A high level of CD46 expression in homozygotic transgenic mice was obtained with spleen cells having approximately 75% of the level found on human peripheral blood mononuclear cells. CD46 was detected in all tissues examined by immunohistochemistry, radioimmunoassay and Western blotting, showing that these mice were suitable for transplantation and measles virus infection studies. It also indicated that the transgene included the important regulatory elements of the CD46 promoter. Transgenic spleen cells were significantly protected in vitro from human complement activated by either the classical or alternative pathways and from alternative pathway rat complement. Furthermore, transgenic mouse hearts transplanted to rats regulated complement deposition in an in vivo model of antibody-dependent hyperacute xenograft rejection. Similar to human lymphocytes, transgenic lymphoblasts could be infected in vitro with measles virus; infected cells expressed viral proteins and produced infectious viral particles. The data demonstrate the suitability of this minigene for obtaining high-level CD46 expression sufficient for enhanced resistance of transgenic cells to complement attack and for obtaining wide tissue distribution of CD46, analogous to human tissues and, therefore, useful for comparative studies.     

MUC1 peptide epitopes associated with five different H-2 class I molecules

We have previously described the induction of murine CD8⁺ major histocompatibility complex (MHC) class I-restricted cytotoxic T cells (CTL) recognizing the 20-amino acid repeat region of the human mucin 1 (MUC1) variable number of tandem repeats region (VNTR), a mucin greatly increased in expression in breast cancer and proposed as a target for immunotherapy. In that study, CTL could detect MUC1 peptides associated with the MHC of all nine strains examined, and we now report the different epitopes presented by five different MHC class I molecules. The epitopes were defined in CTL assays using peptide-pulsed phytohemagglutinin blasts or MHC class I-transfected L cells as targets; in addition, peptide binding assays and T cell proliferation studies were performed. Within the 20-amino acid VNTR, nine potential epitopes could be defined. The epitopes for the four MHC class I molecules [K^b (three epitopes), D^d, L^d and K^k] were closely related, all containing the amino acids PDTRPAP. For D^b, three epitopes were identified, all containing APGSTAP. Most of the epitopes did not contain a consensus motif for the particular MHC class I allele, and bound with low ‘affinity’, compared with known high-affinity peptides. CD8⁺ T cell proliferation also occurred to the same MHC class I-presented epitopes. Finally, when conventional anchor residues were introduced into the peptides, peptide binding increased, whereas CTL recognition was either retained (K^b) or lost (D^b) depending on the epitope.     

Identification and characterization of aquaporin-2 water channel mutations causing nephrogenic diabetes insipidus with partial vasopressin response

Congenital nephrogenic diabetes insipidus (NDI) is a rare disease caused most often by mutations in the vasopressin V2 receptor (AVPR2). We studied a family which included a female patient with NDI with symptoms dating from infancy. The patient responded to large doses of desmopressin (dDAVP) which decreased urine volume from 10 to 4 I/day. Neither the parents nor the three sisters were polyuric. The patient was found to be a compound heterozygote for two novel recessive point mutations in the aquaporin-2 (AQP2) gene: L22V in exon 1 and C181W in exon 3. Residue Cys181 in AQP2 is the site for inhibition of water permeation by mercurial compounds and is located near to the NPA motif conserved in all aquaporins. Osmotic water permeability (Pf) in Xenopus oocytes injected with cRNA encoding C181W-AQP2 was not increased over water control, while expression of L22V cRNA increased the Pf to approximately 60% of that for wild-type AQP2. Co-injection of the mutant cRNAs with the wild-type cRNA did not affect the function of the wild-type AQP2. Immunolocalization of AQP2-transfected CHO cells showed that the C181W mutant had an endoplasmic reticulum-like intracellular distribution, whereas L22V and wild-type AQP2 showed endosome and plasma membrane staining. Water permeability assays showed a high Pf in cells expressing wild-type and L22V AQP2. This study indicates that AQP2 mutations can confer partially responsive NDI.      

Sequence comparison of human and yeast telomeres identifies structurally distinct subtelomeric domains

We have sequenced and compared DNA from the ends of three human chromosomes: 4p, 16p and 22q. In all cases the pro-terminal regions are subdivided by degenerate (TTAGGG)n repeats into distal and proximal sub- domains with entirely different patterns of homology to other chromosome ends. The distal regions contain numerous, short (<2 kb) segments of interrupted homology to many other human telomeric regions. The proximal regions show much longer (approximately 10-40 kb) uninterrupted homology to a few chromosome ends. A comparison of all yeast subtelomeric regions indicates that they too are subdivided by degenerate TTAGGG repeats into distal and proximal sub-domains with similarly different patterns of identity to other non-homologous chromosome ends. Sequence comparisons indicate that the distal and proximal sub-domains do not interact with each other and that they interact quite differently with the corresponding regions on other, non- homologous, chromosomes. These findings suggest that the degenerate TTAGGG repeats identify a previously unrecognized, evolutionarily conserved boundary between remarkably different subtelomeric domains.      

Induction of B cell apoptosis by co-cross-linking CD23 and sIg involves aberrant regulation of c-myc and is inhibited by bcl-2

A novel system to study the effects of co-cross-linking CD23/FceRII and sIg on murine B lymphocytes utilizes a highly multivalent form of anti- Ig prepared by covalently linking anti-Ig antibodies to a DNP-dextran backbone. CD23-sIg co-cross-linking is accomplished by the addition of DNP-specific monoclonal IgE. Previous studies demonstrated that co- cross-linking CD23 and sIg significantly inhibited mouse B cell proliferation, especially at high doses of the multivalent anti-Ig. Interestingly, examination of early activation signals reveals no difference in B cells subjected to co-cross-linking conditions as compared to B cells activated with anti-Ig alone. Total cellular protein tyrosine phosphorylation levels are unchanged by co-cross- linking. Analysis of B cell mRNA reveals that co-cross-linking the receptors does not alter the expression levels of ornithine decarboxylase 8 h after stimulation as compared to the controls. In contrast, levels of the proto-oncogene c-myc were significantly elevated 1 h after inducing B cell activation under co-cross-linking conditions. However, it remains unclear whether this aberrant c-myc regulation plays any role in inducing apoptosis. In addition, on day 3 after stimulation, the co-cross-linking of CD23 and sIg resulted in the formation of apoptotic B cells, determined by both photomicroscopy of the B cell cultures and FACS analysis of B cell nuclei. B cells obtained from bcl-2 transgenic mice proliferated as well as controls, and failed to undergo apoptosis when CD23 and sIg were co-cross-linked on their surface. These studies indicate that co-cross-linking of CD23 with B cell sIg inhibits B cell proliferation by a mechanism that is distinct from that seen by co-cross-linking of the Fc gamma RII and sIg. In addition, these results suggest a means by which antigen- specific IgE can down-regulate additional B cell activation and IgE synthesis.      

Nationwide Implementation of Hello World: A Dutch Email-Based Health Promotion Program for Pregnant Women

Background

In November 2006, an email-based health promotion program for pregnant women was implemented nationally in the Netherlands. The program consisted of emails containing quizzes with pregnancy-related questions tailored to the number of weeks of pregnancy. Emails were sent out once every 4 weeks, up to a maximum of nine emails.

Objectives

The aims of the study were (1) to assess the recruitment of participants and their representativeness of the Dutch population and (2) to study differences in recruitment, program use, and program appreciation among women with different levels of education.

Methods

Data from 13,946 pregnant women who enrolled during the first year of the program were included. Upon registration, participants were asked how they found out about the program and subsequently received an email questionnaire to assess demographic, lifestyle, and Internet characteristics. Program use was tracked, and participants were classified into five user groups (inactive to very active). Program appreciation (low, intermediate, and high) was assessed twice with an email questionnaire that was sent after the woman had received her third and sixth quiz email. Information about pregnant women and their characteristics was obtained from Dutch registries to assess representativeness of the study population.

Results

About 8% of the pregnant women in the Netherlands enrolled in the program. Immigrants were underrepresented, and women with a low level of education seemed to be slightly underrepresented. Most women knew about the program from a promotional email sent by the organization (32%), followed by the Internet (22%) and midwives (16%). Women with little education were more often inactive users of the program than were highly educated women (15% vs 11%, P < .001), whereas highly educated women were more often very active users compared with women with little education (25% vs 20%, P< .001). However, women with less education were more likely than women with more education to have a high appreciation of the program after receiving three quiz emails (52% vs 44%, P = .001).

Conclusions

In this real-life setting, pregnant women can be reached through an email-based health promotion program. Selective engagement by education level remains a challenge.     

Modulation of deltaF508 cystic fibrosis transmembrane regulator trafficking and function with 4-phenylbutyrate and flavonoids

Over 70% of patients with cystic fibrosis have the DeltaF508 mutation. This protein is a partially functional chloride (Cl-) channel that is prematurely degraded in the endoplasmic reticulum. Specific members of the flavonoid class of compounds have been shown to increase Cl- conductance of wild-type and DeltaF508 cystic fibrosis transmembrane regulator (CFTR). Although flavonoid effects on CFTR processing are unknown, evidence of effects on heat shock proteins, specifically those that have been shown to interact with CFTR, led us to believe that there would be an effect on CFTR processing through modulation of CFTR-chaperone interactions. We sought to determine (i) the effect of apigenin, genistein, kaempferol, and quercetin on CFTR processing in IB3-1 cells (F508/W1282X) and (ii) whether sequential treatment with 4-phenylbutyrate (4-PBA) to increase CFTR processing and flavonoid to directly stimulate CFTR would increase Cl- conductance. Our results show no significant effect on CFTR processing as measured by immunoblotting with 1 microM or 5 microM of apigenin, genistein, kaempferol, or quercetin. However, despite no effect on CFTR processing as determined by immunoblot, immunofluorescence demonstrated a favorable change in the intracellular distribution of CFTR with 24 h treatments of apigenin, kaempferol, and genistein. Furthermore, we observed an increase in Cl- conductance as measured by Cl- efflux in cells that were treated for 24 h with 4-PBA and then assayed with forskolin and 1 microM or 5 microM genistein, and also with cells treated for 24 h with either 4-PBA, 5 microM apigenin, or 1 microM quercetin. Thus, a combination of chronic treatment with 4-PBA or select flavonoids, followed by acute flavonoid exposure, may be beneficial in cystic fibrosis.     

In vivo biocompatibility of a plasma-activated, coronary stent coating

Bare metal and drug-eluting coronary stents suffer an inherent lack of vascular cell and blood compatibility resulting in adverse patient responses. We have developed a plasma-activated coating (PAC) for metallic coronary stents that is durable, withstands crimping and expansion, has low thrombogenicity and can covalently bind proteins, linker-free. This has been shown to enhance endothelial cell interactions in?vitro and has the potential to promote biointegration of stents. Using the rabbit denuded iliac artery model, we show for the first time that PAC is a feasible coating for coronary stents in?vivo. The coating integrity of PAC was maintained following implantation and expansion. The rate of endothelialization, strut coverage, neointimal response and the initial immune response were equivalent to bare metal stents. Furthermore, the initial thrombogenicity caused by the PAC stents showed a reduced trend compared to bare metal stents. This work demonstrates a robust, durable, non-cytotoxic plasma-based coating technology that has the ability to covalently immobilize bioactive molecules for surface modification of coronary stents. Improvements in the clinical performance of implantable cardiovascular devices could be achieved by the immobilization of proteins or peptides that trigger desirable cellular responses.     

Binding of the cell adhesive protein tropoelastin to PTFE through plasma immersion ion implantation treatment

The interaction of proteins and cells with polymers is critical to their use in scientific and medical applications. In this study, plasma immersion ion implantation (PIII) was used to modify the surface of polytetrafluorethylene (PTFE), enabling the covalent binding of a cell adhesive protein, tropoelastin, without employing chemical linking molecules. Tropoelastin coating of untreated or PIII treated PFTE simultaneously promoted and blocked cell interactions respectively, i.e. PIII treatment of the PTFE surface completely inverses the cell interactive properties of bound tropoelastin. This activity persisted over long term storage of the PIII treated surfaces. The integrin binding C-terminus of tropoelastin was markedly less solvent exposed when bound to PIII treated PTFE than untreated PTFE, accounting for the modulation of cell adhesive activity. This presents a new methodology to specifically modulate cell behavior on a polymer surface using a simple one step treatment process, by adjusting the adhesive activity of a single extracellular matrix protein.