Some endocrine traits of transgenic rabbits. I. Changes in plasma and milk hormones.

The aim of these studies was to compare some endocrine and non-endocrine characteristics of transgenic (carrying mammary gland-specific mWAP-hFVIII gene construct) and non-transgenic rabbits. The concentrations of corticosterone, progesterone, testosterone, estradiol, insulin-like growth factor I (IGF-I) and human factor VIII (hFVIII) in the blood plasma of adult females (9 months of age, third generation transgenic animals), adult males, and young females (1-2 months of age, fourth generation of transgenic animals), as well as in the milk of lactating adult females, were analyzed by using RIA. In addition, litter size and body mass of pups born by transgenic and non-transgenic females from the third generation were compared. Transgenic animals were compared with their non-transgenic siblings (the same genetic and epigenetic background). Transgenesis did not influence plasma hFVIII, but significantly increased corticosterone (in all animals), reduced IGF-I (in adult males and females), testosterone and estradiol, (in young females) and altered progesterone (increase in adult males and decrease in adult females) concentrations in blood plasma. In addition, transgenic females had higher milk concentrations of testosterone, but not progesterone or IGF-I than their non-transgenic sisters. These endocrine changes were not associated with changes in litter size. Transgenic male (but not female) pups have smaller body mass than control animals. These observations demonstrate the influence of transgenesis per se on the animal growth and endocrine system (secretion of reproductive and stress steroid hormones as well as growth factors) over four generations.

Human major group rhinoviruses downmodulate the accessory function of monocytes by inducing IL-10.

Human rhinoviruses (HRVs) are the predominant cause of the common cold. Although this disease is per se rather harmless, HRV infection is considered to set the stage for more dangerous pathogens in vivo. Here we demonstrate that HRV-14, a member of the major group HRV family, can efficiently inhibit antigen-induced T-cell proliferation and T-cell responses to allogeneic monocytes. HRV-14 triggered a significant downregulation of MHC class II molecules on monocytes. Moreover, supernatants from monocytes cultured in the presence of HRV-14 strongly reduced the allogeneic T-cell stimulatory property of untreated monocytes and monocyte-derived dendritic cells (md-DCs), whereas Epstein Barr virus-transformed B-lymphoblastoid cells were not sensitive. Analysis of the supernatant revealed that HRV-14 induced the production of significant amounts of the immunosuppressive cytokine IL-10. The important T-cell stimulatory cytokine IL-12 or the proinflammatory cytokines IL-1beta or TNF-alpha were not detected or were only minimally detected. Finally, monocytes pretreated with HRV-14 were greatly inhibited in their production of IL-12 upon stimulation with IFN-gamma/LPS. These observations suggest that altered cytokine production in mononuclear phagocytes upon interaction with HRV downmodulates appropriate immune responses during the viral infection.

Expression of human thrombomodulin cofactor activity in porcine endothelial cells.

BACKGROUND: Xenograft rejection may predispose to vascular thrombosis because of putative cross-species' functional incompatibilities between natural anticoagulants present on the donor endothelium and host activated coagulation factors. For example, porcine thrombomodulin expressed on porcine aortic endothelial cells (PAEC) does not provide the expected thrombomodulin (TM)-cofactor activity for human protein C in the presence of human thrombin. In addition, TM may be down-regulated after cellular activation. Our aim was to express human TM cofactor activity in PAEC and to study the proinflammatory effect of tumor necrosis factor-alpha (TNF-alpha) on stable expressed human thrombomodulin in vitro. METHODS AND RESULTS: Retroviral transduction of PAEC with the gene encoding for human thrombomodulin (hTM) resulted in expression of high levels of specific TM cofactor activity on PAEC (0.62 microg/ml activated protein C/10(5) cells). High-level expression of hTM resulted in a 620-fold higher activation of human protein C in the presence of human thrombin when compared with mock-transduced PAEC (0.0001 microg/ml/10(5) cells; P<0.001). Transduced PAEC expressing hTM also bound more human thrombin than control PAEC, as determined by inhibition of thrombin-induced platelet activation (P<0.05). We noted that exposure to TNF-alpha significantly reduced exogenous hTM cofactor activity on transduced PAEC in a time- and dose-dependent fashion; this occurred despite the relatively stable expression of hTM mRNA and hTM antigen in these cells. Treatment of transduced PAEC with selected antioxidants could protect against the loss of hTM cofactor activity directly associated with the oxidative stress induced by TNF-alpha activation responses. CONCLUSIONS: Our data show that the functional deficiency of the anticoagulant protein C pathway in PAEC may be corrected by viral transduction of these cells. As analysis of the hTM function showed modulation under conditions of cellular activation, we suggest that expression of hTM mutants resistant to oxidation may have greater therapeutic utility in the genetic modification of porcine xenografts.

Tumor necrosis factor-induced expression of porcine glycoproteins gp65 and gp100 recognized by human xenoreactive natural antibodies.

In the pig-to-primate model of xenotransplantation, graft rejection is initiated by binding of the recipient's xenoreactive natural antibodies (XNA), mainly of the IgM type, to antigens constitutively expressed on donor endothelial cells (EC). As a consequence of XNA binding and complement fixation, the EC become activated, which is considered to be a major mechanism promoting hyperacute as well as later phases of graft rejection. It is not clear whether binding of XNA to activated EC also contributes to delayed rejection. We asked whether EC activation by cytokines results in the expression of other novel surface antigens recognized by XNA which might become relevant in progressive stages of graft rejection. We activated porcine aortic EC and smooth muscle cells with tumor necrosis factor (TNF), interleukin 1, or lipopolysaccharide and studied expression of new XNA-binding antigens. Expression of two glycoproteins, gp65 and gp100, was strongly induced by recombinant human TNF in EC but not in smooth muscle cells. Notably, gp100 expression was specific to TNF activation, whereas gp65 could also be induced by interleukin 1 or lipopolysaccharide. Cell surface labeling indicated that gp65 is expressed on the plasma membrane. Recognition of XNA-binding antigens on resting EC occurs via alpha-galactosyl epitopes. In contrast, gp65 and gp100 were recognized independently of this epitope. Our data show that gp65 and gp100 represent selective cytokine-induced markers on EC that may have importance in a porcine-to-primate model of xenotransplantation. Conceivable functions of gp65 and gp100 are discussed.

Structure of the human alpha 1-microglobulin-bikunin gene.

Alpha 1-Microglobulin (protein HC) and bikunin (formerly HI-30, urinary trypsin inhibitor, inhibitor subunit of inter-alpha-(trypsin) inhibitor) are abundant serum glycoproteins. They belong to two distinct protein families, the lipocalin family, a family of transport proteins for small hydrophobic molecules and the Kunitz-family of proteinase inhibitors. Mature alpha 1-microglobulin and bikunin result from a common precursor. Now we have isolated and sequenced the human gene coding for this precursor protein. The gene consists of 10 exons which span 1.3 kb and 9 introns with an aggregate length of about 16.5 kb. The largest intron (6.5 kb) separates exon 6 (coding for the C-terminal sequence of alpha 1-microglobulin) from exon 7 (coding for a linker peptide and the N-terminal peptide of bikunin). Repetitive DNA sequences of the Alu-type occur downstream of the polyadenylation site, within introns 4 and 6, and upstream of the putative promoter region which has been defined by sequence comparison and transcription start site determination. The gene also contains several sequence motifs reminiscent to known enhancer sequences.

Complementary DNA and deduced amino acid sequences of procine alpha 1-microglobulin and bikunin.

Analysis of complementary DNA for porcine alpha 1-microglobulin and bikunin indicates that both proteins result from proteolytic processing of a common precursor similar to that found in man. Complete primary structures of these proteins are deduced from the nucleic acid sequence and partially confirmed by peptide sequencing.

The domain structure of the inhibitor subunit of human inter-alpha-trypsin inhibitor reflects the exon structure of its gene.

The gene coding for the inhibitor subunit of the human plasma protein complex inter-alpha-trypsin inhibitor has been cloned. The exon structure of the gene corresponds with the organization of the protein in two distinct inhibitor domains. The exons coding for the inhibitor domains each comprise the complete information for a typical Kunitz-type proteinase inhibitor structure. In contrast to bovine aprotinin, the mature inhibitor protein contains amino acid residues flanking the inhibitor domains on both sides. These amino acid residues are encoded by additional exons.