[Severe accidental hypothermia : Treatment using an intravascular temperature management catheter]. / Schwere akzidentelle Hypothermie : Behandlung mithilfe eines intravaskulären Katheters zum Temperaturmanagement.

Different techniques have been reported for the treatment of severe accidental hypothermia. In this case, we successfully used an intravascular catheter temperature management system which has been developed to induce reversible therapeutic hypothermia in patients following resuscitation. In our patient, the initial core temperature was 26.7 °C, and the temperature management system allowed for successful rewarming without complications with a maximum rate of about 1 °C/h.

Generation of transgenic mice and germline transmission of a mammalian artificial chromosome introduced into embryos by pronuclear microinjection.

We have generated transgenic mice by pronuclear microinjection of a murine satellite DNA-based artificial chromosome (SATAC). As 50% of the founder progeny were SATAC-positive, this demonstrates that SATAC transmission through the germline had occurred. FISH analyses of metaphase chromosomes from mitogen-activated peripheral blood lymphocytes from both the founder and progeny revealed that the SATAC was maintained as a discrete chromosome and that it had not integrated into an endogenous chromosome. To our knowledge, this is the first report of the germline transmission of a genetically engineered mammalian artificial chromosome within transgenic animals generated through pronuclear microinjection. We have also shown that murine SATACs can be similarly introduced into bovine embryos. The use of embryo microinjection to generate transgenic mammals carrying genetically engineered chromosomes provides a novel method by which the unique advantages of chromosome-based gene delivery systems can be exploited.

Developing efficient strategies for the generation of transgenic cattle which produce biopharmaceuticals in milk.

At the close of the millennium, a revolution in the treatment of disease is taking shape due to the emergence of new therapies based on human recombinant proteins. The ever-growing demand for such pharmaceutical proteins is an important driving force for the development of safe and large-scale production platforms. Since the efficacy of a human protein is generally dependent on both its amino acid composition as well as various post-translational modifications, many recombinant human proteins can only be obtained in a biologically active conformation when produced in mammalian cells. Hence, mammalian cell culture systems are often used for expression. However, this approach is generally known for limited production capacity and high costs. In contrast, the production of (human) recombinant proteins in milk of transgenic farm animals, particularly cattle, presents a safe alternative without the constraint of limited protein output. Moreover, compared to cell culture, production in milk is very cost-effective. Although transgenic farm animal technology was still in its infancy a decade ago, today it is on the verge of fulfilling its potential of providing therapeutic proteins that can not be produced otherwise in sufficient quantities or at affordable cost. Since 1989, we have been at the forefront of this development, as illustrated by the birth of Herman, the first transgenic bull. In this communication, we will present an overview of approaches we have taken over the years to generate transgenic founder animals and production herds. Our initial strategies were based on microinjection; at the time the only viable option to generate transgenic cattle. Recently, we have adopted a more powerful approach founded on the application of nuclear transfer. As we will illustrate, this strategy presents a breakthrough in the overall efficiency of generating transgenic animals, product consistency, and time of product development.

Production of recombinant human type I procollagen homotrimer in the mammary gland of transgenic mice.

The large scale production of recombinant collagen for use in biomaterials requires an efficient expression system capable of processing a large (> 400 Kd) multisubunit protein requiring post-translational modifications. To investigate whether the mammary gland of transgenic animals fulfills these requirements, transgenic mice were generated containing the alpha S1-casein mammary gland-specific promoter operatively linked to 37 Kb of the human alpha 1(I) procollagen structural gene and 3' flanking region. The frequency of transgenic lines established was 12%. High levels of soluble triple helical homotrimeric [(alpha 1)3] type I procollagen were detected (up to 8 mg/ml) exclusively in the milk of six out of 9 lines of lactating transgenic mice. The transgene-derived human procollagen chains underwent efficient assembly into a triple helical structure. Although proline or lysine hydroxylation has never been described for any milk protein, procollagen was detected with these post-translational modifications. The procollagen was stable in milk; minimal degradation was observed. These results show that the mammary gland is capable of expressing a large procollagen gene construct, efficiently assembling the individual polypeptide chains into a stable triple helix, and secreting the intact molecule into the milk.

Recombinant human acid alpha-glucosidase: high level production in mouse milk, biochemical characteristics, correction of enzyme deficiency in GSDII KO mice.

Glycogen storage disease type II (GSDII) is caused by lysosomal acid alpha-glucosidase deficiency. Patients have a rapidly fatal or slowly progressive impairment of muscle function. Enzyme replacement therapy is under investigation. For large-scale, cost-effective production of recombinant human acid alpha-glucosidase in the milk of transgenic animals, we have fused the human acid alpha-glucosidase gene to 6.3 kb of the bovine alphaS1-casein gene promoter and have tested the performance of this transgene in mice. The highest production level reached was 2 mg/ml. The major fraction of the purified recombinant enzyme has a molecular mass of 110 kDa and resembles the natural acid alpha-glucosidase precursor from human urine and the recombinant precursor secreted by CHO cells, with respect to pH optimum, Km, Vmax, N-terminal amino acid sequence and glycosylation pattern. The therapeutic potential of the recombinant enzyme produced in milk is demonstrated in vitro and in vivo. The precursor is taken up in a mannose 6-phosphate receptor-dependent manner by cultured fibroblasts, is converted to mature enzyme of 76 kDa and depletes the glycogen deposit in fibroblasts of patients. When injected intravenously, the milk enzyme corrects the acid alpha-glucosidase deficiency in heart and skeletal muscle of GSDII knockout mice.

High-level expression of bovine alpha s1-casein in milk of transgenic mice.

The bovine alpha s1-casein gene, isolated from a cosmid library, was introduced into the murine germline. Transgene expression occurred in all transgenic mice, and was confined to the lactating mammary gland. Half of the mouse lines (five out of ten) expressed at relatively high expression levels (> 1 mg ml-1). The highest levels of expression were obtained with a transgene containing 14.2 kb of 5' flanking sequence, in two cases expression levels comparable to (10 mg ml-1) or well above (20 mg ml-1) alpha s1-casein levels in bovine milk were obtained. Transcription initiation occurred at the same site in the bovine alpha s1-casein gene in transgenic mouse as in the cow. A marked induction of expression occurred at parturition rather than at mid-pregnancy, and thus resembled the bovine rather than the murine developmental expression pattern. Bovine alpha s1-casein specific immunoblotting and RIA were developed for characterization and quantification of the recombinant protein. Using these assays, the properties of the recombinant protein could not be distinguished from those of the natural bovine protein. In spite of the high-level tissue-specific and correctly regulated developmental expression of the transgene, expression levels were integration-site dependent. This may indicate that not all cis-acting regulatory elements involved in bovine alpha s1-casein expression were included in the transgene.

Characterization of recombinant human lactoferrin secreted in milk of transgenic mice.

Human lactoferrin (hLF) is an iron-binding protein involved in host defense against infection and severe inflammation. Transgenic mice were produced harboring either hLF cDNA or genomic hLF sequences fused to regulatory elements of the bovine alphaS1 casein gene. Recombinant hLF expressed in the milk of transgenic mice (transgenic hLF) was compared with natural (human milk-derived) hLF. Immunological identity of the two forms was shown by double antibody immunoassays and the absence of an anti-hLF antibody response in transgenic mice on hyperimmunization with natural hLF. Mono S cation-exchange chromatography and N-terminal protein sequencing of transgenic and natural hLF revealed identical cationicity and N-terminal sequences. SDS-polyacrylamide gel electrophoresis and absorbance measurements of purified transgenic hLF showed this protein was 90% saturated with iron, whereas natural hLF is only 3% saturated. The pH-mediated release of iron from transgenic hLF was not different from that of iron-saturated natural hLF. Unsaturated transgenic hLF could be completely resaturated upon addition of iron. Slight differences in mobility between transgenic and natural hLF on SDS-polyacrylamide gel electrophoresis were abolished by enzymatic deglycosylation. Binding of transgenic and natural hLF to a range of ligands, including bacterial lipopolysaccharide, heparin, single-stranded DNA, Cibacron blue FG 3A, and lectins, was not different. Based on these observations, we anticipate that (unsaturated) rhLF and natural hLF will exert similar, if not identical, antibacterial and anti-inflammatory activity in vivo.

Transgenic mice carrying chimeric or mutated type III intermediate filament (IF) genes.

Mice carrying chimeric, truncated or mutated genes encoding intermediate filament (IF) proteins type III do not show any detectable severe pathology. However, upon (over)expression of the transgene in the eye lens all animals develop lens opacification (cataract). At the cellular level the loss of visual acuity is preceded by interference with the terminal differentiation of lens fibre cells, plasma membrane damage, distorted assembly of the IF cytoskeleton and perturbation of the cytoskeleton-membrane complex. The degree of expression is paralleled by the extent of the damages.

Structure and expression of the mouse casein gene locus.

The analysis of yeast artificial chromosomes (YACs) containing the complete mouse casein gene locus revealed the presence of five casein genes, alpha-, beta-, gamma-, delta-, and kappa-casein, in this order, in the locus. The alpha- and beta-casein genes are only 10 kb apart and have convergent transcriptional orientations. The distance between the beta-casein gene and the alpha s2-like gamma-casein gene is about 70 kb, and these genes have divergent transcriptional orientations. The gamma- and delta-casein genes, both encoding a alpha s2-like casein, are linked within 60 kb and convergently transcribed. The kappa-casein gene is located about 100 kb from the delta-gene. Except for the presence of the delta-casein gene, the organization of the mouse casein locus resembles that of the bovine locus, including the transcriptional orientation of the genes. In contrast to the other casein genes, which are strongly induced at mid-lactation, expression of the delta-casein gene is abruptly induced upon parturition. Comparative analysis of alpha s2-like sequences from various species suggests that the ancestral alpha s2-like gene duplicated around the time of radiation of the rodent and artiodactylid ancestors.

Muscle-specific expression of a dominant negative desmin mutant in transgenic mice.

To extend our knowledge of the functions of desmin and vimentin intermediate filaments in the developing organism, a construct encoding a truncated desmin subunit driven by the desmin promoter (pDDV), was introduced into the murine germ line. The resulting mutant desmin subunit was assembly-incompetent and capable of disrupting both preexisting desmin and vimentin filaments in a dominant negative fashion in transfected C2C12 muscle cells and in transgenic mouse muscle tissue. Expression of the pDDV was tissue-specific in transgenic mice. High level expression of pDDV occurred in a small percentage of desmin-containing muscle cells. Immunohistochemical staining of muscle tissue showed a diffuse desmin pattern instead of the dots and clumps into which mutant desmin typically accumulates in undifferentiated C2C12 muscle cells in tissue culture. Disruption of the endogenous desmin filaments in Sartorius muscle results in ultrastructural abnormalities.

Expression of cDNA-encoded human acid alpha-glucosidase in milk of transgenic mice.

Enzyme replacement therapy is at present the option of choice for treatment of lysosomal storage diseases. To explore the feasibility of lysosomal enzyme production in milk of transgenic animals, the human acid alpha-glucosidase cDNA was placed under control of the alpha S1-casein promoter and expressed in mice. The milk contained recombinant enzyme at a concentration up to 1.5 micrograms/ml. Enzyme purified from milk of transgenic mice was internalized via the mannose 6-phosphate receptor and corrected enzyme deficiency in fibroblasts from patients. We conclude that transgenically produced human acid alpha-glucosidase meets the criteria for therapeutic application.

Disruption of vimentin intermediate filaments in transgenic mice by expression of a dominant negative mutant desmin subunit.

To investigate putative functions of vimentin intermediate filaments in the context of intact tissues and the developing organism, a construct (pVDV), driven by the vimentin promoter and encoding a truncated desmin subunit, was introduced into the murine germ line. The mutant desmin was assembly-incompetent and capable of disrupting preexisting vimentin filaments in a dominant negative fashion, both in transgenic mouse tissues and in fibroblast cultures derived from these mice. Mutant desmin expression strongly enhanced vimentin turnover. In tissues of some transgenic mouse lines, high level expression of pVDV occurred in 10 to 40% of vimentin-containing cells and, surprisingly, in 1 to 10% of the skeletal and tongue muscle cells. Immunohistochemical staining of muscle tissue showed a diffuse staining pattern instead of the punctated aggregates into which mutant desmin typically accumulates in other cell types. The overexpression of pVDV and the concomitant disruption of the endogenous vimentin filament network and enhanced vimentin turnover in a significant percentage of cells did not cause detectable developmental abnormalities.

Glycosylated and unglycosylated human lactoferrins both bind iron and show identical affinities towards human lysozyme and bacterial lipopolysaccharide, but differ in their susceptibilities towards tryptic proteolysis.

We studied the role of N-glycosylation of human lactoferrin (hLF) with respect to properties that are relevant to its antibacterial and anti-inflammatory activities. A human kidney-derived 293(S) cell line that constitutively expresses recombinant hLF (rhLF) was produced. The reactivity towards various antibodies of rhLF that had been expressed in the absence or presence of tunicamycin (which blocks N-linked glycosylation) did not differ from that of natural (human milk-derived) hLF. Cation-exchange chromatography and N-terminal protein sequencing showed identical cationic properties and an intact N-terminal sequence for rhLF and natural hLF. SDS/PAGE of rhLF expressed in the presence of tunicamycin revealed a protein with the same M(r) as that of enzymically deglycosylated natural hLF. Both glycosylated and unglycosylated rhLF appeared to be completely saturated with iron. The affinity of natural hLF, glycosylated and non-glycosylated rhLF for both human lysozyme (Kd 4.5 x 10(-8) M) and bacterial lipopolysaccharide did not differ. SDS/PAGE of hLF species subjected to trypsin indicated that unglycosylated rhLF was much more susceptible to degradation. Furthermore, this analysis suggests that N-glycosylation heterogeneity in natural hLF and rhLF resides in the C-lobe. Thus our results provide no argument for differential antibacterial and/or anti-inflammatory activity of natural and (glycosylated) rhLF and suggest that a major function of glycosylation in hLF is to protect it against proteolysis.

Expression analysis of the individual bovine beta-, alpha s2- and kappa-casein genes in transgenic mice.

To identify cis-acting regulatory elements involved in the regulation of expression of the casein genes, the bovine beta-, alpha s2- and kappa-casein genes were isolated from cosmid libraries and introduced into the murine germline. Bovine casein expression was analysed at the RNA and protein level. The bovine beta-casein gene, including 16 kb of 5'- and 8 kb of 3'-flanking region, appeared to be expressed in all 12 transgenic mouse lines analysed. In 50% of these lines expression levels in milk exceeded 1 mg/ml. Three lines displayed expression levels comparable with or well above (20 mg/ml) the beta-casein levels in bovine milk. Transgene expression was restricted to the mammary gland. Strong induction of expression occurred at parturition and thus resembled the bovine rather than the murine pattern. In spite of this high-level tissue-specific and developmentally regulated expression, beta-casein expression levels were integration-site-dependent, suggesting that not all elements involved in regulation of expression were included in this beta-casein clone. Neither the bovine alpha s2- nor the kappa-casein gene, including 8 kb and 5 kb of 5'- and 1.5 kb and 19 kb of 3'-flanking sequences respectively, were properly expressed in transgenic mice. However, they were transcribed in stably transfected mouse mammary epithelial cells. This indicates that regulatory elements required for high-level, mammary gland-specific expression are not present in the alpha s2- and kappa-casein clones used in this study and are probably located elsewhere in the casein gene locus.

Expression of human lactoferrin in milk of transgenic mice.

The expression of human lactoferrin (hLF) in the milk of transgenic mice is described. Regulatory sequences derived from the bovine alpha S1-casein gene were fused to the coding sequence of the hLF cDNA and several lines of transgenic mice were generated. Human LF RNA was detected exclusively in the mammary gland of lactating females and only after the onset of lactation. No aberrant RNA products could be detected using northern blotting and primer extension analysis. The hLF concentrations in the milk ranged from less than 0.1 to 36 micrograms ml-1. Human LF thus expressed did not differ from human milk derived LF, with respect to molecular mass and immunoreactivity with monoclonal and polyclonal antibodies.

Abnormal incisor-tooth differentiation in transgenic mice expressing the muscle-specific desmin gene.

Immunocytochemistry and electron microscopic observations on the incisor-tooth organ of transgenic mice expressing the muscle-specific desmin gene under the direction of the vimentin promoter, reveal that the expression of the hybrid transgene occurs both in mesenchymal cells and differentiating odontoblasts. The muscle-specific desmin, as estimated by fluorescence intensity, is more expressed in immature mesenchymal cells than in postmitotic differentiated odontoblasts. The expression of the transgene generates alteration of the odontoblast-intermediate filament network and interferes with the secretory activity of both odontoblasts and ameloblasts. Our results are consistent with the hypothesis that odontoblasts have inductive properties on the differentiation of ameloblasts and that intermediate filaments among other factors play the role of cell and tissue organizer.

Formation of 7 alpha- and 7 beta-hydroxylated bile acid precursors from 27-hydroxycholesterol in human liver microsomes and mitochondria.

In a search for enzymes involved in the formation of bile acids from 27-hydroxycholesterol in humans, the metabolism of this and other side-chain oxygenated steroids was studied in human liver microsomes and mitochondria. The microsomal fraction contained enzyme(s) catalyzing 7 alpha-hydroxylation of 27-hydroxycholesterol and 3 beta-hydroxy-5-cholestenoic acid, whereas the 7 alpha-hydroxylation of cholesterol and 3 beta-hydroxy-5-cholenoic acid was low. Only small amounts of 7 beta-hydroxylated products were formed. Purification and subfractionation of microsomal protein yielded a fraction of cytochrome P-450, which required NADPH and NADPH-cytochrome P-450 reductase and catalyzed 7 alpha-hydroxylation of the side-chain oxygenated 3 beta-hydroxy-delta 5-C27-steroids but was inactive toward cholesterol. Added cholesterol did not inhibit the observed enzymatic activity. The results provide evidence that this enzyme is different from cholesterol 7 alpha-hydroxylase. The mitochondrial fraction contained enzyme(s) that catalyzed an isocitrate-dependent 7 alpha-hydroxylation of 3 beta-hydroxy-5-cholestenoic acid. The activity was much lower with 27-hydroxycholesterol. The mitochondrial fraction also catalyzed the oxidation of the 27-hydroxy group and contained a 3 beta-hydroxy-delta 5-steroid dehydrogenase active on 7 alpha-hydroxylated C27-steroids. The metabolic end product of the reactions catalyzed by these enzymes was 7 alpha-hydroxy-3-oxo-4-cholestenoic acid. A considerable fraction of the 7 alpha-hydroxy-delta 5 intermediates was also converted to the corresponding 7 beta-hydroxysteroids, probably by way of the 7-oxosteroids, suggesting the presence of an epimerizing enzyme in the mitochondrial fraction.

Identification of two silencers flanking an AP-1 enhancer in the vimentin promoter.

We have studied the 5' upstream sequences required for the transcriptional regulation of the hamster gene encoding the intermediate filament protein, vimentin. Although vimentin is regarded as the intermediate filament protein of mesothelial tissue, it is also produced in most cultured cells. The human mammary carcinoma cell line, MCF-7, belongs to the exceptions. It contains no vimentin, and the complete upstream promoter region is inactive in this particular cell line. By using transient transfection of chimeric constructs into MCF-7 and HeLa cells, and subsequent chloramphenicol acetyltransferase assays, we were able to show the presence of two negative control regions flanking a double AP-1 enhancer element. Our data indicate that these elements exert their effect irrespective of orientation and position, suggesting that they are silencers. In vitro footprinting assays, gel mobility assays and Southwestern (protein-DNA) blotting revealed the presence of trans-acting factors interacting with both silencer elements. The silencing effect was particularly pronounced in MCF-7 cells, although DNA-binding proteins are present in HeLa cells as well.