Ralf Steinborn

Central role for type I interferons and Tyk2 in lipopolysaccharide-induced endotoxin shock

Toll-like receptor-4 activation by lipopolysaccharide (LPS) induces the expression of interferon-β (IFN-β) in a MyD88-independent manner. Here we report that mice devoid of the JAK protein tyrosine kinase family member, Tyk2, were resistant to shock induced by high doses of LPS. Basal and LPS-induced expression of IFN-β and IFN-α4 mRNA in Tyk2-null macrophages were diminished. However, Tyk2-null mice showed normal systemic production of nitric oxide and proinflammatory cytokines and the in vivo response to tumor necrosis factor (TNF) was unperturbed. IFN-β–null but not STAT1-null mice were also resistant to high dose LPS treatment. Together, these data suggest that Tyk2 and IFN-β are essential effectors in LPS induced lethality.

Specificity of signaling by STAT1 depends on SH2 and C‐terminal domains that regulate Ser727 phosphorylation, differentially affecting specific target gene expression

Complete activation of signal transducer and activator of transcription 1 (STAT1) requires phosphorylation at both Y701 and a conserved PMS727P sequence. S727 phosphorylation of STAT1 in interferon‐γ (IFN‐γ)‐treated mouse fibroblasts occurred without a need for p38 mitogen‐activated protein kinase (MAPK), extracellular signal‐regulated kinases 1 and 2 or c‐Jun kinases, and required both an intact SH2 domain and phosphorylation of Y701. In contrast, UV irradiation‐induced STAT1 phosphorylation on S727 required p38MAPK, but no SH2 domain– phosphotyrosine interactions. Mutation of S727 differentially affected IFN‐γ target genes, at the level of both basal and induced expression. Particularly strong effects were noted for the GBP1 and TAP1 genes. The PMS727P motif of STAT3 was phosphorylated by stimuli and signaling pathways different from those for STAT1 S727. Transfer of the STAT3 C‐terminus to STAT1 changed the stimulus and pathway specificity of STAT1 S727 phosphorylation to that of STAT3. Our data suggest that STAT C‐termini contribute to the specificity of cellular responses by linking individual STATs to different serine kinase pathways and through an intrinsically different requirement for serine phosphorylation at different target gene promoters.

Proviral load determination of different feline immunodeficiency virus isolates using real-time polymerase chain reaction: Influence of mismatches on quantification

Lentiviruses are associated not only with immunodeficiency but also with malignancies. The mechanisms involved in tumorigenesis are still not fully understood. Cats infected with feline immunodeficiency virus (FIV) in the wild represent one model in which the role of viral load in the pathogenesis can be studied, since tumors, especially lymphomas, are quite often observed in cats infected with FIV. To be able to compare the viral load data among cats infected with different FIV isolates, the method used to obtain the viral load has to be unaffected by isolate‐specific differences. This is especially true for the real‐time polymerase chain reaction (PCR), a new method for viral load determination, since nucleotide sequence mismatches have been used for allelic discrimination with this method. To investigate the influence of these mismatches on PCR efficiency, we have used an FIV‐specific real‐time PCR and determined the influence of nucleotide sequence variation in several characterized FIV isolates as well as unknown isolates from naturally infected cats. We could demonstrate that minor mismatches, such as point mutations in the primer or the probe region, decrease overall PCR efficiency but do not abolish the quantification, in contrast to major mismatches of three or four nucleotides, which lead to complete inhibition of the real‐time PCR detection. Based on these results, it will be possible to design real‐time PCR systems allowing the quantification of a broad range of isolates, which is a prerequisite for the investigation of the impact of viral load in tumorigenesis.

Mitochondrial DNA heteroplasmy in cloned cattle produced by fetal and adult cell cloning

Mammals have been cloned from adult donor cells. Here we report the first cases of mitochondrial DNA (mtDNA) heteroplasmy in adult mammalian clones generated from fetal and adult donor cells. The heteroplasmic clones included a healthy cattle equivalent of the sheep Dolly, for which a lack of heteroplasmy was reported.

Adult cloning in cattle: potential of nuclei from a permanent cell line and from primary cultures.

Nuclear transfer was used to evaluate the developmental potential of nuclei from a spontaneously immortalized bovine mammary gland epithelial cell line (MECL) and from primary cultures of mammary gland cells (PMGC) and ear skin fibroblasts (PESF) established from 3‐year‐old cows. Cell proliferation was investigated by incorporation and detection of 5‐bromo‐2′‐deoxyuridine (BrdU). The proportion of cells in S‐phase was significantly (P < 0.05) higher for MECL cells than for PMGC and PESF, both in the presence of serum (90% vs. 28% and 15%) and following serum starvation (27% vs. 6% and 3%). Nuclei from PESF supported the development of reconstructed embryos to the blastocyst stage significantly better than those of PMGC (60% vs. 26%; P < 0.05). Embryos reconstructed with cells from MECL failed to develop to blastocysts. After transfer of embryos derived from PMGC and PESF, respectively, 2/2 and 5/12 recipients were pregnant on day 42. On day 90, the corresponding pregnancy rates were 2/2 and 3/12. One live calf derived from a PMGC was born at day 287 of gestation. Another live PESF‐derived calf was delivered by caesarean section at day 286 of gestation. Our study suggests that nuclei from primary cultures of adult cells can be successfully reprogrammed by nuclear transfer, whereas nuclei from a permanent cell line failed to support the development of nuclear transfer embryos. Mol. Reprod. Dev. 54:264–272, 1999.

Production of Type I IFN Sensitizes Macrophages to Cell Death Induced by Listeria monocytogenes

Type I IFNs (IFN-α/β) modulate innate immune responses. Here we show activation of transcription factor IFN regulatory factor 3, the synthesis of large amounts of IFN-β mRNA, and type I IFN signal transduction in macrophages infected with Listeria monocytogenes. Expression of the bacterial virulence protein listeriolysin O was necessary, but not sufficient, for efficient IFN-β production. Signaling through a pathway involving the type I IFN receptor and Stat1 sensitized macrophages to L. monocytogenes-induced cell death in a manner not requiring inducible NO synthase (nitric oxide synthase 2) or protein kinase R, potential effectors of type I IFN action during microbial infections. The data stress the importance of type I IFN for the course of infections with intracellular bacteria and suggest that factors other than listeriolysin O contribute to macrophage death during Listeria infection.

Potential of fetal germ cells for nuclear transfer in cattle.

The developmental potential of bovine fetal germ cells was evaluated using nuclear transfer. Male and female germ cells at three stages of fetal development from 50‐ to 57‐, 65‐ to 76‐ or 95‐ to 105‐day‐old fetuses were fused to enucleated oocytes 2 to 4 hr prior to activation with 7% ethanol (5 min) followed by 5 hr culture in 10 μg/ml cycloheximide and 5 μg/ml cytochalasin B. The in vitro development of nuclear transfer embryos derived from germ cells was compared with those derived from embryonic cells (blastomeres from day 5 or day 6 embryos). Blastocyst rate (38%) obtained with germ cells from 50‐ to 57‐day‐old fetuses tended to be higher than when using germ cells from 65‐ to 76‐ or 95‐ to 105‐day‐old fetuses (23% and 20%, respectively). Within each stage of fetal development, the proportion of blastocysts derived from male germ cells tended to be higher than that obtained with female germ cells, but due to the high variation between individual fetuses this difference was not significant. With the post activation procedure used in this study, germ cells from 50‐ to 57‐day‐old fetuses supported the development of nuclear transfer embryos to the blastocyst stage significantly (P < 0.05) better than nuclei of embryonic cells (38% vs. 3%). After transfer of blastocysts derived from germ cells of 50‐ to 57‐ and 65‐ to 76‐day fetuses, respectively, 45% (5/11) and 50% (3/6) recipients were pregnant on day 30. The corresponding pregnancy rates on day 90 were 36% (4/11) and 17% (1/6). One live male calf was delivered by cesarean section at day 277 of gestation. Our results show that nuclei of bovine fetal germ cells may successfully be reprogrammed to support full‐term development of nuclear transfer embryos. Mol. Reprod. Dev. 52:421–426, 1999.

Non-balanced mix of mitochondrial DNA in cloned cattle produced by cytoplast-blastomere fusion

We have investigated the transmission of parental mitochondrial DNA (mtDNA) in three clones of born cattle obtained by intraspecific cytoplast‐blastomere fusion. Using allele‐specific TaqMan PCR a low level transmission of blastomere mtDNA (DB mtDNA) into the cloned offspring was detected, thereby generating a heteroplasmic population of mtDNA. The amount of DB mtDNA was 13% and 18% in two animals of a clone which derived from a 24‐cell morula and 0.6% and 0.4% in two calves of clonal origin derived from a 92‐cell morula. These values are in accordance with the tendency expected for neutral mtDNA segregation that the fewer cell divisions that have occurred in the donor embryo, the higher the amount of DB mtDNA. We also found a strong decrease of DB mtDNA which was about three orders of magnitude in the third clone derived from a 52‐cell morula stage.

Novel functions of tyrosine kinase 2 in the antiviral defense against murine cytomegalovirus.

We have recently reported that tyrosine kinase 2 (Tyk2)-deficient mice have a selective defect in the in vivo defense against certain viruses. In our current study we show that Tyk2 is essential for the defense against murine CMV (MCMV). In vivo challenges with MCMV revealed impaired clearance of virus from organs and decreased survival of mice in the absence of Tyk2. Our in vitro studies demonstrate that MCMV replicates to dramatically higher titers in Tyk2-deficient macrophages compared with wild-type cells. We show an essential role of type I IFN (IFN-αβ) in the control of MCMV replication, with a prominent role of IFN-β. MCMV infection leads to the activation of STAT1 and STAT2 in an IFN-αβ receptor 1-dependent manner. Consistent with the role of Tyk2 in IFN-αβ signaling, activation of STAT1 and STAT2 is reduced in Tyk2-deficient cells. However, lack of Tyk2 results in impaired MCMV-mediated gene induction of only a subset of MCMV-induced IFN-αβ-responsive genes. Taken together, our data demonstrate a requirement for Tyk2 in the in vitro and in vivo antiviral defense against MCMV infection. In addition to the established role of Tyk2 as an amplifier of Jak/Stat signaling upon IFN-αβ stimulation, we provide evidence for a novel role of Tyk2 as a modifier of host responses.

Pronounced Segregation of Donor Mitochondria Introduced by Bovine Ooplasmic Transfer to the Female Germ-Line

Abstract Ooplasmic transfer (OT) has been used in basic mouse research for studying the segregation of mtDNA, as well as in human assisted reproduction for improving embryo development in cases of persistent developmental failure. Using cattle as a large-animal model, we demonstrate that the moderate amount of mitochondria introduced by OT is transmitted to the offsprings oocytes; e.g., modifies the germ line. The donor mtDNA was detectable in 25% and 65% of oocytes collected from two females. Its high variation in heteroplasmic oocytes, ranging from 1.1% to 33.5% and from 0.4% to 15.5%, can be explained by random genetic drift in the female germ line. Centrifugation-mediated enrichment of mitochondria in the pole zone of the recipient zygotes ooplasm and its substitution by donor ooplasm led to elevated proportions of donor mtDNA in reconstructed zygotes compared with zygotes produced by standard OT (23.6% ± 9.6% versus 12.1% ± 4.5%; P < 0.0001). We also characterized the proliferation of mitochondria from the OT parents—the recipient zygote (Bos primigenius taurus type) and the donor ooplasm (B. primigenius indicus type). Regression analysis performed for 57 tissue samples collected from the seven OT fetuses at different points during fetal development found a decreasing proportion of donor mtDNA (r2 = 0.78). This indicates a preferred proliferation of recipient taurine mitochondria in the context of the nuclear genotype of the OT recipient expressing a B. primigenius indicus phenotype.