Development of competitive mRNA PCR for the quantification of interleukin-6-responsive junB oncogene expression.

The transcription factor junB belongs to the jun family of protooncogenes. The appearance of junB mRNA in hepatic cells is an extremely early and sensitive marker of the action of proinflammatory cytokines including interleukin-6. In this study, a competitive reverse transcription (RT)-PCR assay has been developed that is suitable for the quantitative determination of junB mRNA expression. This nonisotopic assay compared to other methods (e.g., Northern blot) is a fast and convenient way to determine the expression of the junB gene and thus the immediate concentration- and time-dependent action of interleukin-6. Because interleukin-6 and interleukin-6-type cytokines play a highly important regulatory role in various pathophysiologically important processes, such as hepatic acute-phase reaction, the quantitative assay of junB mRNA completes the scale of laboratory approaches in inflammation and among other pathological conditions.

Adenovirus-triggered innate signalling pathways.

Adenoviruses are important infectious agents and also emerging vectors in different biomedical applications. These viruses elicit a strong innate and adaptive immune response, which influences both the course of disease and the success of the applied vectors. Several Toll-like Receptor (TLR)-dependent and -independent mechanisms contribute to these responses. Understanding of the involved viral and cellular factors is crucial for the treatment of various adenovirus diseases and the optimal design of adenovirus vector applications. Here we summarize our current understanding of the complex nature of adenovirus-induced innate immune mechanisms.

Characterization of transgenic mice containing adenovirus early region 3 genomic DNA.

Human adenoviruses (Ad) contain a complex transcription region (E3) which codes for proteins that interact with several arms of the immune system. However, E3 genes are not essential for replication in tissue culture. An E3-encoded 19,000-molecular-weight (19K) glycoprotein (gp19K) binds to the class I major histocompatibility complex (MHC) in the endoplasmic reticulum and prevents MHC transport to the cell surface. Three other E3 proteins are involved in the inhibition of apoptosis by tumor necrosis factor alpha. The entire E3 genomic DNA was utilized to produce transgenic mice to study the effect of the E3 proteins on pathogenesis of various infectious agents and to investigate the in vivo synthesis and processing of the multiple E3 mRNAs and proteins. There was basal expression of the E3 promoter in the thymus, kidneys, uterus, and testes and at all levels of the gastrointestinal tract. In addition, the E3 promoter of the transgene could be activated in some other organs, including the liver, by infection of these animals with an E3-deficient Ad (Ad7001) which contains a functional E1A region. Transactivation in vivo could also be demonstrated by infusion of bacterial lipopolysaccharide. There appeared to be differential ratios of expression between several of the E3 mRNAs in transgenic lung fibroblasts and primary kidney cells cultured from the transgenic animals. This observation suggested that there was differential mRNA splicing that was organ specific. These transgenic animals should provide a useful model for studying the effects of the E3 proteins on the immune system and on diseases affected either by control of MHC or by selected functions of tumor necrosis factor that are inhibitable by Ad E3 proteins.

Prolonged survival of pancreatic islet allografts mediated by adenovirus immunoregulatory transgenes.

The adenovirus (Ad) early region 3 (E3) genes code for at least four proteins that inhibit the host immune responses mediated by cytotoxic T lymphocytes and tumor necrosis factor alpha. To evaluate the potential use of these immunoregulatory viral functions in facilitating allogeneic cell transplantation, the Ad E3 genes were expressed in pancreatic beta cells in transgenic mice under control of the rat insulin II promoter. Transgenic H-2b/d (C57BL/6 x BALB/c) islets, expressing the Ad E3 genes, remained viable for at least 94 days after transplantation under the kidney capsule of BALB/c (H-2d) recipients. Nontransgenic H-2b/d control islets were rejected as anticipated between 14 and 28 days. Histological analysis of the transplanted transgenic islets revealed normal architecture. Immunohistochemical studies with antisera to islet hormones revealed the presence of both beta and non-beta islet cells, suggesting a propagation of the immunosuppressive effect of Ad proteins from beta cells to other islet cells. The use of viral genes, which have evolved to regulate virus-host interactions, to immunosupress the anti-genicity of donor transplant tissue suggests additional ways for prolonging allograft survival. In addition, these findings have implications for designing Ad vectors for gene therapy.

Monoclonal antibodies to adenovirus type 35 hexon.

A panel of 37 monoclonal antibodies (MAbs) directed against adenovirus type 35 (AV35) hexon was studied by indirect enzyme-linked immunosorbent assay (ELISA) and passive hemagglutination (HA) methods. Nine heterologous hexon types and the homologous type were used to determine the reactivity pattern (RP) of the MAbs and to study the antigenic relationship among the different hexon types. Eleven types of RPs were shown using ELISA and seven types were shown using the HA test. In the case of six MAbs, the RPs were identical in both assay systems; 31 MAbs showed some differences when the results of the two methods were compared. The common epitopes of the different hexon types studied seem to be characterized as genus, subgenus, intersubgenus, and intertype specificities. The type-specific determinant of AV35 hexon could be detected by several MAbs. The antigenic relationship seems to be closer between the two oncogenic subgenera (A and B) of adenoviruses, whereas the antigenic relationship to AV35 hexon is somewhat looser for subgenera D, and E. Hexon types of subgenus C showed the greatest differences in antigenic structure compared with the AV35 hexon.

Molecular cloning and physical mapping of the DNA of human adenovirus type 35.

The prototype strain of the human adenovirus type 35 (AV35) was examined. BamHI, EcoRI, HindIII, KpnI, PstI, and SalI restriction endonucleases were used for the mapping of DNA fragments. Three original maps were constructed, and previously published maps were somewhat modified. A PstI-specific fragment library was also prepared and characterized using the pBR322/E. coli system. Some of the recombinants seem to be applicable for rapid DNA diagnostics, and for the comparative mapping of type- and subgroup-specific DNA sequences. The comparative presentation of physical maps of subgroup B human adenoviruses might improve the efficiency of genotyping of adenoviruses using restriction endonucleases.

Mouse adenovirus type 1 causes a fatal hemorrhagic encephalomyelitis in adult C57BL/6 but not BALB/c mice.

Mouse adenovirus type 1 (MAV-1) produces a lethal disease in newborn or suckling mice characterized by infectious virus and viral lesions in multiple organs. Previous reports of MAV-1 infection of adult mice generally described serologic evidence of infection without morbidity or mortality. However, our current results demonstrate that MAV-1 causes a fatal illness in adult C57BL/6(B6) mice (50% lethal dose, [LD50], 10(3.0) PFU) but not in adult BALB/c mice at all of the doses tested (LD50, > or = 10(5.0) PFU). Adult (BALB/c x B6)F1 mice were intermediately susceptible (LD50, 10(4.5) PFU). Clinically, the sensitive B6 mice showed symptoms of acute central nervous system (CNS) disease, including tremors, seizures, ataxia, and paralysis. Light microscopic examination of CNS tissue from the B6 animals revealed petechial hemorrhages, edema, neovascularization, and mild inflammation in the brain and spinal cord. Analysis by electron microscopy showed evidence of inflammation, such as activated microglia, as well as swollen astrocytic endfeet and perivascular lipid deposition indicative of blood-brain barrier dysfunction. Outside of the CNS, the only significant pathological findings were foci of cytolysis in the splenic white pulp. Assessment of viral replication from multiple tissues was performed by using RNase protection assays with an antisense MAV-1 early region 1a probe. The greatest amounts of viral mRNA in MAV-1-infected B6 animals were located in the brain and spinal cord. Less viral message was detected in the spleen, lungs, and heart. No viral mRNA was detected in BALB/c mouse tissue, with the exception of low levels in the heart. Viral titers of organ tissues were also determined and were concordant with RNase protection findings on the brain and spinal cord but failed to demonstrate significant infectious virus in additional organs. Our experiments demonstrate that MAV-1 has a striking tropism for the CNS that is strain dependent, and this provides an informative in vivo model for the study of adenoviral pathogenesis.

Characterization of an intermediate adenovirus strain.

An intermediate strain of human adenovirus of subgenus D was investigated by type specific serological reactions and restriction endonuclease analysis. The latter method showed the strain identical to the prototype strain of human adenovirus type 9 as well as did serum neutralization tests. In contrast with the previous methods haemagglutination inhibition tests showed the strain related to both the prototypes strains of human adenovirus 9 and 13.

Multiple enlargements in the right inverted terminal repeat of the DNA of canine adenovirus type 2.

The Manhattan strain of canine adenovirus type 2 (CAV 2) was examined. Restriction endonuclease analysis and blot hybridization experiments revealed the heterogeneity of the viral DNA. At least 9 unequally expanded species of the viral genome have been recognized. This diversity is caused by different enlargements in the right inverted terminal repeat (ITR) of the virus. The differences between the individual enlargements were shown to be the different multiples of 150 base pairs. Relatedness of CAV 2 DNA to the DNA of bovine adenovirus type 2 (BAV 2) and human adenovirus type 2 (HAV 2) has also been observed during DNA hybridization experiments.

Relationship of E1 and E3 regions of human adenovirus 35 to those of human adenovirus subgroups A, C and D.

Cloned PstI fragments of human adenovirus 35 (AV35) genome were compared with the DNA of representatives of human adenovirus subgroups A (type 12), B (type 7), C (types 1, and 5), D (type 8), and E (type 4), using blot hybridization techniques. The E1b region of AV35 was found to be more distantly related to those of other subgroups than E1a regions sequences and examined by others. DNA hybridization was observed only between E1b of AV35 and the DNA of AV4, thus the recombinant constructed might be applied as B-subgroup-specific diagnostic probe. Common nucleotide sequences were detected within the E3 regions of serotypes 1, 4, 5, 7, 8, and 35. On the basis of inter-subgroup homology, and PstI-fragments it may be concluded, that the structure of E3 sequences of AV7 and AV35 DNA are closely related to those of AV3 DNA sequenced by Signäs et al. [18]. E4 regions were compared only of serotypes representing subgroups B, C, and D. These sequences were subgroup specific, similarly to E1b regions.

Soluble interleukin 6 (IL-6) receptor influences the expression of the protooncogene junB and the production of fibrinogen in the HepG2 human hepatoma cell line and primary rat hepatocytes.

Interleukin 6 (IL-6) belongs to a family of cytokines using receptors sharing a common signal-transducing chain, gp130 and containing a specific ligand-binding chain (IL-6R alpha). It was shown that both the membrane-bound and the soluble form (sIL-6R) of this ligand specific receptor chain occurs naturally. The soluble form of IL-6 receptor was found to be able to associate with the membrane-bound gp130 and to generate active IL-6 receptor complex capable of inducing signal transduction. This study on a human hepatoma cell line and primary rat hepatocytes examined how the effectiveness of IL-6 is modified by the presence of soluble IL-6 receptor and whether the sIL-6R in the absence of IL-6 acts on hepatocytes. The authors studied the gene expression of junB, a member of the Jun family of transcription factors, and the production of fibrinogen in response to IL-6 and sIL-6R. The data show that in hepatic cells, endogeneously expressing IL-6R, the IL-6 induced junB and fibrinogen expression is inhibited by the presence of sIL-6R. In addition we found that sIL-6R alone (in the absence of IL-6) induced junB mRNA expression, but had no effect on fibrinogen production.