Constitutive expression of antimicrobial peptide PR-39 in transgenic mice significantly enhances resistance to bacterial infection and promotes growth.

Use of huge amounts of antibiotics in farm animal production has promoted the prevalence of antibiotic-resistant bacteria, which poses a serious threat to public health. Therefore, alternative approaches are needed to reduce or replace antibiotic usage in the food animal industry. PR-39 is a pig-derived proline-rich antimicrobial peptide that has a broad spectrum of antibacterial activity and a low propensity for development of resistance by microorganisms. To test whether ubiquitous expression of PR-39 in transgenic (TG) mice can increase resistance against bacterial infection, we generated TG mice that ubiquitously express a pig-derived antimicrobial peptide PR-39 and analyzed their growth and resistance to infection of the highly pathogenic Actinobacillus pleuropneumoniae (APP) isolated from swine. The growth performance was significantly increased in TG mice compared with their wild-type (WT) littermates. After the APP challenge, TG mice exhibited a significantly higher survival rate and significantly lower tissue bacterial load than WT littermates. Furthermore, the tissue lesion severity that resulted from APP infection was milder in TG mice than that in their WT littermates. This study provides a good foundation for the development of PR-39-expressing TG animals, which could reduce the use of antibiotics in the farm animal industry.

Transcervical Inoculation with Chlamydia trachomatis Induces Infertility in HLA-DR4 Transgenic and Wild-Type Mice.

Chlamydia trachomatis is the leading cause of infection-induced infertility in women. Attempts to control this epidemic with screening programs and antibiotic therapy have failed. Currently, a vaccine to prevent C. trachomatis infections is not available. In order to develop an animal model for evaluating vaccine antigens that can be applied to humans, we used C. trachomatis serovar D (strain UW-3/Cx) to induce infertility in mice whose major histocompatibility complex class II antigen was replaced with the human leukocyte antigen DR4 (HLA-DR4). Transcervical inoculation of medroxyprogesterone-treated HLA-DR4 transgenic mice with 5 × 105C. trachomatis D inclusion forming units (IFU) induced a significant reduction in fertility, with a mean number of embryos/mouse of 4.4 ± 1.3 compared to 7.8 ± 0.5 for the uninfected control mice (P < 0.05). A similar fertility reduction was elicited in the wild-type (WT) C57BL/6 mice (4.3 ± 1.4 embryos/mouse) compared to the levels of the WT controls (9.1 ± 0.4 embryos/mouse) (P < 0.05). Following infection, WT mice mounted more robust humoral and cellular immune responses than HLA-DR4 mice. As determined by vaginal shedding, HLA-DR4 mice were more susceptible to a transcervical C. trachomatis D infection than WT mice. To assess if HLA-DR4 transgenic and WT mice could be protected by vaccination, 104 IFU of C. trachomatis D was delivered intranasally, and mice were challenged transcervically 6 weeks later with 5 × 105 IFU of C. trachomatis D. As determined by severity and length of vaginal shedding, WT C57BL/6 and HLA-DR4 mice were significantly protected by vaccination. The advantages and limitations of the HLA-DR4 transgenic mouse model for evaluating human C. trachomatis vaccine antigens are discussed.

Spontaneous bacterial and fungal infections in genetically engineered mice: Is Escherichia coli an emerging pathogen in laboratory mouse?

The impact of particular microbes on genetically engineered mice depends on the genotype and the environment. Infections resulting in clinical disease have an obvious impact on animal welfare and experimentation. In this study, we investigated the bacterial and fungal aetiology of spontaneous clinical disease of infectious origin among the genetically engineered mice from our institution in relation to their genotype. A total of 63 mice belonging to 33 different mice strains, from severe immunodeficient to wild-type, were found to display infections as the primary cause leading to their euthanasia. The necropsies revealed abscesses localized subcutaneously as well as in the kidney, preputial glands, seminal vesicles, in the uterus, umbilicus or in the lung. In addition, pneumonia, endometritis and septicaemia cases were recorded. Escherichia coli was involved in 21 of 44 (47.72%) of the lesions of bacterial origin, whereas [Pasteurella] pneumotropica was isolated from 19 of 44 (43.18%) cases. The infections with the two agents mentioned above included three cases of mixed infection with both pathogens. Staphylococcus aureus was considered responsible for five of 44 (11.36%) cases whereas Enterobacter cloacae was found to cause lesions in two of 44 (4.54%) mice. Overall, 16 of the 44 (36.36%) cases of bacterial aetiology affected genetically engineered mice without any explicit immunodeficiency or wild-type strains. The remaining 19 cases of interstitial pneumonia were caused by Pneumocystis murina. In conclusion, the susceptibility of genetically modified mice to opportunistic infections has to be regarded with precaution, regardless of the type of genetic modification performed. Beside the classical opportunists, such as [Pasteurella] pneumotropica and Staphylococcus aureus, Escherichia coli should as well be closely monitored to evaluate whether it represents an emerging pathogen in the laboratory mouse.

A Lactobacillus rhamnosus strain induces a heme oxygenase dependent increase in Foxp3+ regulatory T cells.

We investigated the consequences of feeding with a Lactobacillus species on the immune environment in GALT, and the role of dendritic cells and heme oxygenase-1 in mediating these responses. Feeding with a specific strain of Lactobacillus rhamnosus induced a significant increase in CD4+CD25+Foxp3+ functional regulatory T cells in GALT. This increase was greatest in the mesenteric lymph nodes and associated with a marked decrease in TNF and IFNγ production. Dendritic cell regulatory function and HO-1 expression was also increased. The increase in Foxp3+ T cells could be prevented by treatment with a heme oxygenase inhibitor. However, neither inhibition of heme oxygenase nor blockade of IL-10 and TGFß prevented the inhibition of inflammatory cytokine production. In conclusion Lactobacillus feeding induced a tolerogenic environment in GALT. HO-1 was critical to the enhancement of Foxp3+ regulatory T cells while additional, as yet unknown, pathways were involved in the down-regulation of inflammatory cytokine production by T cells.

Transgenic mice carrying the human poliovirus receptor: new animal models for study of poliovirus neurovirulence.

Recombinant viruses between the virulent Mahoney and attenuated Sabin 1 strains of poliovirus type 1 were subjected to neurovirulence tests using a transgenic (Tg) mouse line, ICR-PVRTg1, that carried the human poliovirus receptor gene. The Tg mice were inoculated intracerebrally with these recombinant viruses and observed for clinical signs, histopathological lesions, and viral antigens as parameters of neurovirulence of the viruses. These parameters observed in the Tg mice were different for different inoculated viruses. Dose-dependent incidences of paralysis and of death were observed in the Tg mice inoculated with any viruses used. This indicates that values of 50% lethal dose are useful to score a wide range of neurovirulence of poliovirus. The neurovirulence of individual viruses estimated by the Tg mouse model had a strong correlation with those estimated by monkey model. Consequently, the mouse tests identified the neurovirulence determinants on the genome of poliovirus that had been identified by monkey tests. In addition, the mouse tests revealed new neurovirulence determinants, that is, different nucleotides between the two strains at positions 189 and 21 and/or 935 in the 5'-proximal 1,122 nucleotides. The Tg mice used in this study may be suitable for replacing monkeys for investigating poliovirus neurovirulence.

Mouse mammary tumor virus-induced tumorigenesis in sag transgenic mice: a laboratory model of natural selection.

Transgenic mice that expressed the superantigen protein encoded in the C3H exogenous mouse mammary tumor virus long terminal repeat deleted their V beta 14+ T cells during the shaping of their immune repertoire and showed no evidence of virus production in their mammary glands after infection by milk-borne C3H exogenous virus. However, they developed mammary gland tumors that had newly integrated copies of C3H exogenous virus, although the latency of tumor formation was much longer than in their nontransgenic littermates that retained their V beta 14+ T cells. After four generations, infectious C3H virus was completely eliminated from the transgenic mouse pedigree. These data support the hypothesis that endogenous mouse mammary tumor proviruses are retained in the genome as protection against exogenous virus infection and subsequent tumorigenesis and show that there may be natural selection against the virus in vivo.

Herpes simplex virus pathogenesis in transgenic mice is altered by the homeodomain protein Hox 1.3.

The DNA sequence TAAT is the core binding motif for the mouse homeodomain protein Hox 1.3 (proposed new name, Hoxa-5). These sequences are present within the multiple TAATGARAT regulatory motifs in the promoters of the immediate-early genes which control herpes simplex virus type 1 replication. To investigate the role of this homeodomain protein in the regulation of herpes simplex virus gene expression and pathogenesis, transgenic mice containing a mouse Hox 1.3 cDNA under the control of the virus- and interferon-inducible Mx 1 promoter were generated. After infection of transgenic mice with herpes simplex virus, Hox 1.3 RNA and protein were expressed at the sites of virus replication. In these transgenic mice, herpes simplex virus replication, spread of virus through the host, and virus-induced mortality were markedly enhanced. Increased spread and replication of herpes simplex virus were also observed in cultured fibroblasts from transgenic mice. This finding suggests that in vivo, Hox 1.3 may increase viral spread by increasing viral replication at the level of the individual infected cells. These results demonstrate that expression of a transgene encoding a single host protein, Hox 1.3, alters the pathogenesis of experimental herpes simplex virus infection. We conclude that a protein that belongs to a class of DNA-binding proteins which are best known for their role in regulating embryonic development may also regulate herpesvirus pathogenesis.

Poliovirus-sensitive transgenic mice as a new animal model.

Transgenic mice susceptible to poliovirus infection were produced by introducing a human gene encoding cellular receptors for poliovirus into the mouse genome. Expression of receptor mRNAs in tissues of the transgenic mice was analysed using Northern blot hybridization. The results indicate that the human gene is expressed in many tissues of the transgenic mice just as in human tissues, and that the amount of the receptor mRNAs varies from tissue to tissue. The transgenic mice inoculated with poliovirus by any of the routes tested in this study show clinical symptoms similar to those in humans and monkeys, although the sensitivity of the mice depended on the inoculation route. In any route, the virulent Mahoney strain of type 1 poliovirus is much more virulent than the attenuated Sabin 1 strain in the transgenic mice. These observations suggest that the transgenic mice become an excellent new animal model for studying molecular mechanisms of pathogenesis of poliovirus and for assessing oral poliovirus vaccines.

Poliovirus attenuation and pathogenesis in a transgenic mouse model for poliomyelitis.

A transgenic mouse model for poliomyelitis has been used to study aspects of poliovirus attenuation and pathogenesis. Transgenic mice expressing the cell receptor for poliovirus (TgPVR mice) develop poliomyelitis after inoculation with poliovirus by a variety of routes. TgPVR mice have been used to identify genomic sequences responsible for the attenuation phenotype of the P1/Sabin and P2/Sabin vaccine strains. Primary cell cultures derived from TgPVR mice differentiate between neurovirulent and attenuated virus strains, indicating that it may be possible to use these cultures to determine the functional basis of attenuation. Studies in TgPVR mice indicate that poliovirus tropism is not controlled solely by expression of PVR, and that poliovirus spreads from muscle to the central nervous system by neural pathways.

Transdominant inhibition of herpes simplex virus growth in transgenic mice.

A mutant allele (X25) of an essential regulatory protein, ICP4, encoded by herpes simplex virus (HSV) has been shown to have a transdominant, negative effect on the activity of the wild-type protein, resulting in the inhibition of virus growth in vitro. The X25 protein appears to exert its transdominant effect by sequestering functional ICP4 monomers into nonfunctional, heterodimeric complexes (A. Shepard, P. Tolentino, and N. A. DeLuca, 1990, J. Virol. 64, 3916-3926). In order to assess the antiviral potential of X25 in vivo, four transgenic mouse lines were generated bearing 1 to 10 copies of a DNA fragment encoding the mutant allele. Monolayers of embryonic cells prepared from each of the lines expressed the transgenic X25 protein. When challenged via the eye, every line exhibited at least some enhanced resistance to HSV infection. In the best line, transgenic animals exhibited a statistically significant (> 95% confidence) 5- to 13-fold lower eye swab titer relative to their nontransgenic littermates at Day 1 postinfection. A similar reduction in titer was observed in the trigeminal ganglia at Day 3 postinfection. These results indicate that the X25 protein is able to exert a significant antiviral effect in vivo.

The induction of cataracts by HIV-1 in transgenic mice.

OBJECTIVE: To elucidate the tissue specificity of the expression of HIV-1 genes in an animal and its pathological effects on these tissues. DESIGN AND METHODS: Transgenic mice carrying a defective HIV-1 genome were bred in order to overcome the host-range barrier of this virus. RESULTS: mRNA specific to the transgene was detected in the eyes and the spleen, and, in smaller quantities, in the thymus and the brain. Interestingly, many of the transgenic mice developed cataracts at 3-6 months of age. Swelling and vacuolation of the lens fiber cells were marked, but the epithelial cells of the lens were less affected. HIV antigens were detected in the lens fiber cells and the retina by immunological staining. Accumulation of large amounts of p24 Gag antigen was demonstrated in the affected lens by immunoblot analysis, while negligible Env or other viral proteins was detected. Although accumulation of the Gag protein was also detected in the skin and the brain, no apparent abnormality was observed in these tissues. CONCLUSIONS: Preferential expression of the HIV genes in the eyes, skin, brain and lymphoid tissues was demonstrated. The accumulation of the Gag protein is suggested to have detrimental effects on lens fiber cells, causing cataracts.

HBV production in transgenic mice.

We produced transgenic mice by microinjecting a partially duplicated copies of hepatitis B virus (HBV) gene into fertilized eggs of C57BL/6 mice. One mouse was a high producer of HBV surface antigen (HBsAg) and HBV e antigen (HBeAg) in the serum. All offspring carrying HBV DNA were positive for both antigens in the serum. The HBV DNA was expressed in liver- and kidney-specific manner. The normal process of HBV replication, including the packaging of the pregenome 3.5-kb RNA into a nucleocapsid, the reverse-transcription of the complete minus strand DNA, and the release of Dane particles into the serum before the completion of synthesis of plus strand, occurred in the liver of these transgenic mice. These results suggest that the species specificity of HBV infection is not due to the inability to replicate in nonnatural host but to the lack of receptors or factors needed for virus adsorption and internalization. The founder mouse is now 19 months of age but shows no clinical or pathological change, suggesting that HBV itself is not cytopathic.

Development of transgenic mice containing woodchuck hepatitis virus DNA.

We produced transgenic mice containing woodchuck hepatitis virus DNA with tandem repeat structure capable of producing virus and viral antigens. Poly(A)+ RNAs probably corresponding to pregenome and viral antigens were detected in their liver. These mice have now been healthy for one year. However, there is the possibility of inducing immunologically mediated hepatitis. We anticipate these transgenic mice may present a useful model system for studying the significance of chronic hepatitis in hepatocellular carcinoma development.

Transgenic mice as future tools in risk assessment.

Historically, mice have served a routine and useful purpose in the research, development, and testing of biologicals, chemicals, and drugs for efficacy, toxicity, and carcinogenic risk. The literature is replete with examples using mice to study organic compounds both in short-term tests involving tumor initiation and promotion and in long-term experiments dealing with fertility, reproduction, and teratology. During the past two decades, a virtual explosion of advances has occurred in modern biology that includes the discoveries of retroviruses, oncogenes, DNA restriction enzymes, nucleotide sequence analyses, and microinjection techniques. Fusion of these milestones in genetic, molecular, and cell biology with recent developments in mouse embryology has opened novel avenues and methods of experimentation as significant additions to the risk assessment armamentarium that currently uses both prokaryotes and eukaryotes. Some promising directions afforded by transgenic mice as powerful future tools in risk assessment will be summarized below.

Use of transgenic animals to study human retroviruses.

The purpose of transgenic technology is to introduce a gene into the germline of an animal in order to investigate its proper expression in the appropriate cell types and its effect on cellular functions. This technology has been used to explore a wide variety of genetic and biological issues including stage- and tissue-specific gene expression, development, immunology, oncology, and gene therapy. The intent of this article is to discuss the value of gene transfer technology for the study of mechanisms of viral pathogenesis. We will focus mainly on our experience with two experimental systems involving human T-lymphotropic virus Type 1 (HTLV-1) and human immunodeficiency virus Type 1 (HIV-1).

Activation of endogenous retroviral genomes in Mov strains of mice.

A collection of Mov mouse strains, each carrying in their germ line a Moloney murine leukaemia virus (M-MuLV) proviral genome at a different chromosomal location, was used to study expression of endogenous retroviruses. No M-MuLV-specific RNA was detected in the non-viraemic Mov strains studied, indicating that less than two copies of RNA are transcribed per cell. Virus expression was seen in three viraemic Mov strains. In Mov-3 mice the provirus was activated shortly after birth, whereas proviruses in Mov-9 and Mov-14 were activated at different stages of embryogenesis. The results suggest that the chromosomal position influences proviral expression during development. The first appearance of virus particles was not accompanied by detectable amounts of viral transcripts, suggesting that viraemia is a consequence of provirus activation in a small, as yet unidentified, population of cells, followed by virus spread and infection of susceptible cells.