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P311 is an 8-kDa protein that is expressed in many brain regions, particularly the hippocampus, cerebellum and olfactory lobes, and is under stringent regulation by developmental, mitogenic and other physiological stimuli. P311 is thought to be involved in the transformation and motility of neural cells; however, its role in normal brain physiology is undefined. To address this point, P311-deficient mice were developed through gene targeting and their behaviors were characterized. Mutants displayed no overt abnormalities, bred normally and had normal survival rates. Additionally, no deficiencies were noted in motor co-ordination, balance, hearing or olfactory discrimination. Nevertheless, P311-deficient mice showed altered behavioral responses in learning and memory. These included impaired responses in social transmission of food preference, Morris water maze and contextual fear conditioning. Additionally, mutants displayed altered emotional responses as indicated by decreased freezing in contextual and cued fear conditioning and reduced fear-potentiated startle. Together, these data establish P311 as playing an important role in learning and memory processes and emotional responses.

The murine cytomegalovirus (MCMV) monoclonal antibody 5C7:6 was used in Western analysis to probe MCMV infected murine embryo cells (MEC). This antibody recognizes three virus specific polypeptides of 130, 105, and 95 kDa and pulse–chase experiments demonstrated that these three proteins, although antigenically related, are distinct. The 105- and 95-kDa species were expressed with early kinetics, whereas the 130-kDa protein was synthesized as a true late. By screening a λgt11 MCMV cDNA library, the gene encoding these proteins was identified as the M25 open reading frame previously reported by Dallas et al. (Dallas, P. B., Lyons, P. A., Hudson, J. B., Scalzo, A. A., and Shellam, G. R., 1994, Virology 200, 643–650). Immunofluorescent studies monitored the location of pM25, present in the nucleus at 15 h after infection, condensing around the periphery of the nucleus at 18 h, before finally accumulating in the cytoplasm. Immunoelectron microscopy detected gold particles associated with the viral tegument of enveloped virions located in the cytoplasm and extracellular space but not with naked nucleocapsids. Western analysis of MCMV purified virions depicted the presence of the 130-kDa protein, the predominant M25 species, in mature virus particles. Together these findings provide compelling evidence that the 130-kDa M25 polypeptide is a component of the viral tegument.

Crohn's disease (CD) is a chronic, immune-mediated, inflammatory disorder of the intestine that has been linked to numerous susceptibility genes, including the immunity-related GTPase ( IRG) M ( IRGM). IRGs comprise a family of proteins known to confer resistance to intracellular infections through various mechanisms, including regulation of phagosome processing, cell motility, and autophagy. However, despite its association with CD, the role of IRGM and other IRGs in regulating intestinal inflammation is unclear. We investigated the involvement of Irgm1, an ortholog of IRGM, in the genesis of murine intestinal inflammation. After dextran sodium sulfate exposure, Irgm1-deficient [Irgm1 knockout (KO)] mice showed increased acute inflammation in the colon and ileum, with worsened clinical responses. Marked alterations of Paneth cell location and granule morphology were present in Irgm1 KO mice, even without dextran sodium sulfate exposure, and were associated with impaired mitophagy and autophagy in Irgm1 KO intestinal cells (including Paneth cells). This was manifested by frequent tubular and swollen mitochondria and increased LC3-positive autophagic structures. Interestingly, these LC3-positive structures often contained Paneth cell granules. These results suggest that Irgm1 modulates acute inflammatory responses in the mouse intestine, putatively through the regulation of gut autophagic processes, that may be pivotal for proper Paneth cell functioning.

Monoclonal antibodies were isolated from the spleen of a latently infected mouse to identify possible antigenic markers for latent murine cytomegalovirus infection. One antibody, AM3, was selected for further study. Characterization of the antigen recognized by AM3 confirmed that it is the early-late phosphoprotein pp50. The antigen was readily detected by immunoautoradiography in the spleens of acutely and latently infected mice. Low levels of the AM3/pp50 transcript were also detected in latently infected tissues by in situ hybridization. Presence of AM3/pp50 mRNA, as well as IE-1 transcripts, was confirmed by reverse transcription-polymerase chain reaction in 3 of 5 latently infected spleens. The expression of both immediate-early and early-late classes of viral genes suggests that persistent infection may be a component of the MCMV life cycle operationally defined as latency and that this gene is not specific for latency.

The spleens of mice latently infected with murine cytomegalovirus were examined by reverse transcription followed by the polymerase chain reaction to assay for transcriptional activity of the viral immediate early 1 gene. Transcripts were detected in 6 of 8 animals at a level between 50 and 500 copies in 200 ng of total cellular RNA. This level appears to be roughly 10-fold higher than that of viral genome even though viral DNA is more readily detectable. These data suggest that a significant fraction of latent murine cytomegalovirus is transcriptionally active in the spleen, which may be indicative of an abortive infection or possibly low-level persistence or a transient reactivation.

The polymerase chain reaction (PCR) was used to detect human immunodeficiency virus type 1 (HIV-1) proviral sequences (gag and env) in nucleated cells from the cerebrospinal fluid (CSF) of 31 HIV-1-positive patients, and the results were compared with clinical and radiologic evidence of neurologic disease. Provirus was detected in 21 patients, of whom 20 had neurologic abnormalities. Provirus was not detected in another 6, all of whom were neurologically normal. No neurologic disease has developed in 4 of these 6 patients for whom 12.8 months of follow-up is available. PCR of CSF nucleated cells from HIV-positive patients provides early, rapid, direct evidence of neurologic involvement.

We cloned six mycobacterial antigens into a mammalian expression vector as fusion proteins with the enhanced green fluorescent protein (EGFP). Plasmid DNA was injected intramuscularly, and the injection sites were examined 1 week later. Expression of each antigen-EGFP fusion protein was visualized as green fluorescence in muscle tissue sections. A plasmid expressing EGFP alone and a plasmid with a frameshift mutation served as positive and negative controls. Visualization of fluorescent protein in vivo was 100% specific when compared to in vitro results. In vivo sensitivity was only 37% based on individual injection sites, but increased to 100% when results from multiple injection sites were combined for each plasmid. EGFP alone was expressed in a higher proportion of myocytes than the antigen-EGFP fusion proteins (P < 0.001). There was a trend toward an inverse correlation between protein size and the proportion of myocytes with visible fluorescence (r = -0.68; P = 0.09). We compared antibody subtypes generated to Mycobacterium bovis antigen 85A, when it was expressed alone or as a fusion protein. Inclusion of EGFP modified the immune response toward a Th1 response, as indicated by the ratio of antigen 85A-specific IgG2a to IgG1 generated by each plasmid (antigen 85A alone 0.73 +/- 0.18 versus antigen 85A-EGFP 1.82 +/- 0.57, mean +/- S.D.; P < 0.01), though the magnitude of the antibody isotype shift was modest. Direct visualization of antigen-EGFP fusion proteins provided a simple and rapid method to confirm in vivo antigen expression.

Mycobacterium avium causes disseminated disease in humans with AIDS, paratuberculosis in ruminants, lymphadenopathy in swine, and tuberculosis in birds. We constructed DNA vaccines expressing mycobacterial antigens as fusion proteins with enhanced green fluorescent protein (EGFP). Plasmids p65K-EGFP, p85A-EGFP, and p85B-EGFP expressed the M. avium 65-kDa antigen, the Mycobacterium bovis BCG 85A antigen, and the M. avium 85B antigen, respectively, as EGFP fusion proteins. We visualized protein expression directly in cultured murine fibroblasts and intact muscle. p65K-EGFP expressed fusion protein in a diffuse cytoplasmic pattern, and p85A-EGFP and p85B-EGFP produced a speckled pattern. We vaccinated C57BL/6 mice with three doses of plasmid DNA and then challenged them intraperitoneally with M. avium . Negative controls received saline, and positive controls received one dose of BCG vaccine. Mice in all groups developed disseminated infection with a high burden of organisms. Compared to negative controls, mice vaccinated with p85A-EGFP had an eightfold reduction in spleen M. avium CFU at 4 weeks after infection and a fourfold reduction at 8 weeks, reductions similar to those generated by BCG vaccine. Mice vaccinated with p65K-EGFP had a fourfold CFU reduction at 4 weeks and no effect at 8 weeks. This is the first report of DNA vaccines expressing foreign antigens as fusion proteins with EGFP and the first report of successful DNA vaccination against M. avium .

The hypothesis that replication-competent mouse cytomegalovirus (MCMV) is detectable in severe combined immunodeficient (scid) mice after implantation of latently infected tissue was examined. Sections of latently infected salivary gland from 5 BALB/c mice were implanted into 20 C.B-17 scid mice. Recipient scid mice were sacrificed weekly for 5 weeks, and MCMV infection was detected in target organs using culture and DNA polymerase chain reaction (PCR). All donors were negative by standard culture but positive by DNA PCR. Replicating MCMV was recovered from 9 of 15 recipient scid mouse salivary gland, lung, liver, or spleen samples at postoperative weeks 2-5. No virus was recovered from recipient scid mice at weeks 1 or 12. Transplantation of latently infected tissues into scid mice resulted in rapid reactivation and dissemination of the virus. Further study of this model promises insight into the mechanisms of CMV latency and reactivation.