Biomedical subjects
Publications and source records attributed to J W Hartley.
The four biologically distinct classes of murine leukemia viruses (MuLVs) are described and the potential for expression of each as infectious virus in laboratory mice is discussed. Commonly used in vitro methods for MuLV detection and identification are outlined.
Mice infected with LP-BM5 murine leukemia virus (MuLV) develop a syndrome denoted as murine AIDS. Macrophages harvested from the peritoneal cavities of these mice at 4 or 9 wk postinoculation with LP-BM5 MuLV were analyzed by Northern hybridization for the presence of the defective LP-BM5 virus and their ability to synthesize various cytokines upon induction with Newcastle disease virus (NDV) or (LPS). Neither IFN-alpha or IFN-beta was found to be constitutively expressed in LP-BM5-infected macrophages and in NDV induction studies, and the levels of biologically active IFN-alpha and its mRNA were found to be lower in LP-BM5 MuLV-infected macrophages than in the macrophages from uninfected controls. Similarly, after NDV or LPS induction, the levels of TNF mRNA and TNF protein were significantly lower in LP-BM5-infected macrophages than in macrophages from uninfected mice. The LP-BM5 MuLV-infected macrophages constitutively expressed low levels of IL-1 beta, and when induced with LPS, the relative levels of IL-1 beta were significantly higher in infected than in uninfected macrophages. Although no constitutive expression of IL-6 was detected, the levels of IL-6 mRNA induced with NDV were higher in LP-BM5 MuLV-infected macrophages than in controls. Thus, we found alterations in the expression of selected cytokines in macrophages from mice inoculated with LP-BM5 MuLV rather than a general deregulation of all cytokine expression. These results show that macrophages infected with the defective LP-BM5 virus respond differently to NDV- or LPS-stimulation and suggest that aberrant expression of certain cytokine genes may play a role in the immunopathologic condition in mice with murine AIDS.
Mice of certain strains are highly sensitive to development of a severe immunodeficiency disease following inoculation as adults with LP-BM5 murine leukemia viruses (MuLV) whereas others are extremely resistant. These strain-dependent differences in response to infection have been shown to be genetically determined with resistance to disease being, in general, associated with homozygosity for Fv-1n and H-2 haplotypes a and d and sensitivity with homozygosity for Fv-1b and other H-2 haplotypes including b, s, and q. The Fv-1b, H-2r strain RIIIS/J (RIIIS) was found to be highly resistant to disease even though B10.RIII(71NS)/J (B10.RIII), also H-2r, was very sensitive, thus excluding a role for H-2 in the resistance of RIIIS. The characteristics of RIIIS resistance were evaluated in studies of infected (B10.RIII x RIIIS) F1, F2 and reciprocal backcross mice. Resistance to disease was shown to be semidominant and determined by more than one gene, although a preponderant influence of a single gene was suggested. Studies of segregating populations showed that resistance was not associated with or linked to polymorphisms of the V beta complex or genes in proximity to the Emv-2 locus on chromosome 8. However, there was almost complete concordance between absence of disease in infected mice and inhibition of ecotropic virus spread. These results demonstrate that genes other than Fv-1 or H-2 can profoundly influence the development of retrovirus-induced immunodeficiency and replication of ecotropic viruses.
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LP-BM5 murine leukemia virus, a derivative of Duplan-Laterjet virus, contains a mixture of replication-competent B-tropic ecotropic and mink cell focus-inducing (MCF) viruses and a defective genome that is the proximal cause of a syndrome, murine AIDS (MAIDS), characterized by lymphoproliferation and immunodeficiency. The defective (BM5d) and ecotropic components of this mixture were molecularly cloned, and complete (BM5d) or partial (ecotropic) nucleotide sequences were determined. BM5d closely resembled the Du5H genome cloned from the Duplan virus, featuring a highly divergent p12 sequence in the gag open reading frame. In MAIDS-sensitive C57BL/6 mice, BM5d was detected in tissues within 2 weeks of infection but was absent from tissues of the MAIDS-resistant strain, A/J, 12 weeks after infection. B-cell-lineage tumors from mice with MAIDS contained and expressed BM5d, and clonal integrations of this genome were variably associated with clonal expansions of B cells in infected mice. Finally, mRNA crosshybridizing with a probe for BM5d was present in spleen but not kidney cells of uninfected B6 mice.
Host genetic determinants of resistance or susceptibility to a retrovirus-induced immunodeficiency syndrome, termed MAIDS, were evaluated in Fv-1b mice infected with the mixture of ecotropic, MCF, and defective murine leukemia viruses designated LP-BM5 murine leukemia viruses. Genes of the MHC were shown to exert a major influence on the development of disease and the extent of virus spread in mice infected as adults. Strains bearing the b, f, k, q, r, and s haplotypes were moderately to highly susceptible to MAIDS whereas mice of the d haplotype were the most resistant. Resistance to disease was strongly associated with inhibition of mink cell focus-inducing virus spread and, to a lesser extent, with inhibition of ecotropic virus expression. Mapping studies localizing resistance associated with the d haplotype to H-2Dd were confirmed by the demonstration that B6 mice carrying this gene as a transgene or by recombination were resistant to disease. Penetrance of resistance to disease associated with expression of H-2Dd was markedly influenced by MHC genes mapping to the left of H-2D and by non-MHC loci such that some strains bearing this gene were highly susceptible to MAIDS. The combined effects of MHC and background genes among 40 strains examined yielded a remarkably wide spectrum of disease phenotypes with the onset of advanced disease ranging from 10 wk to 72 wk postinfection. Resistance to disease in moderately to highly resistant strains was shown to develop with age. Unexpectedly, the disease resistant phenotype was found to be a recessive trait.
Transcriptional enhancers of replication-competent mouse C-type retroviruses are potent determinants of the distinct disease-inducing phenotypes of different viral isolates and can also strongly influence the incidence and latent period of disease induction. To study the contribution of individual protein-binding sites to viral pathogenicity, we introduced mutations into each of the known nuclear factor-binding sites in the enhancer region of the Moloney murine leukemia virus and injected viruses with these mutations into newborn NFS mice. All viruses induced disease. Viruses with mutations in both copies of the leukemia virus factor a (LVa) site, leukemia virus factor c (LVc) site, or in just the promoter proximal copy of the glucocorticoid response element (GRE) had a latent period of disease onset and disease specificity indistinguishable from that of the wild-type Moloney virus. Viruses with mutations in two or three of the GREs, in both copies of the leukemia virus factor b (LVb) site, in two of the four nuclear factor 1 (NF1) consensus motifs, or in both copies of the conserved viral core element showed a significant delay in latent period of disease induction. Strikingly, viruses with mutations in the core element induced primarily erythroleukemias, and mutations in the LVb site also resulted in a significant incidence of erythroleukemias. These and other genetic and biochemical studies suggest models for how subtle alterations in the highly conserved structure of mouse C-type retrovirus enhancers can produce a dramatic effect on disease specificity.
A line of transgenic mice that express the human adenovirus type 12 E1A and E1B genes under the regulatory control of the mouse mammary tumor virus long terminal repeat was studied. Mice from this line develop olfactory neuroblastomas at approximately 6 months of age. Large numbers of type C retrovirus (ecotropic murine leukemia virus) particles were found in the tumor rosettes. No similar examples of virus activation were identified in tumors from other transgenic experiments. Examination of spontaneous olfactory neuroblastomas from three domestic cats also demonstrated retrovirus in tumor rosettes.
We have developed and used a system for recording and analyzing the electrocardiogram (ECG) of the horse during exercise. The system consists of a commercial ECG transmitter telemetering a Z lead (base-apex) ECG from an exercising horse. The received data are then remodulated at an audio frequency and stored on a audio cassette recorder. Exercise protocols of up to 10 minutes are digitized using a Macintosh II computer. For rhythm analysis, a computer program to identify the various waves of the ECG uses a modification and refinement of the integrated-squared-derivative (ISD) technique. This technique consists of frequency selective digital filtering and differentiation followed by integration of the squared derivative. The waves of the ECG are identified using adaptive thresholding of the ISD and the derivative of the ECG. Thus far, we have been able to successfully identify the Q, R, and the end of T in ECGs severely contaminated by exercise artifact. The system can place the absolute time of occurrence of the Q, R, and end of T in a text file for export to other analysis programs such as spreadsheets and statistics packages.
OBJECTIVE: To define an outbreak of bacteraemia due to coagulase negative staphylococci highly resistant to ciprofloxacin in a leukaemia unit, investigate the source and mode of spread of the outbreak strain, and assess control measures. DESIGN: The outbreak strain was characterised by five different typing methods. Surveillance of patients, staff, and environment was carried out during the outbreak and five months after control measures were introduced. SETTING: A unit with 10 beds for adults with leukaemia and patients receiving bone marrow transplants. The outbreak occurred during a trial of ciprofloxacin for empirical treatment of neutropenic fevers. INTERVENTIONS: Ciprofloxacin was withdrawn from use in the unit and daily bathing with chlorhexidine gluconate solution started. Main outcome measure--The absence of bacteraemia due to the outbreak strain for five months after control measures. RESULTS: During the study 49 patients developed 21 episodes of bacteraemia due to the outbreak strain, which was ciprofloxacin resistant (minimum inhibitory concentration greater than or equal to 128 mg/l), susceptible to phage 155 A9C, and SII biotype and had characteristic immunoblot and DNA fingerprint features. There was a high amount of colonisation of patients but not staff with this strain, which was also wide spread in the environment. The control measures led to rapid resolution of the outbreak and disappearance of the strain from the unit. CONCLUSIONS: In areas where coagulase negative staphylococcal infections are common doctors must be aware of the possibility of cross infection with single strain, and the availability of more discriminatory methods of typing will facilitate the identification and control of such episodes.
California wild mouse-derived ecotropic virus Cas-Br-M induces a spongiform encephalopathy and a wide variety of hematopoietic neoplasms on inoculation of neonatal mice. We isolated a MCF virus [Ns-6(186) MCF] from a thymic T-cell lymphoma developing in a NFS mouse inoculated with Cas-Br-M virus. Biologically cloned NS-6(186) MCF virus, in contrast to previously studied MCF viruses, was found to induce thymic or nonthymic T-cell lymphomas with high efficiency in the absence of ecotropic helper virus. Comparison of the restriction endonuclease maps derived from Cas-Br-M and NS-6(186) MCF revealed differences only in the env region, between 5.8 and 7.8 kb from the 5' end. Two biologically active molecular clones of the NS-6(186) MCF (clone 15 with two LTRs and clone 19 with 1 LTR) were studied. Although both clones exhibited similar in vitro activities, clone 15-derived virus induced only T-cell lymphomas with short latency whereas clone 19-derived virus induced a wide variety of neoplasms with a significantly longer latency. Nucleotide sequence analysis established that the U3 region of each of the two LTRs of clone 15 has a 53-bp duplication which includes "enhancer elements," but that the single LTR of clone 19 has no such duplication.
C57BL/6 mice infected with a mixture of murine leukemia viruses (MuLV) develop a syndrome characterized by lymphoproliferation and profound immunodeficiency. Analyses of this viral mixture (LP-BM5 MuLV) showed that it includes replication-competent ecotropic and mink cell focus-inducing MuLV and defective viruses with genome sizes of 3.8-6.5 kilobases. The ecotropic and mink cell focus-inducing MuLV biologically cloned from the mixture did not induce disease, whereas viral preparations containing the ecotropic MuLV and 4.8-kilobase defective virus were active. Cells producing the 4.8-kilobase defective virus expressed an unusual gag-encoded polyprotein of Mr 60,000.
Cas NS-1 is an acutely transforming murine retrovirus that induces pre-B and pro-B cell lymphomas. Molecular cloning showed it was generated from the ecotropic Cas-Br-M virus by sequential recombinations with endogenous retroviral sequences and a cellular oncogene. The oncogene sequence shows no homology with known oncogenes but some similarity to the yeast transcriptional activator GCN4. A 100-kDa gag-cbl fusion protein, with no detectable kinase activity, is responsible for the cellular transformation. The cellular homologue of v-cbl, present in mouse and human DNA, is expressed in a range of hemopoietic lineages.
The nondefective Moloney and Friend murine leukemia viruses induce T-cell lymphomas and erythroleukemias, respectively, after being injected into newborn NFS mice. In previous studies, we showed that the distinct disease specificities of the two viruses could be switched by exchanging a small segment, about 200 nucleotides in length, encompassing their enhancer regions. This segment included the direct repeat sequence and an adjacent GC-rich region of about 20 nucleotides defined in studies of Moloney murine sarcoma virus enhancer-promoter function (L. A. Laimins, P. Gruss, R. Pozzatti, and G. Khoury, J. Virol. 49:183-189, 1984). The direct repeats of Friend and Moloney viruses are identical in a central core sequence of 32 nucleotides but have sequence differences on either side of this core as well as in their GC-rich segments. To determine whether disease specificity resides in part or in all of the direct repeat and GC-rich region, we constructed recombinants between Friend and Moloney viruses within this segment and tested them for their disease-inducing phenotypes. We found that disease specificity, in particular the ability of Friend virus sequence to confer erythroleukemogenicity on Moloney virus, is encoded throughout the region in at least three separable segments: the 5' and 3' halves of the direct repeat and the GC-rich segment. When just one of these segments (either both 5' halves of the direct repeat, both 3' halves, or just the GC-rich segment) from Friend virus was substituted into a Moloney virus genome, it conferred only a negligible or low incidence of erythroleukemia (less than or equal to 5% to between 10 and 15%). Any two segments together were considerably more potent (35 to 95% erythroleukemia), with the most effective pair being the two halves of the direct repeat. Individual segments and pairs of segments were considerably more potent determinants when they were matched with a genome of the same origin. Thus, although sequences outside the enhancer region are minor determinants of disease specificity when the enhancer is derived entirely from either Friend or Moloney virus, they can play a significant role when the enhancer is of mixed origin. Some recombinant enhancers conferred a long latent period of disease induction. This was particularly striking when the 5' halves of each copy of the direct repeat sequence were derived from Moloney virus and the 3' halves were derived from Friend virus.(ABSTRACT TRUNCATED AT 400 WORDS)
C57BL mice (Fv-1b) develop a severe immunodeficiency disease following inoculation as adults with LP-BM5 murine leukemia virus (MuLV), a derivative of Duplan-Laterjet virus which contains B-tropic ecotropic and mink cell focus-inducing MuLVs and a putative defective genome which may be the proximal cause of disease. The stages of development of this disease were defined for C57BL mice on the basis of lymphadenopathy and splenomegaly; histopathological changes consistent with B-cell activation; and alterations in expression of cell surface antigens affected by proliferation of T cells, B cells, and macrophages. By using this disease profile as a standard, the response of adult mice of various inbred strains and selected F1 hybrids was compared. We show that although the strains which are highly sensitive are of the Fv-1b genotype (i.e., permissive for B-tropic MuLVs), certain Fv-1b strains, e.g., BALB/c and A/J, are resistant to murine acquired immunodeficiency syndrome, whereas certain Fv-1n strains (permissive for N-tropic MuLVs but restrictive for B-tropic MuLVs), notably P/N, BDP, and AKR, show moderate sensitivity and (C57BL/6 x CBA/N)F1 mice (Fv-1n/b and thus dually restrictive) are of relatively high susceptibility. The results of virus recovery tests suggest that apparently anomalous sensitivity, based on predicted Fv-1 restriction, may reflect MuLV induction and/or mutation to provide a helper virus for which the host is permissive.
Selected conserved amino acids in the putative RNase H domain of reverse transcriptase (RT) were modified in a molecularly cloned infectious provirus and in a Moloney murine leukemia virus RT expression vector by site-directed mutagenesis. Substitution of either of two conserved aspartic acid residues in proviral DNA prevented production of infectious particles in transfected NIH 3T3 cells, and the same modifications depressed RT-associated RNase H activity by more than 25-fold with little or no effect on polymerase activity.
The susceptibility to tumors induced by raf and raf/myc retroviruses was investigated in BALB/c, C57BL/6, (BALB/c x C57BL/6)F1 and (BALB/c x C57BL/6) backcross mice. Newborn mice were susceptible to neoplasms generated by both viruses, but resistance to raf-induced leukemia developed rapidly in all mice as they matured. Older C57BL/6 mice were also resistant to raf/myc lymphomas, whereas BALB/c mice remained susceptible to the virus at all ages, indicating that different genes control susceptibility to raf and raf/myc tumors. From these data and the susceptibility of C x B recombinant inbred strains, it appears that very few genes (perhaps even a single gene) may govern susceptibility to raf/myc lymphomas and that resistance is the dominant trait.
A portable apparatus for performing repeated, brief coronary artery occlusions automatically in long-term chronic studies involving dogs and larger animals is described. The battery-operated, back-pack-carried device uses a motorized syringe driven by a digital-programmable timing circuit to inflate a coronary artery occluder for durations of 10 s to 3 min at intervals from 14 s to 16 h. A single battery charge produces 450 occlusions. The generated pressure is adjustable to 1,000 mmHg maximum. A separate fail-safe circuit monitors system operation to open a pressure-relief valve if occlusions exceed a preset duration.