Further evidence for deletion of envelope glycoprotein (gp69/71) sequences in formation of Moloney-murine sarcoma virus.

Moloney-murine sarcoma virus (S+L- strain of M-MSV) has been nonproductively cloned in murine and non-murine host cells (S+L- cells) and the expression of Moloney leukaemia virus (M-MuLV) 30000 mol. wt. core protein (p30) and envelope glycoprotein (gp69/71) were studied by radioimmunoassay. Antigenic determinants of the M-MuLV p30 were associated with the sarcoma virus genome in these non-productively transformed cell clones studied, while the determinants of M-MuLV gp69/71 were not. The absence of envelope-associated glycoprotein expression in sarcoma virus transformed cells was confirmation of biological studies demonstrating that rescued sarcoma virions acquire envelope-associated properties of host range, neutralization and interference from rescuing helper virus, and further evidence that the M-MuLV gp69/71 sequences have been deleted during the formation of the M-MSV. During the course of these studies, it was also found that S+L- dog cells were releasing into culture supernatant large amounts of the p30 antigenic determinant, apparently as a soluble antigen.     

Depression of Rauscher leukemia virus envelope glycoprotein gp71 binding by lymphoid cells during leukemogenesis in mice.

The availability of membrane receptors for the 71,000-dalton envelope glycoprotein (gp71) of Rauscher murine leukemia virus on splenic and thymic cells from BALB/c mice during Rauscher murine leukemia virus-induced leukemogenesis was determined utilizing a radiolabeled gp71 binding assay. Shortly after infection, the relative cellular [125I]gp71 binding level decreased, first with splenic cells (at day 7 to 10 after infection) and later with thymic cells (at day 10 to 20 after infection). The dependency of the reduction of binding on the replication of the inoculated virus was demonstrated by regression analyses using cellular gp71 binding level as the dependent variable and infectious virus titer, as well as viral gp71 and p30 levels, of spleens and thymuses from infected mice as independent variables. With each independent variable, the reduction of gp71 binding for both cell types was highly dependent (P less than 0.01) on the level of virus detected in their respective organ. In the early stages of leukemogenesis, the [125I]gp71 binding level declined to approximately 20 to 30% of control values. During this period the rate of reduction of binding was very rapid and, in general was similar for both splenic and thymic cells. Further progression of the disease resulted in little or no further reduction in binding. The application of this technique to monitor host ecotropic virus synthesis and to study cell surface virus receptor control mechanisms in vivo is discussed.     

Structural analysis of the major envelope glycoprotein (gp70) of the amphotropic and ecotropic type C viruses of wild mice.

Virus-associated and free serum gp70s from wild mice (Mus musculus) were isolated by immunoaffinity column chromatography and were analyzed by two-dimensional tryptic peptide mapping procedures. Our results show that the gp70s of wild mouse ecotropic and amphotropic MuLVs are structurally divergent. The gp70s of wild mouse ecotropic viruses are more similar in structure to the Gross-AKR MuLV subgroup than to the FMR subgroup. The gp70s of the amphotropic wild mouse viruses are highly conserved in structure and are closely related to those of one group of xenotropic MuLVs. Non-virion-associated free serum gp70 of wild mice is homologous to that of inbred laboratory mouse strains.     

An ultrastructural study of the glomerular slit diaphragm in New Zealand black/white mice.

Glomerular epithelial slit alterations and their relation to proteinuria have not been studied in detail in New Zealand Black/White (NZB/W) mice. The kidneys of proteinuric and nonproteinuric female NZB/W mice and normal Swiss albino mice were perfusion-fixed with tannic acid-glutaraldehyde and studied by light and electron microscopy. Semiquantitative studies were performed on full montages of glomeruli enlarged 10,000 times. Fine structural alterations of the epithelial slits, with emphasis on the slit diaphragm, were studied on semiserial thin sections. Proteinuric NZB/W mice with features of membraneous nephropathy exhibited: (1) wedging of electron-dense deposits below the slit diaphragm, (2) enlargement and distortion of interpedicel spaces, (3) displacement, folding, and stacking of slit diaphragms, (4) formation of occluding junctional complexes in residual slits, and (5) variable loss of foot processes. Similar alterations were not observed in controls or nonproteinuric NZB/W mice, including animals having complexes inglomerular mesangia but not in epithlialslits. These studies show that in NZB/W mice, abnormal protein excretion is associated with structural modification of the slit pore and suggest a role for such a component in the process of protein ex     

Properties of mouse leukemia viruses. XVI. Suppression of spontaneous fetal leukemias in AKR mice by treatment with broadly reacting antibody against the viral glycoprotein gp 71.

AKR mice were treated early in life with antibodies prepared in a goat against the major glycoprotein gp71 of Friend murine leukemia virus (FL V) and evaluated over a period of up to 2 years for various parameters associated with AKR thymoma/lymphoma. If both the mothers and offspring were given the antibody, suppression of AKR disease was observed to the degree that the 50% incidence of leukemia was delayed by about 1 year. A lesser effect was found if the treatment was initiated at an age of 3 days and application of the antibody to either mothers alone, or treatment of the mice at later times (39 days) was not successful. Thus, it was concluded that the critical period for treatment is between birth and the first few days of life. Postmortem examination of animals that were successfully treated during this early period revealed a significant proportion exhibiting a unique leukemic pattern. In addition, several died from nonleukemic neoplasms as well as other causes. Levels of virus or antiviral antibodies were also determined at various times in individual mice which had been segregated in family groups. The results demonstrated that overall, successful antibody treatment resulted in (1) suppression of virus and (2) development of significant levels of antiviral antibodies. In contrast, control mice exhibited the opposite pattern: high levels of virus and no detectable antibody. Moreover, it could be shown that antibody treatment reduced the incidence of MCF-like recombinant virus isolation. A hypothesis is presented which suggests that the main function of the administered antibody is to disturb a key event occurring during the early stages of life of the AKR mouse, which is of critical importance for the development of leukemia from 6 months of age onwards.     

Quantitative flow cytometry of mouse mammary tumor virus envelope glycoprotein (gp52): alternative measures of hormone-mediated change in a viral cell surface antigen.

An immunofluorescence procedure with C3H mouse mammary tumor cells (Mm5mt/cl) has incorporated flow cytometry to provide a fluorescence-based measurement of changes in the mouse mammary tumor virus (MMTV) cell surface glycoprotein (gp52). A comparison of mean channel fluorescence intensity (delta mean) of cell populations stained with immune sera and NRS permitted a gp52-specific signal to be measured for controls and cells treated with 10(-6) M dexamethasone (Dex). Three different methods have been developed to quantitatively compare gp52-related fluorescence on control and hormone-treated cells. First, delta mean, measured as a gp52-specific difference in channel number was 169-209 for control cells and 299-341 for Dex-treated cells. These fluorescence measurements with 4 different sera demonstrated gp52-specific increases due to Dex treatment of 141, 130, 143, and 115 channels. A second method of gp52 quantitation determined the percentage shift in staining populations over NRS and specified channel intensity markers. Dex treatment resulted in a 6.9 to 32.4% shift over channel 508 (NRS marker) and a more marked shift of 45.5 to 49.2% over channel 676 (control cell marker). A third methodology utilized fluorescein bead standards to calculate molecules of equivalent soluble fluorescein (MESF). These MESF determinations permitted hormonal effects to be measured as fold increases over controls. Dex induction of gp52 for C3H and GR mammary tumor cells ranged from 1.5 to 9.1 fold increases. Alternative steroid treatments and antibody directed against the internal cytoplasmic MMTV P27 provided negative controls for measurements of changing gp52 levels.     

Properties of mouse leukemia viruses: IX. Active and passive immunization of mice against friend leukemia with isolated viral GP71 glycoprotein and its corresponding antiserum

Mice could be protected against Friend leukemia virus-induced leukemia by inoculation with the major viral glycoprotein GP₇₁ or its antiserum.     

Expression of viral envelope glycoprotein and transformation genes in cells transformed by a defective Kirsten murine sarcoma virus.

Concurrent expression of transformation properties and virion envelope glycoproteins has been observed as a property of several clones of normal rat kidney cells transformed by, but not producing, Kirsten murine sarcoma virus. The present studies were carried out to determine whether a genetic linkage exists between the viral sarcoma and envelope genes in these cells. Several alternative models for the possible structure and origin of the sarcoma and envelope genes were considered. One possibility, that the viral envelope gene was derived from an endogenous rat virus, was studied by characterization of the interference properties of the transformed cells. The sarcoma virus genome of envelope-positive clones was efficiently rescued by woolly monkey and murine xenotropic but not by murine ecotropic viruses. Thus, the interference properties of cells producing the envelope glycoprotein are analogous to those of a cell producing murine ecotropic virus, indicating that the envelope was of murine viral origin. In these experiments it was also found that sarcoma viruses rescued from envelope-positive cells upon superinfection with primate and xenotropic murine viruses could transform host cells for both xenotropic and ecotropic viruses, indicating that these superinfecting viruses became phenotypically mixed with the ecotropic envelope expressed in transformed, envelope-positive cells. Possible linkage between the envelope and transformation genes was analyzed by the frequency of concurrent rescue of sarcoma and envelope genes. Transfer of the Kirsten sarcoma viral genome to uninfected cells upon rescue by superinfection with woolly monkey virus showed a high frequency of apparent segregation of the transformation and envelope genes [from 29 to 57% for (KSV env⁺)NRK-6]. The model supported by the present data is that the transformed, envelope-positive cells were infected with a virus which contained both the envelope and the sarcoma genes.     

The envelope glycoprotein of HIV-1 gp120 and human complement protein C1q bind to the same peptides derived from three different regions of gp41, the transmembrane glycoprotein of HIV-1, and share antigenic homology

Gp41, the transmembrane glycoprotein of HIV-1, has been shown to be non-covalently associated with gp120. We have shown that it also binds human C1q. To analyze the interaction site(s) of gp41 with these two molecules, we established an enzyme-linked immunosorbent assay (ELISA) system using recombinant soluble gp41 [amino acids (aa) 539–684] and peptides thereof. In the cell-external part of gp41 three sites (aa 526–538, aa 590–613 and aa 625–655) were found to bind both gp120 and C1q. That gp120 and C1q use the same sites was evidenced by the fact that these proteins competed with each other for the same sites in recombinant soluble gp41 and gp41 peptides. It could be demonstrated by ELISA, that rabbit antibodies against human C1q recognized gp120, and rabbit antibodies against gp120 cross-reacted with C1q. Rabbit anti-gp120, HIV-1-positive human sera and anti-gp120 obtained from such sera agglutinated sensitized sheep erythrocytes with human C1q (EAC1q). These data suggest that in addition to functional homology between C1q and gp120 structural homology between these two molecules exists. This molecular mimicry might become the basis for immunologically relevant autoimmune phenomena.     

Human MoAbs produced from normal, HIV-1-negative donors and specific for glycoprotein gp120 of the HIV-1 envelope.

Human MoAbs of IgM class were developed against three regions of the HIV-1 envelope. Uninfected donor lymphocytes were immunized in vitro with recombinant protein pB1. Four out of five antibodies were directed to different parts of the V3 region, which contains a major neutralizing site. Two out of these antibodies were directed to more than one amino acid sequence, indicating reactivity to discontinuous sites. Two of the human MoAbs inhibited viral spread between cells in tissue culture, interpreted as reactivities to conserved amino acid sequences exposed during viral maturation. No MoAb neutralized virus, which may be explained by the relatively low avidity of the antibodies. One MoAb was directed to a region containing amino acids participating in CD4 binding. This technique appears to allow formation of antibodies with fine specificities other than those obtained in infected hosts.     

Stimulation of antibody response in the gastrointestinal mucosa of immunodeficient mice by oral treatment with bacterial antigens. An immunoperoxidase study

The immunodeficient (nude) mice were chosen as a model to verify the in vivo stimulating activity of bacterial antigens on the humoral immune response. By using an immunoperoxidase technique, the Ig+ cell content in the gastro-intestinal mucosa of mice was evaluated after oral treatment with a mixture of bacterial antigen fractions (trade name Colopten). Treatment for 15 days was able to induce a significant increase in the proportions of Ig+ cells in both the jejunum and ileum. In contrast, the number of Ig+ cells was significantly increased after 30 days of treatment throughout the gastro-intestinal tract. Based on the staining intensity, a semiquantitative evaluation of the Ig content of the cells was made. Strongly stained Ig+ cells were localized into the gastro-intestinal mucosa during treatment and appeared to be the prominent lymphoid cell population in the small bowel after prolonged administration of Colopten. The morphological analysis of tissues showed that after treatment Ig+ cells tended to be collected within the mucosa rather than being isolated as in untreated animals. Therefore, these results demonstrate that oral administration of Colopten was able to elicit a local humoral immune response in an animal model for severe immunodeficiency.     

5-Azacytidine inhibits the lpr gene-induced lymphadenopathy and acceleration of lupus-like syndrome in MRL/MpJ-lpr/lpr mice.

MRL/MpJ-lpr/lpr mice spontaneously develop a lupus-like autoimmune disorder characterized by massive proliferation of T cells and rapidly fatal immune complex glomerulonephritis. We evaluated the therapeutic effect of 5-azacytidine (5AC), a cytidine analogue known as an inhibitor of DNA methylation, in MRL/MpJ-lpr/lpr mice. Intraperitoneal injection of 5AC (50 micrograms, twice a week) starting from 6 weeks of age retarded the development of lymphadenopathy and autoimmune syndrome. Its beneficial effects included: (a) increased life-span, (b) diminution of lymphadenopathy and splenomegaly, (c) reduction in circulating levels of autoantibodies such as anti-DNA and rheumatoid factors, and (d) suppression of lupus glomerulonephritis. However, similar treatment in BALB/c mice did not affect the development of IgG anti-human IgG antibody responses. These results suggest that the protective effect of 5AC is related to the inhibition of the lpr gene-induced T cell proliferation, thereby suppressing the autoimmunity-accelerating effect mediated by the lpr gene.     

Immunoglobulin and complement deposits in the lungs of New Zealand Black/White mice

Summary Forty-four New Zealand Black/White (NZB/W) F1 hybrid mice were studied for evidence of immune complex deposition in the lungs and kidneys. Positive immunofluorescence was seen in the lungs of 27 mice. The pattern of lung fluorescence was granular in association with capillary walls in 16 mice and intracellular in 12. The incidence of granular fluorescence was increased with age and was seen in 80% of the lungs of mice over 13 months old.There was a correlation between capillary wall fluorescence in the lungs and the kidneys (P<0.001) and between lung cellular fluorescence and renal mesangial fluorescence (P<0.001). The role of immune complex deposition in human pulmonary disease is discussed.     

An immunoperoxidase study of epithelial marker antigens in ulcerative colitis with dysplasia and carcinoma.

An immunoperoxidase technique was applied to formalin and Helly fixed paraffin wax sections from cases of ulcerative colitis complicated by dysplasia and carcinoma for carcinoembryonic antigen and components of the colonic secretory immunoglobulin system--namely, secretory component, IgA, and J chain. Sections from both resection specimens and mucosal biopsies were available. Intensity of immunostaining was assessed qualitatively. There was appreciable variation in expression of carcinoembryonic antigen and secretory component antigens. Carcinoembryonic antigen stained heavily in dysplasia and carcinoma while these tissues showed only focal light staining for secretory component. Normal tissue stained heavily for secretory component. The variation in staining intensity for both carcinoembryonic antigen and secretory component in inflamed and regenerative mucosa precluded their use as a reliable diagnostic aid in discriminating these tissues from true dysplasia. Loss of secretory component production or transport or both may be incurred during malignant change, but it should not be assessed as an isolated index of epithelial maturity. The relation with mucosal plasma cells warrants further study to determine more fully the factors affecting tissue secretory component expression.     

Macrophage clearance function and immune complex disease in New Zealand Black/White F1 hybrid mice.

Macrophage clearance function in NZB, NZW, NZB/W, Ajax and B10D2 new mice was assessed by measurement of the rate of clearance (KPVP) of intravenously-injected 125I-labelled polyvinylpyrrolidone (PVP). There were significant strain and age-related variations in KPVP. In particular there was a marked fall in KPVP in NZB/W mice with increasing age. This fall was most apparent in female NZB/W and preceded the age at which renal disease usually develops in these animals. We suggest that ineffective macrophage function and production of low affinity antibody contribute to the early development of immune complex glomerulonephritis in these mice.     

Distribution of LDH-1 in normal, ischemic, and necrotic myocardium. An immunoperoxidase study

To study the distribution of lactate dehydrogenase (LDH-1) (H4) in normal, ischemic, and necrotic myocardium using the peroxidase-antiperoxidase technic, the authors studied formalin-fixed paraffin-embedded sections of human (n = 11) and canine (n = 28) myocardium. All normal control myocardium showed positive immunostaining for LDH-1 (H4). In infarcts 10 hours or more old, the histologically necrotic myocardium (by triphenyl tetrazolium chloride staining) (TTC) showed markedly diminished immunostaining. In 24-dogs ischemia was induced in a closed-chest model using a balloon-tipped catheter inflated in the left anterior descending coronary artery. In dogs with 3 hours or more of occlusion, myocardium that was necrotic by TTC staining, light and/or electron microscopy, showed diminished staining for LDH-1, while normal, control myocardium stained intensely. In four dogs, ischemia was induced by a controlled perfusion apparatus by which left main coronary flow was reduced by 50%. Ischemia without necrosis was documented by demonstration of glycogen loss with no light or electron microscopic evidence of necrosis. These ischemic fibers stained intensely for LDH-1, as did controls. Thus, by immunoperoxidase staining, LDH-1 can be demonstrated in normal human and canine myocardium. In experimental models of ischemia in dogs, tissue that was ischemic but not necrotic showed no diminished staining. LDH-1 loss can be detected in necrotic myocardium as early as 3 hours after coronary artery occlusion.     

Effects of interleukin 2 and large envelope glycoprotein (gp 120) of human immunodeficiency virus (HIV) on lymphocyte proliferative responses to cytomegalovirus.

Lymphocytes from many HIV-infected asymptomatic individuals or patients with AIDS-related conditions (ARC) and from all AIDS patients were unable to proliferate in vitro in response to UV-inactivated cytomegalovirus (CMV). The addition of recombinant IL2 (rIL2) restored proliferative responses of lymphocytes from most HIV-infected asymptomatic individuals and ARC patients to levels similar to those of HIV-seronegative (HIV-) CMV-seropositive (CMV+) individuals. In contrast, rIL2 augmented CMV-specific lymphocyte proliferation of only 33% (6/18) of AIDS patients. Proliferative responses to CMV with or without rIL2 did not correlate well with the levels of CD4+ lymphocytes, HIV antigen levels or ratios of CD4+ and CD8+ lymphocytes. Proliferative responses to CMV were inhibited by relatively high concentrations (greater than or equal to 10 micrograms/ml) of recombinant HIV envelope glycoprotein (rgp120) and this immunosuppression was completely overcome by rIL2. These results indicate that defects in antigen-driven lymphocyte responses of HIV-infected individuals are not simply the result of reduced numbers of CD4+ lymphocytes but are influenced by defects in IL2 pathways and by immunosuppressive effects of HIV gp120.