The intercalating drugs quinacrine and proflavine induced increases in single-stranded DNA detected in the nuclei of mouse BALB/c 3T3 1--13 cells. The denatured DNA was detected by fluorescein-labeled antinucleoside antibodies, which bind to single-stranded but not double-stranded DNA. Exposure of cells to the potent mutagen proflavine increased the fraction of immunoreactive nuclei from 0.65 to 0.8. With the weaker mutagen quinacrine, higher concentrations were needed to induce increases in immunoreactivity. Both intercalating drugs rapidly induced morphological transformation in mouse 3T3 cells. Treatment with proflavine resulted in higher transformation frequencies than were found with quinacrine. Significant increases in cell transformation frequency were observed at the concentrations which induced high levels of immunoreactivity. These results suggest that DNA strand separation is itself, or at least accompanies, an early step in cell transformation by intercalating drugs.

Cultured post-fused human skeletal muscle monolayers exposed to WSN influenza A virus were analyzed by scanning and transmission electron microscopy. At 12–14 h post-inoculation (p.i.), affected mononuclear cells retracted from the cell surface, but remained anchored to the substrate by taut filar processes. Retraction was accompanied by shortening of microvilli, appearance of hemispherical cytoplasmic protrusions and corrugation of the surface proper. These changes were more pronounced at 24 and 48 h p.i. The rounded, moribund mononuclear cells eventually detached from the substratum. Surface alterations were accompanied by the intracellular appearance of electron-dense nuclear inclusions (often associated with the nucleolus) and paracrystalline ribosome-studded cytoplasmic bodies, which increased in size and number with time.

Cultured post-fused human skeletal muscle monolayers exposed to WSN influenza A virus were analyzed by scanning and transmission electron microscopy. At 12-14h post-inoculation (p.i.), affected mononuclear cells retracted from the cell surface, but remained anchored to the substrate by taut filar processes. Retraction was accompanied by shortening of microvilli, appearance of hemispherical cytoplasmic protrusions and corrugation of the surface proper. These changes were more pronounced at 24 and 48h p.i. The rounded, moribund mononuclear cells eventually detached from the substratum. Surface alterations were accompanied by the intracellular appearance of electron-dense nuclear inclusions (often associated with the nucleolus) and paracrystalline ribosomestudded cytoplasmic bodies, which increased in size and number with time. In myotubes, distinct surface alterations appeared later (24h p.i.). Early myotube retraction was accompanied by accentuation of the longitudinally oriented surface pleats and appearance of "blebs" followed by cell-rounding. At 48-72 h, many myotubes detached from the substratum. The surfaces of those still adhering appeared corrugated. Intranuclear and cytoplasmic inclusions accumulated, and budding virions, often filamentous, could be demonstrated at the plasmalemma of mononuclear cells and myotubes. Late (end-stage) cytopathic effects included clumping of chromatin, breakdown of the nuclear envelope, disappearance of cortical and endoplasmic cytofilaments, mitochondrial swelling, and vesiculation of surface membranes. The lesions leading to cell injury and cell death appeared to be due to massive accumulation of virus-induced products that altered cellular metabolism, with physical and functional abnormalities of surface membranes.

The labeling index was estimated in human tumor biopsies with a new method in which antinucleoside antibody and immunoperoxidase staining are used. Excellent agreement of the [3H]thymidine autoradiography labeling index and the antinucleoside immunoreactivity labeling index was obtained in cell aspirates from solid tumor and fluid specimens taken before radiation or chemotherapy as well as in serial tumor samples taken during therapy. The new method was also applied to biopsies of solid tumor specimens, for which the [3H]thymidine autoradiography method is not generally applicable. Fifteen pretreatment human mammary adenocarcinomas were examined. The mean labeling index was 10.8%, in good agreement with results of others who used [3H]thymidine autoradiography. Biopsies from eight patients with head and neck cancers were examined by the new method to learn whether their pretreatment labeling indices were predictive of a good response to a combination chemotherapy regimen. Before application of antinucleoside antibody, frozen sections must be treated with ribonucleases. We found that, with some specimens, antinucleoside antibody immunoperoxidase labeling indices were spuriously higher without the ribonuclease pretreatment; probably this was due to interference from immunoreactive nuclear RNA. These results confirm previous studies with cell cultures and animal tumor models, indicating that the new method is a reliable way to estimate the proportion of cells in DNA synthesis in biopsies of human tumors. Results may be obtained in 2 hr by a relatively simple method with potentially wide application. Correlation of pretreatment labeling indices with tumor responses to therapy and scheduling of chemotherapy or radiation therapy after clinical synchronization of the tumor cells are some of the potential clinical applications.

The evolution of the renal tubulo-interstitial (TI) lesions of SWR/J mice neonatally infected with lymphocytic choriomeningitis (LCM) virus was studied by immunofluorescence, light, and electron microscopy. By immunofluorescence, viral antigens were observed in the cytoplasm of TI cells and in the walls of interstitial vessels throughout the experimental period, i.e., from 20 days to 1 year after infection. In contrast, comparable deposits of mouse IgG and C3 were not demonstrable in TI structures. By light and electron microscopy, interstitial cellular infiltration, tubular cell atrophy and degeneration, and, in the late stages, peritubular, periglomerular, and perivascular fibrosis were observed. The severity of TI lesions did not correlate with that of immune complex glomerulonephritis. The immunopathologic data presented here are consistent with the hypothesis that the persistence of LCM antigen in TI structures throughout the course of the disease elecits a cell-mediated immune reaction.

We found that fused human muscle in culture supports neurotropic influenza A viral infection, as demonstrated by viral growth experiments, hemadsorption, observation of cytopathic changes and detection of intracellular viral antigen. The time of peak virion production and the appearance of cytopathic effects in these experiments were similar to previously described characteristics in influenza A-susceptible organotypic cultures of other tissues. Cytopathological changes occurred earlier in mononucleated cells than in myotubes and included cell-rounding; cytoplasmic retraction, granularity and vacuolization; ribonucleic acid-containing cytoplasmic inclusions; nucleolar enlargement; and clumping of chromatin. Immunofluorescent staining demonstrated early nuclear fluorescence, followed by spread of viral antigen into the cytoplasm. A subpopulation of mononucleated cells, shown by cloning studies to be myoblasts, showed no cytopathic effects or evidence of intracellular viral antigen and was presumably resistant to neurotropic influenza A infection. The adverse effects of influenza A on fused human muscle cells in tissue culture contrast with the inability of the virus to replicate in skeletal muscle after animal inoculation in acute experiments. Therefore, host factors may affect attempts to produce an animal model of human muscle disease with this virus.

The labeling index determined by [3H]thymidine autoradiography in cells from clinical specimens was compared with the percentage of the cells showing nuclear reactivity to immunoperoxidase-labeled antinucleoside antibodies. This nuclear immunoreactivity is specific for denatured or single-stranded DNA's and is detectable almost exclusively during DNA synthesis. Results with the two methods showed excellent agreement. The new method allowed rapid accurate assessment of S phase in the tumor cells from freshly isolated aspirated specimens as well as frozen sections, suggesting general applicability to estimation of the labeling index without autoradiography.

Immunoreactivity to fluorescein-labelled antinucleoside antibodies was induced in HeLa cell nuclei by the carcinogen N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). DNA strand breaks induced by MNNG were detected by measuring the influence of MNNG on the sedimentation in alkaline sucrose gradients of a fast sedimenting DNA-Containing complex. Dissociation of the complex and induction of immunoreactivity both proved to be sensitive, dose-dependent indicators of MNNG action in HeLa cells. Since antibodies directed against nucleosides react only with denatured or single-stranded DNA, it appears likely that MNNG-induced immunoreactivity results from exposure of single-stranded regions of DNA, providing a means for the rapid identification of carcinogen action by immunofluorescent techniques. N-Acetoxy-2-acetylaminofluorene also induced immunoreactivity and led to the dissociation of the DNA complex in HeLa cells. Seven other chemicals with known carcinogenic properties also induced immunoreactivity in this test.

The influence of anoxia on X-ray damage in HeLa cells was studied by observing effects on nuclear immunoreactivity to antinucleoside antibodies and on the sedimentation in alkaline sucrose gradients of their DNA “complexes”. The fraction of G1 HeLa cells which was immunoreactive to fluorescein labeled antinucleoside antibodies increased from control levels of 11% ± 3.5 S.E. to 71% ± 5.7 S.E. after 1 000 rads in air. In anoxia 1 000 rads increased this fraction to only 42% ± 3.1 S.E. After 1 000 rads in air the return to normal G1 levels of immunoreactivity required 90 min, but it required only 30 min after radiation in anoxia. If cells were held at 0 °C for 35 min before anoxic irradiation the rapid return to control levels of immunoreactivity during postradiation incubation at 37 °C was not observed. Cold shock did not increase the proportion of cells initially made immunoreactive by 1 000 rads in anoxia. Anoxia reduced the effect of 1 000 rads on the sedimentation properties of the DNA complex. Cold shock prior to anoxic radiation retarded the faster reconstitution of the DNA complex otherwise observed after anoxic radiation.

Fluorescein-labeled antibodies (FA) to human immunoglobulins, complement, fetal proteins and other antigens were used to search for these immunologic reactants in malignant and benign neoplasia as well as in control tissues. The FA reacted with five or more target antigens in 42 of 59 (70%) malignant tumors and 21 of 23 (40%) benign tumors. The remaining neoplasia and normal tissues showed positive staining with fewer than five specific antibodies. IgM, IgA, B1C, fetal proteins and HSV were the antigens found most frequently. This indicates that neoplasia exhibits immunologic activity more frequently than normal tissue.

Angiotensin-converting enzyme catalyzes cleavage of dipeptides from the COOH-termini of a large number of oligopeptides. It is a glycoprotein with a molecular weight of approximately 130,000 to 140,000 and contains a single, large polypeptide chain and one molar equivalent of bound zinc. It is located in blood vessel walls of many organs and is probably exposed on the luminal surface. It is also present in blood and certain parenchymal cells. Its two currently recognized important substrates are angiotensin I and bradykinin, and its action on each may be viewed as vasopressor. Thus, it catalyzes the formation of angiotensin II, a potent vasopressor agent, and the inactivation of bradykinin, a powerful vasodepressor molecule. The lung is important in regulating the level of angiotensin II in the systemic arterial circulation due to its strategic location, its large vascular bed and the fact that enzymatically generated angiotensin II is minimally metabolized in the pulmonary circulation. Studies with venom peptide inhibitors suggest that the enzyme may represent an important therapeutic target in renin-dependent hypertension. The enzyme is apparently accessible to exogenous anticatalytic antibody so that immunologic regulation of its activity may ultimately be feasible in vivo.

Fluorescein-labeled antibody to rabbit pulmonary angiotensin-converting enzyme localized in the vascular endothelium of rabbit lung, liver, adrenal cortex, pancreas, kidney, and spleen. Epithelial cells of the renal proximal tubules were the only parenchymal cells among the organs studied that demonstrated immunoreactivity.

Interstitial immune complex nephritis in patients with systemic lupus erythematosus (SLE). Renal tissues from 45 patients with SLE nephritis, 34 patients with idiopathic membranous nephropathy (IMN) and 77 patients with minimal glomerular disease (MGD) were studied by light, immunofluorescence and electron microscopy. Interstitial nephritis characterized by focal or diffuse infiltration of inflammatory cells, tubular damage and interstitial fibrosis was observed in 66% of SLE patients. Fluorescein-conjugated antibodies to immunoglobulins or complement or both were bound to peritubular capillaries, interstitium and tubular basement membranes (TBM) in 53% of patients with a granular pattern corresponding to opaque deposits seen by light or electron microscopy or both. Antibodies reactive with thymidine or cytosine or both were bound to interstitial structures in 19% of patients tested and showed the same granular distribution. Interstitial cellular infiltration was rare and deposits of immunoglobulins and complement were rare or absent in IMN and MGD, whereas deposits of DNA products were never observed. The findings are consistent with the interpretation that in patients with SLE nephritis immune deposits, presumably containing DNA-anti-DNA complexes, localize in peritublular capillaries, TBM and interstitum, thereby producing an inflammatory reaction which contributes to development and evolution of renal diseases.

Arrest of HeLa cells in G2 after ionizing radiation is accompanied by persistent nuclear immunoreactivity to antinucleoside antibodies. The reactivity declined to the normal G2 level during escape from arrest and subsequent cell division.

The glomeruli of 48 renal allografts from rats injected with phytohaemagglutinin (PHA), as well as those of 51 control kidneys, were studied by light, electron and immunofluorescence microscopy. Glomerular endothelial and mesangial lesions associated with lymphocyte and blast cell infiltration were detected by electron microscopy in about 50% of the allografts performed between Carworth rats or between Long Evans and Lewis rats, when the animals were injected with PHA 1–3 days after renal transplantation (Tx). The same pathology rarely occurred when PHA was injected prior to Tx or when renal grafting was performed between Fisher and Lewis rats with subsequent PHA stimulation. Deposits of rat IgG and/or C3 in glomerular structures were not detectable by immunofluorescence. The changes suggest that in rejection glomerulitis of rats injected with PHA the lymphocytes are the effector cells, the endothelium is the target structure, and the lesions are produced by the synergistic effect of allograft rejection and PHA stimulation. The morphogenesis of these changes is comparable to that of certain in vitro systems of cytotoxicity.

Membranous and/or proliferative pneumonitis, similar in certain features to human interstitial pneumonitis, developed in rabbits making hyperactive antibody response to daily injections of bovine serum albumin (BSA) administered in multiple large doses sufficient to maintain the state of relative antigen-antibody equivalence. The pulmonary lesions were associated with deposition in alveolar capillary walls and interstitium of antigen, host globulin and complement, presumably in immune complexes. In some rabbits chronic interstitial pneumonitis, characterized by thickening of alveolar capillary walls, interstitial fibrosis and deposition of fibrinogen, was observed. The production of immune complex pneumonitis seems to depend on the degree of the antibody response because rabbits developing chronic serum sickness with low doses of BSA, rabbits with acute serum sickness as well as nonresponders showed no pulmonary alterations. This observation is comparable to that described by Dixon in his studies on experimental immune complex glomerulonephritis. It is conceivable that the pulmonary pathology shown here is produced by formation of larger amounts of complexes which may persist longer at critical levels in the circulation than in rabbits immunized with a single daily injection of BSA. In conclusion this study suggests: first, that experimental chronic serum sickness can be used as a model, not only for glomerulonephritis, but also for experimental systemic disease, comparable to human systemic diseases produced by circulating antigen-antibody complexes; and second, that the pathogenesis proposed here offers an alternative to using antilung basement membrane pneumonitis for the experimental approach to the study of human lung immunopathology.

Neurotropic influenza virus (WS-N strain) causes cytolytic changes in organotypic neural cultures derived from embryonic mouse hypothalamus. Initially, nerve cells develop enlarged, refractile nucleoli. In later stages, both nerve and glial cells undergo necrotic changes. These alterations are accompanied by synthesis of progeny virus and formation of intracellular viral antigen. Electron microscopic observations of infected cultures reveal virus particles with the morphology and dimensions of influenza virions. Organotypic neural cultures may prove useful in studies of influenza virus neurotropism.

Brains of six patients with postencephalitic parkinsonism were examined for influenza virus antigen by direct immunofluorescence. Tissue sections were exposed to fluorescein-conjugated globulin directed against the NWS, WSN, WS, and PR8 strains of influenza A0, the ribonucleoprotein of the WSN strain, the swine strain of influenza A, the Japan strain of influenza A2, the Lee strain of influenza B, measles, and herpes simplex virus. Intranuclear fluorescent antigen was detected in hypothalamic and midbrain sections prepared from all six cases after staining with antibody to the two neurotropic influenza A0strains (NWS and WSN) and to the ribonucleoprotein of the WSN strain. No fluorescence was observed after staining of similar sections prepared from the brains of five patients with idiopathic Parkinson disease with labeled influenza NSW antiserum.

HeLa cells, incubated with camptothecin during the G 1 phase of the cell cycle, show nuclear fluorescence with fluorescein-labeled antinucleoside antibodies. If the G 1 cells are washed free of the drug, the cells no longer demonstrate nuclear fluorescence. Since these antibodies react only with single-stranded DNA, the positive staining in camptothecin-treated G 1 cells suggests that the drug induces denatured regions in DNA. Fluorescent antinucleoside antibodies may be a useful technique for the observation of drug-induced changes in DNA during the G1 phase of the cell cycle.