Quantitative assay for transformation of 3T3 cells by herpes simplex virus type 2.

The interaction of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) with Swiss/3T3 cells was investigated. Virus-induced cytopathic effects developed in the absence of production of infectious virus. HSV-2 inactivated with UV light (2, 4, 6, and 8 min) also induced cell death in the absence of virus replication. Cell death was not detectable after infection by HSV-2 that had been inactivated by UV irradiation for 10, 12, and 14 min. 3T3 cells infected with UV-inactivated virus (10 and 12 min) continued to replicate past the contact-inhibited monolayer normally associated with these cells. Infection of 3T3 cells with UV-irradiated USV-2 also induced the development of transformed foci. Transformed cells with an epithelioid of fibroblastoid morphology were identified and isolated. All HSV-2-transformed cell lines contained HSV-2-specific antigens detectable by immunofluorescence techniques. The maximum frequency of HSV-2-induced transformation was 3 times 105 PFU per transformed focus, and the observed transformation could be inhibited by pretreatment of the virus with specific antiserum. No type C particles were detected within five cell culture passages after transformation by HSV-2. Type C virus particles were detected after 10 cell culture passages of the HSV-2-transformed cell lines.     

Combined protein and DNA measurements by the ninhydrin-Schiff and Feulgen techniques.

Feulgen nuclear staining with pararosanilin-SO2 was combined with the ninhydrin-Schiff technique. The aldehyde groups converted from primary amino groups are stained with an acriflavine-Schiff reaction. This results in a red nuclear fluorescence and a bright yellow cytoplasmic and nuclear fluorescence. The combined fluorescence staining facilitates cytofluorometric determination of total protein and DNA in the same cell. The ninhydrin-Schiff reaction is affected by the fixation procedure and the duration of the ninhydrin reaction. Investigations with a model system showed that proportionality between the fluorescence intensity of acriflavine and the amount of protein stained by the procedure was obtained after fixation with a fixation mixture suggested by B?hm et al. (1968) and a reaction with ninhydrin at 37 degrees C for 10 h. The ninhydrin-Schiff reaction has no effect on the fluorescence intensity of cells previously treated with pararosanilin-Feulgen staining and it is not affected itself by this previous procedure. Testing this double fluorescence staining on cytology specimens taken from patients with gastric carcinoma and uterine cervial carcinoma, cancer cells were shown to have markedly increased protein and DNA contents compared with those of normal cells.     

Expression of an early, nonstructural antigen of herpes simplex virus in cell transformed in vitro by herpes simplex virus.

Hyperimmune rabbit antiserum to an early, nonstructural herpes simplex virus type 2 (HSV-2)-induced polypeptide (VP143) reacted in immunofluorescence tests with a variety of cell lines transformed by HSV-2. Cytoplasmic fluorescence was observed in 10 to 50% of HSV-2-transformed cells, whereas no fluorescence was observed in cells transformed by other oncogenic DNA viruses or by a chemical carcinogen. VP143-specific reactivity could be absorbed from anti-VP143 serum with HSV-2-transformed cells but not with cells transformed by other agents. When HSV-2-transformed cells were synchronized in mitosis and examined at various times postmitosis for VP143-specific fluorescence, the expression of VP143 was shown to be cell cycle dependent.     

Presence of herpes simplex virus-related antigens in transformed L cells.

Antiserum prepared against herpes simplex virus type 1 (HSV-1)-infected L cells, i.e., lytic antiserum, was shown by an indirect immunofluorescence test to stain 90 percent of HSV-transformed L or HeLa cells. Immunofluorescence in these cells was always most intense in the perinuclear cytoplasmic region. Similar results were obtained with antiserum prepared against HSV-transformed L cells. These data indicate that HSV-transformed cells (both L and HeLa) express HSV-related antigens. Antiserum prepared against HSV-1-transformed L cells, i.e., transformed-cell antiserum, was found to agglutinate purified HSV type 1 virions but failed to neutralize infectivity. This suggests that HSV-1 structural antigens are expressed in HSV-1-transformed L cells. Immunodiffusion studies showed that at least two HSV-related antigens could be demonstrated with antigens from HSV-1-transformed L cells and transformed-cell antiserum. These two antigens were shown to be present in all clonal lines of HSV-1-transformed cells examined, six L cell lines and one HeLa cell line. Therefore, we conclude that transformation of cells by HSV-1, which is known to be associated with acquisition of viral thymidine kinase, must also be associated with the presence of these two antigens. We performed experiments showing that there are species of HSV-related antibody in HSV-transformed cell antiserum that could not be absorbed out with antigens from HSV-infected L cells. Antibodies present in lytic antiserum were completely removed by antigen preparations from cells lytically infected with HSV-1. Also, lytic antiserum failed to block HSV-related staining of transformed L cells in a direct immunofluorescence test. These results are compatible with one of two notions: either (i) certain genes are expressed during transformation that are not expressed during lytic infection, or (ii) these genes are expressed to a much more reduced extent during lytic infection than in transformed cells.     

Estimation of Type-Specific Neutralizing Antibody to Herpes Simplex Virus Type 2 in Uterine Cervical Cancer Patients by a New Absorption Method

A simple method of estimating type-specific neutralizing antibody to type 2 herpes simplex virus (HSV-2) was devised with the use of the microneutralization system. Serially diluted serum was mixed in the well with a constant amount of type 1 virus (HSV-1), and after 3 days' incubation at 37 C, the plate was irradiated with ultraviolet light. The absorbing HSV-1 consisted of culture fluid plus an extract of infected Vero cells not especially concentrated. The well then received indicator HSV-1 or HSV-2, and after being left at 37 C for 1 hr a suspension of dispersed Vero cells was dropped into the wells, following our standard neutralization procedure. Preliminary tests with rabbit antisera showed that even a low level of HSV-2 antibody was detected by this method, unless an exceptionally high titer of HSV-1 antibody originally coexisted with the HSV-2 antibody. Sera from acutely infected persons testified to the specificity of the antibody so detected. It was revealed by means of the new technique that the rate of HSV-2 antibody was significantly higher in uterine cervical cancer patients than in control women. There was no correlation between the clinical stage of cervical cancer and the presence of HSV-2 antibody.     

Factors affecting the immunoperoxidase demonstration of intracellular immunoglobulins and J chain from cytocentrifuged cell smears

Immunohistochemical methods were used to study 1) the optimum fixation conditions for the preservation of human J chain and immunoglobulin (Ig) immunoreactivity and 2) the relation of J chain synthesis by plasmablasts and plasma cells to Ig synthesis in cell smears of cultured human peripheral blood lymphocytes stimulated with pokeweed mitogen (PWM). J chain was demonstrated using the indirect immunoperoxidase method, and intracellular Ig was demonstrated with the unlabeled antibody--enzyme method. In the sequential double staining procedure, J chain was demonstrated using the indirect immunoperoxidase method followed by the demonstration of Ig with the direct immunofluorescence method. Optimum preservation of J chain immunoreactivity was obtained with fixation in neutral buffered formalin at 22 degrees C for 5 min followed by immediate immunoperoxidase staining. False negative results were seen when the slides were stained 2 weeks after fixation. In PWM-stimulated smears, J chain appeared on day three, simultaneously with or after the onset of Ig synthesis. In double stained smears most IgG-positive cells also showed immunoreactivity for J chain from the third day on.     

Detection of herpes simplex virus type 2 glycoproteins expressed in virus-transformed rat cells.

Rat embryo fibroblasts transformed by herpes simplex virus type 2 (HSV-2) were assayed for the expression of certain virus-specific glycoproteins on the surface membranes. Monospecific antisera to HSV-2-specific glycoproteins, designated gAgB, gC, and gX, were used in membrane immunofluorescence studies with HSV-2-transformed cell lines tREF-G-1, tREF-G-2, and a tumor-derived rat fibrosarcoma cells line produced in syngeneic rats inoculated with tREF-G-1 cells. Analysis of the three HSV-2-transformed cell lines showed that antisera to the gAgB and gX glycoproteins were reactive with these cells. In contrast, no significant reactivity was observed when anti-gC serum was reacted with the HSV-2-transformed cell lines. All three antiglycoprotein sera reacted positively with rat cells productively infected with HSV-2. Additionally, the HSV-2-transformed and tumor-derived cell lines showed positive internal immunofluorescence after reaction with antiserum to an early, nonstructural viral protein designated VP143 (molecular weight, 143,000). Infectivity of HSV-2 in standard plaque assays was neutralized by hyperimmune rat antisera to tREF-G-2 or rat fibrosarcoma cells and to HSV-2 virions and by sera from rats bearing the fibrosarcoma. Adsorption of rat-anti-HSV-2 serum with tREF-G-2 or rat fibrosarcoma cells reduced neutralizing activity to 10 and 12%, respectively, compared with 90% neutralization by antiserum adsorbed with nontransformed rat embryo fibroblast cells and 100% neutralization with unadsorbed antiserum. In summary, HSV-2-transformed rat cells retained and expressed genetic information necessary for the production of HSV-2 glycoproteins and a nonstructural protein after high passage in tissue culture or in the syngeneic host.     

Regulation of persistent infection with herpes simplex virus in vitro by hydrocortisone.

About 1% of Raji cells showed sensitivity to herpes simplex virus type 2 (HSV-2) infection when tested by infectious center assays or immunofluorescence tests, and the percentage did not change during cell passage. The addition of hydrocortisone to Raji cells persistently infected with HSV-2 caused a marked increase in virus production and in the number of HSV-producing cells. In the case of HSV-1, it was shown that the addition of hydrocortisone was required to maintain persistent infection. These observations suggest that control of replication of HSV-1 and HSV-2 in these cells is regulated by different mechanisms.     

Oncogenic Transformation of Hamster Embryo Cells After Exposure to Inactivated Herpes Simplex Virus Type 1

The in vitro transformation of hamster embryo fibroblasts by herpes simplex virus type 1 (HSV-1) after exposure of the virus to UV irradiation is described. Cell transformation was induced by 2 out of 12 strains of HSV-1 that were tested for transforming potential. Cells transformed by the KOS strain of HSV-1 were not oncogenic when injected into newborn Syrian hamsters. However, cells transformed by HSV-1 strain 14-012 induced tumors in 47% of the newborn hamsters injected. HSV-specific antigens were found in the cytoplasm of cells transformed by both virus strains. Sera from tumor-bearing hamsters contained HSV-1- and HSV-2-neutralizing antibodies as well as antibodies which reacted specifically with HSV antigens by the indirect immunofluorescence technique. Hamster oncornavirus antigens were not detected by immunofluorescence methods. These observations represent the first evidence of the oncogenic potential of HSV-1.     

Lasar flow cytophotometric immunoperoxidase detection of herpes simplex virus type 2 antigens in infected cultured human cells.

Human cells in culture (HEp-2) were infected with herpes simplex virus type 2 (HSV-2) at multiplicities of infection varying from 0.2 to 10, and fixed 6, 12, 18 and 24 hr after infection. Infection-related antigens were detected by an indirect double antibody (peroxidase conjugated goat anti-rabbit to rabbit anti-herpes simplex virus type 2) immunoenzymatic staining reaction that rendered infection-related antigens visible by light microscopy. A corresponding series of laser flow cytophotometric experiments yielded reproducible large-angle (1-19 degrees) laser-light scattering distributions that depended upon multiplicities of infection and the location of the infection-related antigens in the infected cells.     

A simple and sensitive method for the activity staining of xanthine oxidase

Xanthine oxidase is a commercially-important enzyme. Several biochemical compounds have been quantitated by xanthine oxidase. Xanthine oxidase has been used as an auxiliary enzyme in the staining of several enzymes or tissues, however, there is no direct staining method available for it, on polyacrylamide gels. Partially-purified xanthine oxidase from cow milk was used as the enzyme source for the development of an activity-staining method on polyacrylamide gels. Staining was very sensitive. Detection of 0.02 μU of the enzyme on polyacrylamide gels was possible. Staining of 0.05 μU takes about 1 min whereas staining of 0.5 μU will take less than 5 s. Addition of TEMED is not essential for activity staining but it did increase both the rate and the intensity of the staining. The stained gels must be washed with distilled water, extensively, in order to remove excess unoxidized nitroblue tetrazolium, and must be protected from light, for a clear background and sharp activity-band staining. This method might be useful for quality control of xanthine oxidase obtained from different sources.     

Light scattering measurements on gangliosides: dependence of micellar properties on molecular structure and temperature

Static and dynamic laser light scattering measurements on micellar aqueous solutions of gangliosides GM2, GM1, GD1a are reported. The aggregation number, the hydrodynamic radius and the micellar shape depend on the type of ganglioside and the unsaturation degree of the hydrocarbon chains. At a temperature of 25 degrees C the molecular weights of GM2, GM1 and GD1a are 740,000, 470,000 and 418,000 DA respectively. A significant decrease of micellar size with temperature has been found for saturated GM1 in the region 25 degrees-40 degrees C. The strong sensitivity of the micellar parameters to the ganglioside structure is explained by making reference to some simple model which takes into account geometrical packing considerations. By measuring the scattered light intensity at low ionic strength of the solution (0.1-30 mE) it was possible to evaluate the ganglioside micellar charge, 100 electronic units for GM2, 48 for GM1 and 60 for GD1a.     

Combined protein and DNA measurements by the ninhydrin-Schiff and Feulgen techniques

Summary Feulgen nuclear staining with pararosanilin-SO₂ was combined with the ninhydrin-Schiff technique. The aldehyde groups converted from primary amino groups are stained with an acriflavine-Schiff reaction. This results in a red nuclear fluorescence and a bright yellow cytoplasmic and nuclear fluorescence. The combined fluorescence staining facilitates cytofluorometric determination of total protein and DNA in the same cell.The ninhydrin-Schiff reaction is affected by the fixation procedure and the duration of the ninhydrin reaction. Investigations with a model system showed that proportionality beween the fluorescence intensity of acriflavine and the amount of protein stained by the procedure was obtained after fixation with a fixation mixture suggested by Böhm et al. (1968) and a reaction with ninhydrin at 37° C for 10 h.The ninhydrin-Schiff reaction has no effect on the fluorescence intensity of cells previously treated with pararosanilin-Feulgen staining and it is not affected itself by this previous procedure.Testing this double fluorescence staining on cytology specimens taken from patients with gastric carcinoma and uterine cervial carcinoma, cancer cells were shown to have markedly increased protein and DNA contents compared with those of normal cells.Partly supported by Deutsche Forschungsgemeinschaft (DFG), grant Nr. Bo 395/4     

两种HSV-1及猴B病毒相关抗体测定方法的比较

目的以人单纯疱疹病毒（HSV-1）做为抗原,利用空斑法和IFA法比较猴BV和人HSV-1阳性血清两种不同血清的中和能力的差异,建立一种实用、准确、可靠的病毒毒力的检测方法。方法首先,将HSV-1病毒悬液作连续的10倍稀释,取1 mL接种于已经长成单层的Vero-E6细胞上,用1%甲基纤维素覆盖,待其出现蚀斑后计数,算出病毒悬液中每毫升所含蚀斑单位,即滴定出HSV-1的TC ID50。同时,用免疫荧光方法（IFA）对猴和人疱疹阳性血清进行滴定,得到其血清的效价。其次,用滴定出的病毒液分别与两种阳性血清体外中和后,接种到单层的Vero-E6细胞上,用1%甲基纤维素覆盖,待其出现蚀斑后计数。最后,计算出其蚀斑减少率。结果用1%甲基纤维素作覆盖层的蚀斑数量平均为10-5PFU,能形成115-116个/mL蚀斑,形状呈黍米大小的规则圆形,其蚀斑边缘清晰。IFA滴定的人HSV-1阳性血清与猴BV阳性血清的中和抗体均为1∶80。人HSV-1和猴BV两种阳性血清的空斑减少率均为100%。结论确定了利用1%甲基纤维素做为覆盖层可得到清晰可靠的蚀斑,由此方法检测到用人HSV-1可以代替猴B病毒,筛查猴B病毒抗体。且为将来进行药物筛选和中和实验中利用病毒空斑法建立方便、可靠的方法。     

Sequential paraformaldehyde and methanol fixation for simultaneous flow cytometric analysis of DNA, cell surface proteins, and intracellular proteins.

A cell fixation and permeabilization procedure consisting of sequential paraformaldehyde and methanol was evaluated and found suitable for concomitant flow cytometric quantification of total cellular DNA, immunofluorescence measurements of cell surface proteins, and immunofluorescence measurements of intracellular proteins. Paraformaldehyde/methanol-fixed cells exhibited significantly greater intracellular antitubulin immunofluorescence than cells fixed with paraformaldehyde or methanol alone (p less than 0.002) and significantly greater intracellular antitubulin immunofluorescence than cells fixed with methanol followed by paraformaldehyde (p less than 0.006). With paraformaldehyde/methanol fixation, cell morphology was well preserved and forward and right angle light scatter properties were sufficiently well maintained to permit gating on these parameters. Cell surface marker staining with fluorescent anti-leukocyte antibodies was unaffected by fixation with paraformaldehyde/methanol. Paraformaldehyde effects on the intensity of DNA staining with propidium iodide were dependent on paraformaldehyde concentration and fixation temperature; these effects were least pronounced at low paraformaldehyde concentrations (0.25% or less), and at temperatures lower than 37 degrees C. Paraformaldehyde fixation may result in differences in propidium iodide staining of DNA in some diploid cells, which may produce small spurious aneuploid peaks in normal peripheral blood leukocytes. Paraformaldehyde fixation also produces an apparent increase in the DNA index of aneuploid cell populations in comparison with methanol fixation, particularly when the DNA index exceeds 1.5. Occasionally, this paraformaldehyde fixation-induced effect is useful in identifying biologically distinct near-diploid subpopulations in tumors.     

Properties of Hamster Embryo Fibroblasts Transformed In Vitro After Exposure to Ultraviolet-Irradiated Herpes Simplex Virus Type 2

An in vitro method which led to the transformation of hamster embryo fibroblasts after exposure to herpes simplex virus type 2 (HSV-2) inactivated with ultraviolet irradiation is described. The transformed cells (333-8-9) produced tumors when inoculated into newborn Syrian hamsters but not when injected into weanling Syrian hamsters of the same LSH inbred strain. However, after one in vivo passage, the 333-8-9 cells became highly oncogenic in weanling hamsters. No infectious virus was recovered from these cells. Herpes simplex virus antigens were detected in the transformed cells by the indirect immunofluorescence technique. Sera from tumor-bearing hamsters contained antibody with highly specific neutralizing activity against HSV-2. These studies indicate the continued involvement of the HSV-2 genome in an oncogenic cell line.     

Evidence for receptor-mediated uptake of adenosine deaminase in rabbit kidney

We investigated the subcellular location of adenosine deaminase-complexing protein in the proximal renal tubules of rabbit kidney and its interaction with intravenously infused monomeric calf adenosine deaminase. Cortical tissue from non-infused animals, stained in suspension by the peroxidase-antiperoxidase method for complexing protein and embedded in resin, was examined by transmission electron microscopy. Positive staining indicated the presence of complexing protein on the surface of microvilli in the proximal tubules. Sections (1 micron) of resin-embedded cortex from infused rabbits, stained first for complexing protein and then for adenosine deaminase, were examined by light microscopy. After staining for complexing protein by indirect immunofluorescence, the sections were photographed and then immersed in buffer containing 6 M guanidine hydrochloride plus 2-mercaptoethanol for 3 hr at 60 degrees C to remove bound antibodies. The sections were then stained by the peroxidase-antiperoxidase method for infused enzyme. Vesicle-like apical structures, the basal membrane area and, as previously reported, the brush border of proximal tubule cells were positive for complexing protein. Vesicle-like structures and brush borders positive for complexing protein were also stained for adenosine deaminase. The basal membrane area did not stain. These results support the hypothesis that complexing protein can act as a receptor for adenosine deaminase.     

Flow cytometric analysis of Chlamydia trachomatis interaction with L cells

Immunofluorescent staining and flow cytometric analysis have been investigated as means of studying the early stages of in vitro infection of Chlamydia trachomatis. The lymphogranuloma venereum strain of C. trachomatis was grown in vitro in L cells, fixed in p-formaldehyde, stained with fluorescein isothiocyanate (FITC)-conjugated monoclonal antibody to the chlamydial major outer membrane protein, and analyzed flow cytometrically. Infected cells stained 50-100 times more intensely than uninfected cells, and they could easily be discriminated by flow analysis. The number of infected cells and the fluorescence intensity of individual cells were proportional to the multiplicity of infection. The attachment of purified elementary bodies to L cells could be analyzed by immunofluorescence and flow cytometry. Cells exposed to 0.26 inclusion-forming units/cell could be discriminated from an unexposed population. Flow analysis of purified elementary bodies was possible after fluorescent staining with the aid of a laser-based cytometer and gating on low volume.     

Cytochemical properties of mitochondria in the gastric parietal cell.

The matrix of some mitochondria in gastric parietal cells of rat and guinea pig evidenced affinity for the high iron diamine method which localizes sulfated complex carbohydrates selectively by light and electron microscopy. Such staining has not been observed elsewhere in the stomach. The high iron diamine reactive mitochondria about equaled in number those which were unreactive, and the two groups were indistinguishable morphologically. The distinction was not apparent either when mitochondria were stained by other cytochemical procedures including dialyzed iron for acidic complex carbohydrates, 3-3' diaminobenzidine-H2O2 at pH 6.0 for cytochrome oxidase, and Kominick's pyroantimonate osmium tetroxide for antimonate precipitable cations. The dialyzed iron method stained acid glycoconjugates in the outer intermembrane space in parietal cell mitochondria. These mitochondria stained more strongly with dialyzed iron than have any others examined heretofore with this method and comprised the only reactive mitochondria in the stomach. Parietal cell mitochondria also stained strongly for cytochrome oxidase but those of other gastric cells failed to evidence this reactivity.     

Different levels of reactive endogenous cytochrome c in mitochondria rich cells revealed by diaminobenzidine staining

Light microscopic examination of rat and mouse tissues incubated in a medium containing 3,3'-diaminobenzidine (DAB) and catalase revealed that cells known to possess abundant mitochondria (hepatocytes, cardiomyocytes, renal proximal and distal tubular cells, parietal cells of gastric mucosa, and retinal photoreceptor cells) were stained with different intensity: from moderate (parietal cells, cardiomyocytes, renal distal tubular cells) to weak (hepatocytes, renal proximal tubular cells) or even negative (photoreceptors). When exogenous cytochrome c was added to the incubation medium, all these cells displayed quite uniform, strong staining, indicating a comparable activity of cytochrome oxidase. Since DAB is oxidized directly by cytochrome c which in turn undergoes reoxidation by cytochrome oxidase, the observed differences of staining intensity in the absence of exogenous cytochrome c are postulated to result from different content of reactive endogenous cytochrome c in mitochondria of the investigated cells.