Comparative diagnostic aspects of herpes simplex virus tumor-associated antigens.

Sera from cancer patients and healthy individuals, obtained from two independent sources, were examined for their abilities to react with herpes simplex virus-associated tumor antigens, AG-4 and NVA-TAA (nonvirion antigen-tumor-associated antigen). Both antigens were prepared by infection of HEp-2 cells with herpes simplex virus type 2, and all antigen-antibody interactions were measured by the micro-complement fixation test. Of sera from 16 patients with cancer of the uterine cervix, 81% (P less than 0.01) reacted with NVA-TAA, whereas 78% (P less than 0.001) of 18 sera examined reacted with AG-4. These values differed significantly from those for normal sera, of which 14% reacted with NVA-TAA and 13% with AG-4. Of sera for 8 patients with squamous cell carcinoma of head and neck or vulva, 75% (P less than 0.02) reacted with NVA-TAA, whereas 63% (P less than 0.05) reacted with AG-4. As a group, other cancers (including adenocarcinoma of lung, breast, ovary, and cervix; liposarcoma; sarcoma; melanoma; and carcinoma of the endometrium) did not differ significantly from controls in reactive patterns with AG-4 or NVA-TAA. These studies partly supported the reported preferential reactivity of AG-4 and NVA-TAA with sera of patients with squamous cell carcinoma, especially of the uterine cervix.

Reaction of antigens isolated from herpes simplex virus-transformed cells with sera of squamous cell carcinoma patients.

Antigens isolated from herpes simplex virus type 1, herpes simplex virus type 2, or cytomegalovirus-transformed hamster cells were tested against 66 sera from non-cancer individuals or patients with different types of cancer. By use of the microcomplement fixation procedure to quantify all antigen-antibody interactions, it was observed that 94% (p less than 0.001) of all sera from patients with squamous cell carcinoma reacted with antigens from herpes simplex virus type 1-transformed cells, while 84% (p less than 0.001) of the same sera reacted with antigen preparations from herpes simplex virus type 2-transformed cells. When sera from patients with adenocarcinoma, sarcoma, liposarcoma, and melanoma were tested against these antigens, there was no significant difference in their reactivity from sera of noncancer patients. When sera from all individuals (normal and cancer) were tested against antigens from cytomegalovirus-transformed cells, no significant reaction pattern developed. These studies are the first to describe the isolation of a reactive tumor-associated protein from herpes simplex virus-transformed cells.

Reaction patterns of herpes simplex virus type 1 and type 2 proteins with sera of patients with uterine cervical carcinoma and matched controls.

Serum from 105 individuals with diagnosed uterine cervical cancer and 231 matched controls were examined for their ability to react with a large number of herpes simplex virus type 1 or type 2 (HSV-1, HSV-2) proteins. Radiolabeled HSV-1 or HSV-2 proteins were mixed with test serum and immune complexes were isolated with staphylococcal protein A. Viral proteins in the immune complexes were resolved by polyacrylamide gel electrophoresis and visualized by fluorography. When the frequency of precipitation for cancer and control serum was calculated for each HSV-1 and HSV-2 protein, the results demonstrated that four HSV-1 and 11 HSV-2 proteins were precipitated more frequently by cases than by controls (p less than or equal to 0.05). However, since these results could be influenced by the presence or absence of HSV-2 specific antibodies as well as social, economic, and sexual history, the data were grouped and analyzed according to these parameters. This enabled all significant differences between case and control sera in the precipitation of HSV-1 or HSV-2 proteins to be abolished except for two HSV-2 proteins with molecular weights of 38,000 and 118,000. These two proteins appear to be tumor associated and not merely covariables of past infection or risk factors alone.

The isolation of anti-gum arabic antibodies by affinity chromatography.

Antibodies directed at gum arabic have been induced in rabbits immunized with gum arabic in Freund's complete adjuvant. These antibodies have been isolated in pure form by affinity chromatography on AH-Sepharose 4B containing gum arabic ligands. Oxidation of the susceptable carbohydrate residues of gum arabic with periodate or reduction of the glucuronic acid moieties with carbodiimide and borohydride converted the polysaccharide to products which no longer yielded precipitin reactions with the antibodies. The antibodies are therefore anti-carbohydrate antibodies with specificity for certain carbohydrate units of the gum arabic. Results of chemical modification and inhibition experiments indicate that 4-alpha-L-arabinofuranosyl-D-glucuronic acid units of the polysaccharide are the major immunodeterminant groups.

Differential feulgen-deoxyribonucleic acid hydrolysis patterns of Herpes simplex virus type 1 and type 2 infected cells.

Infection of human embryonic lung cells with herpes simplex virus type 1 (HSV-1) and herpes simplex type 1 (HSV-2) resulted in: (a) qualitative (nuclear cytopathologic) alterations and quantitative (nuclear area) differences in infected compared to control nuclei; (b) increased Feulgen-deoxyribonucleic acid (F-DNA) amounts in infected cells, probably due to viral DNA; (c) higher F-DNA levels in HSV-2 infected cells; and (d) increased rates of F-DNA hydrolysis in viral-infected as compared to uninfected nuclei.

Detection of herpes simplex virus infection using glucose oxidase-antiglucose oxidase immunoenzymatic stain.

Herpes simplex virus (HSV) infected cells have been detected in tissue culture and human cell specimens by an immunoenzymatic staining method using the fungal enzyme glucose oxidase. Infected cells from culture or human specimens appear as dark blue, brown, or red, depending on the tetrazolium salt used in the disclosing reaction, with virtually no staining of uninfected cells. The specificity and sensitivity of this method and of the more commonly used immunoperoxidase method are comparable, but the immunoglucose oxidase method avoids the problems of nonspecific staining by the endogenous peroxidase present in mucosecretions and inflammatory cells. Staining time can be reduced up to 40% of that necessary for the unlabeled immunoperoxidase procedure without compromising the quality of staining results.

Cell electrophoresis research directed toward clinical cytodiagnosis.

The application of cell electrophoresis to cytodiagnosis requires that a scientifically established basis exists for identifying abnormal cells electrophoretically, that research to detect such differences in the cytodiagnostic setting is possible and that a rapid and simple method of cell electrophoresis is adaptable to the clinical setting. Data are presented indicating modifications of electrophoretic mobility due to herpes simplex virus type 1 infection and Rous sarcoma virus transformation of culture cells. A simple apparatus for electrophoretically separating cells on a density gradient and collecting them for subsequent analysis is described, and results of experiments with this apparatus are consistent with those obtained by microscopic electrophoresis. Laser-doppler spectroscopic electrophoresis is suggested as a rapid method adaptable to clinical application.

Resveratrol inhibition of herpes simplex virus replication.

Resveratrol, a phytoalexin, was found to inhibit herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) replication in a dose-dependent, reversible manner. The observed reduction in virus yield was not caused by the direct inactivation of HSV by resveratrol nor inhibition of virus attachment to the cell. The chemical did, however, target an early event in the virus replication cycle since it was most effective when added within 1 h of cell infection, less effective if addition was delayed until 6 h post-infection and not effective if added 9 h post-infection. Resveratrol was also found to delay the cell cycle at S-G2-M interphase, inhibit reactivation of virus from latently-infected neurons and reduce the amount of ICP-4, a major immediate early viral regulatory protein, that is produced when compared to controls. These results suggest that a critical early event in the viral replication cycle, that has a compensatory cellular counterpart, is being adversely affected.

Herpes simplex virus: a possible etiologic agent in some gastroduodenal ulcer disease.

There is increasing evidence that the herpes simplex virus may account for some gastric ulcer disease. To examine this possibility, 62 tissue biopsies from 21 patients were obtained during esophagogastroduodenoscopy for gastroduodenal ulcer disease and from one operative specimen during the procedure for perforation of a gastric ulcer. The samples were collected from the base and rim of the ulcer, as well as from apparently healthy tissue adjacent to the lesion. When the DNA was extracted from these tissues and hybridized to a herpes simplex virus-specific DNA probe, positive results were obtained with 9.5 per cent (2 out of 21) of the patients with benign ulcers. Positive signals were obtained only with ulcer-associated tissues and never with healthy tissue. Hybridization also occurred with DNA from one ulcerative carcinoma in the study. These data suggest that a subset of ulcer disease may be caused by herpes simplex virus or that this virus may be secondarily associating with these lesions.

Characterization of the tumor-associated 38-kd protein of herpes simplex virus type 2.

The BglII N DNA fragment of herpes simplex virus type 2 (HSV 2), which is capable of oncogenically transforming cells in vitro, encodes a 37,800-dalton (38-kd) protein that has been seroepidemiologically associated with uterine cervical carcinoma. Polyclonal monospecific antiserum was produced against electrophoretically purified 38 kd from HSV 2-infected cells and used to identify antigenic and biochemical characteristics of the protein as well as to probe transformed cells for the expression of viral 38 kd. The HSV 2 type specificity of the 38-kd protein, previously shown using anti-HSV 2 serum and monoclonal antibodies, was confirmed using anti-38-kd serum. The 38-kd protein of HSV 2 produced in vivo and in vitro displayed type specificity and showed no evidence of posttranslational processing. The 38-kd protein has a relative isoelectric point of 9.1, is synthesized at a maximum level four hours after infection and appears to be a component of the virion. When 35S-methionine radiolabeled 38 kd was immunoprecipitated by anti-38-kd serum, high-molecular-weight proteins (118-140 kd) were also present. However, if prior to reacting with the anti-38-kd serum the high-molecular-weight proteins were separated from 38 kd with sodium dodecyl sulphate-polyacrylamide gel electrophoresis, the only reaction observed with immunoblot was with 38 kd. Therefore, the observed coprecipitation appears to result from the formation of a complex between the proteins and is not the result of shared antigenic determinants. Cells transformed by inactivated HSV 2 were examined for the expression of the 38-kd protein using immunoenzymatic staining. The viral 38-kd protein was not consistently found, but since the protein is reported to be a component of the viral enzyme complex ribonucleotide reductase, it cannot be excluded from possible HSV 2 transformation.

Cancer cell necrosis by autoschizis: synergism of antitumor activity of vitamin C: vitamin K3 on human bladder carcinoma T24 cells.

Scanning and transmission electron microscopy and fluorescence light microscopy were employed to characterize the cytotoxic effects of vitamin C (VitC), vitamin K3 (VitK3) or a VitC:VK3 combination on a human bladder carcinoma cell line (T24) following 1-h and 2-h vitamin treatment. T24 cells exposed to VitC alone exhibited membranous damage (blebs and endoplasmic extrusions, elongated microvilli). VitK3-treated cells displayed greater membrane damage and enucleation than those treated with VitC as well as cytoplasmic defects characteristic of cytoskeletal damage. VitC:VitK3-treated cells showed exaggerated membrane damage and an enucleation process in which the perikarya separate from the main cytoplasmic cell body by self-excision. Self-excisions continued for perikarya which contained an intact nucleus surrounded by damaged organelles. After further excisions of cytoplasm, the nuclei exhibited nucleolar segregation and chromatin decondensation followed by nuclear karryorhexis and karyolysis. This process of cell death induced by oxidative stress was named autoschizis because it showed both apoptotic and necrotic morphologic characteristics.

Analysis of the thymidine kinase of a herpes simplex virus type 1 isolate that exhibits resistance to (E)-5-(2-bromovinyl)-2'-deoxyuridine.

The mechanism responsible for the decreased sensitivity of a clinical herpes simplex virus type 1 (HSV-1) isolate, HSV-145, to (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) was examined. Measurements of 50% inhibitory doses of several drugs demonstrated that although HSV-145 was sensitive to phosphonoacetic acid, adenine arabinoside and acyclovir, its sensitivity to BVDU and 5-(2-chloroethyl)-2'-deoxyuridine was significantly less than that normally observed for HSV-1. Analysis of the thymidylate kinase (TMP-K) activity of HSV-145 thymidine kinase (TK) demonstrated a decreased level of TMP-K activity when compared to HSV-1 TK. The TMP-K activity of HSV-145 resembled that observed for HSV-2 and the TK-deficient strain HSV-1 TK-7. When the nucleotide sequence of the HSV-145 TK gene was compared to that of the HSV-1 strains C1(101) and SC16 a single nucleotide substitution (G changed to A at base position 502) was detected which would result in the substitution of threonine at amino acid position 168 for alanine. The substitution is the same as that for the laboratory-derived BVDU-resistant virus HSV-1 SC16B3. Collectively, these studies highlight the importance of amino acid conservation at position 168 of the HSV-1 TK in conferring efficient TMP-K activity and BVDU sensitivity.

Inactivation of herpes simplex virus types 1 and 2 by synthetic histidine peptides.

Synthetic homologous histidine peptides were found to directly and irreversibly inactivate herpes simplex virus types 1 and 2 (HSV-1 and HSV-2). The inactivation, which occurred within 1 min of virus exposure to the drug, was independent of temperature but dependent upon the pH and molecular size of the polypeptide. Poly-L-histidine consisting of 24 residues (His-24), with a molecular weight (m.w.) of 3,310, inactivated greater than 99% of the virus present at pH 6.0 and 62% at pH 5.0. Poly-L-histidine consisting of 64 (His-64; average m.w., 8,800) or 75 residues (His-75; average m.w., 10,300) inactivated greater than 99% of virus present at pH 5.0 and 6.0. However, His-24, -64, and -75 were not active against these viruses at pH 7.0 or 8.0. At the concentrations tested, poly-L-histidine of 12 (His-12; m.w., 1,665) or 18 (His-18; m.w., 2,487) residues had no effect on HSV-1 or HSV-2 at any of the pHs tested. When these studies were repeated with other basic homologous polypeptides, poly-L-arginine and poly-L-lysine, various degrees of inactivation were observed that were most pronounced in the neutral-to-alkaline pH range. Once virus was inactivated by poly-L-histidine, the effect could not be reversed in vitro by raising the pH to 7.2 or in vivo by injecting the virus into the neutral environment of an animal. These data suggest that the presence of histidine residues in a peptide of suitable structure may endow that peptide with potent antiviral capabilities.

DNA binding of a 38,000-dalton herpes-simplex-virus-2-specific protein.

Western transfer and immunoenzymatic staining with affinity-purified monospecific antiserum was used to detect a 38-Kd protein that bound to native and denatured DNA cellulose. This protein has previously been shown to be a delayed early herpes simplex virus type-2 specific protein.

Adenovirus-transformed cells restrict Herpes simplex virus replication.

A cell line that normally supports the replication of herpes simplex virus types 1 and 2 became resistant to these viruses after transformation by simian adenovirus 7. Kinetic studies of the mechanism of resistance demonstrated that both herpesviruses were able to attach to the transformed cells and express some early genomic functions, as demonstrated by the presence of low levels of viral thymidine kinase. However, isopycnic centrifugation studies of the abortive system failed to detect viral deoxyribonucleic acid synthesis, whereas indirect immunofluorescent studies of viral proteins revealed that less than 10 per cent of the cells contained these viral macromolecules at any given time. Collectively the data suggest that after transformation by simian adenovirus 7 these cells are altered so as to render them resistant or incapable of supporting the growth of herpes simplex virus types 1 and 2. The results further suggest that the block occurs after viral absorption and prior to viral deoxyribonucleic acid synthesis.

Detection of antibodies specific for herpes simplex virus in human sera by the enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay (ELISA) was used for the detection in human sera of antibody to herpes simplex virus antigens. Development and standardization of the assay suggested that antigenic purity, temperature of reactions, concentration of enzyme-conjugated antiglobulin, and concentrations of test sera are all critical parameters of a successful ELISA procedure. When ELISA titers of 30 human sera were compared to micro-complement fixation titers of the same sera, a significant degree of correlation was observed, but quantitatively ELISA was found to be up to 200 times more sensitive. The sensitivity of the assay and its adaptability to automation should provide an additional method for study or diagnosis of infection with herpes simplex virus.

Steroid hormone alteration of herpes simplex virus type 1 replication.

The effect of steroid hormones on herpes simplex virus type 1 replication was examined. Virus replication studies revealed that various concentrations of prednisolone, hydrocortisone, dexamethasone, or progesterone could decrease virus yields up to a maximum of 99%. Using isopycnic centrifugation in CsCl to separate viral from cell DNA, it was found that virus-specific DNA synthesis was decreased by 30 to 100% depending on the hormone and concentration used. Cell-specific DNA synthesis was also adversely affected, but this did not alter cell viability or plating efficiency.

Flow cytometric detection of herpes simplex virus type 2 infected and transformed cells by immunoenzymatic and by indirect immunofluorescence staining.

The glucose oxidase antiglucose oxidase (GAG) immunoenzymatic staining procedure has been used to detect herpes simplex virus (HSV) antigens microscopically. In this study, the GAG procedure was adapted to cells in suspension, and its potential usefulness in flow cytometry was examined. HSV-2 infected monkey kidney and HSV-2 transformed mouse cells were stained using antisera to HSV-2 or to an HSV-2 specific protein with a molecular weight of 38 Kd, respectively, with the GAG procedure. Flow cytometric analysis of the GAG stained cells was then performed by the measurement of scattered light intensity in the angular intervals 1 degree-2 degrees, 2.5 degrees-19 degrees, and 3 degrees-6 degrees. The greatest scattered light intensity decrement caused by staining occurred in the 3 degrees-6 degrees angular interval, as predicted by previous work. In infected cells, which stain intensely by immunofluorescence, the difference between positively and negatively stained cells was adequate for detecting infected cells using the GAG method; however, this was not the case for the lightly staining transformed cells. The indirect immunofluorescence method of analysis of the same populations was superior to the scattered light method of analysis of the GAG stained infected and transformed cells.

Detection of herpes simplex virus infection of female genitalia by the peroxidase-antiperoxidase method alone or in conjunction with the Papanicolaou stain.

The sensitive peroxidase-antiperoxidase (PXAPX) method individually and in conjunction with the Papanicolaou (PAP) stain was used to detect herpes simplex virus (HSV) in specimens from human female genitalia. Initial studies using a model system of HSV-1 or HSV-2-infected Vero cells established (i) acetone as the most effective fixative, (ii) optimal dilutions of preimmunization and anti-HSV serum for differentiation of infected from noninfected cells, (iii) optimal concentration of 3,3'-diaminobenzidine tetrahydrochloride and H2O2 for maximal staining of infected cells with minimal background reaction, and (iv) removal of endogenous peroxidase with absolute MeOH. These various parameters, once established, were utilized in the PXAPX or PXAPX-PAP on human specimens from the vulva or cervix. In these specimens, examined by standard light microscopy, PXAPX-positive cells were dark brown with a single nucleus or multiple nuclei. By coupling the PAP to the PXAPX, detailed nuclear observations of PXAPX-positive cells were possible and revealed nuclear changes consistent with HSV infection, including syncytium formation, chromatin condensation, and an occasional Cowdry type A inclusion. The PXAPX and PXAPX-PAP correlated (r = 0.742) over a period of 72 h with HSV isolation from these samples.