Biomedical subjects
Publications and source records attributed to B Brichácek.
14 tumours in the ENT region were tested for the presence of human papillomaviruses (HPV) DNA. The dot-blot hybridization technique was used for HPV sequence detection. HPV DNA mixture of types 6b, 11, 16 and 18 served as a probe. The following five samples showed positive reaction: 3 carcinomas of the larynx, 1 carcinoma of the nasal cavity and 1 papilloma of the nasal cavity.
Sera from 75 patients with warts were tested for antibodies against human papillomaviruses (HPVs) in an immunoelectron microscopy test. A group of 40 patients' sera were tested against a pooled suspension of the HPVs extracted from their warts and 90% of the sera were found to be antibody-positive. Prior to serological investigation viruses from a second group of 35 patients were typed by dot blot hybridization with HPV-1 and HPV-2 DNA. According to these results the patients were divided into type 1, type 2 and non-1, non-2 virus carriers. Sera of these patients were first tested for antibodies reactive with their homologous viruses and then for antibodies against type 1 virus. More than 80% of the patients possessed antibodies against their homologous viruses, but only a small minority of sera from type 2 and non-1, non-2 virus carriers were reactive with type 1 viruses. These results suggest that the antibody reactive in the immunoaggregation test is type-specific.
Nine oligopeptides corresponding to segments of different open reading frame (ORF) proteins of human papillomavirus (HPV) 6b and HPV-16 were prepared and tested for reactivity with human sera in enzyme-immunoassay (ELISA). Of these only heptadecapeptide derived from L2 ORF of HPV-6b, and encoded also by L2 ORF of HPV 11, was reactive with some human sera. Over 400 human sera of different origin were tested for the presence of antibody to this antigen. While less than 15% of sera from healthy subjects or cervical carcinoma patients were found antibody positive, sera from the majority of condylomata accuminata (CA) patients were reactive. The antibody titres varied from 1:10 (initial serum dilution) to 1:80; in this respect there was no marked difference between sera from CA patients and the other subjects. The prevalence of antibody was higher among promiscuous than nonpromiscuous women. This is in line with the assumption that sexual intercourse is the most important route of HPV 6 and 11 transmission.
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Epstein-Barr virus (EBV) nuclear antigen type-1 (EBNA-1) was extracted and purified from Raji cells by chromatography on DNA-Sepharose and Blue-dextran Sepharose. Its complexes with plasmid pM765-10 derived from EBV (strain M-ABA) DNA were visualized by electron microscopy. The criteria of specificity were as follows: (1) preferential binding of EBNA-1 to the ori-P region of pM765-10; (2) specific enlargement of EBV DNA/EBNA-1 complexes with anti-EBNA-1 (IR-3) IgG antibody; and (3) resistance of the resulting EBV DNA/EBNA-1/anti-EBNA-1 antibody complexes to treatment with 1.5 M NaCl. The optimal conditions for the formation of EBV DNA/EBNA-1 complexes were 50 to 150 mM NaCl and pH 6.0. A balanced equilibrium of EBNA-1 and pM765-10 was necessary to achieve both a high yield and specificity of EBV DNA/EBNA-1 complexes.
Biopsy specimens from 13 patients with adenocarcinoma of the colon and from 10 patients with endoscopic polypectomies for colon adenoma were examined for the presence of the DNA of cytomegalovirus (CMV), Epstein-Barr virus (EBV), and human papillomavirus (HPV) types 2, 6, 16 and 18. The specific activities of viral DNA probes obtained by nick--translation ranged from 10(7) to 10(8) cpm/micrograms DNA. By Southern blot hybridization with an estimated sensitivity of 10 pg virus DNA which corresponded to 0.05 virus genome equivalents per cell we failed to detect any virus DNA in the biopsy material tested.
Human primary cord-blood lymphocytes were transfected, using the DEAE-dextran technique, with a set of seven largely overlapping clones jointly covering the whole M-ABA Epstein-Barr virus (EBV) genome. Three fragments, cosmids cMB-14 and cM301-99 and plasmid pM966-20, were able to stimulate transient cellular DNA synthesis, blastic transformation, and clumps formation, as well as to prolong the life span from a maximum of 2 weeks in control cultures to up to 6 weeks. The fragments stimulating DNA synthesis also expressed this property in mutual combinations or when combined with cosmid cMSal-A or cM302-21. Their use with any other fragments in cotransfection did not result in further DNA synthesis stimulation. Cosmids cM302-23 and cMSal-B suppressed this effect. Cosmid cM301-99 but not cM302-23 induced transient EBNA-1 formation in about 1% of lymphocytes. Lymphocytes transfected with single fragments or their combinations failed to grow into immortalized cell lines. The results suggest that transient expression of viral functions at levels achievable by transfection is not sufficient for cell immortalization.
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The conditions of in situ hybridization for demonstration of herpesvirus genomes in animal and human tissues were tested using the ORWO(R) K6 emulsion. It was possible to localize herpes simplex virus (HSV) genomes in infected mice organs (brain and liver) as well as Epstein-Barr virus (EBV) genomes in tonsils of patients with infectious mononucleosis and in tumour specimens of patients with nasopharyngeal carcinomas. Immunofluorescence (IF) revealed mostly corresponding results. The in situ hybridization is more favourable due to its higher specifity, but it is more time consuming and expensive. IF seems advantageous for screening of a great number of tissues.
The presence of Epstein-Barr virus genetic material was demonstrated in thin sections of biopsy specimens in 6 of 7 carcinomas of human palatine tonsil by the in situ hybridization test. The biopsy specimens from 4 tonsillar carcinomas exhibited a strong positive reaction; two biopsy specimens were weakly positive. Two tumor-free biopsy specimens were negative. None of the thin sections reacted with 3H-labeled herpes simplex virus DNA probe.
Thin sections of biopsies from five patients with supraglottic laryngeal carcinoma who possessed antibody to early antigen of EB virus were examined for the presence of EB virus nuclear antigen (EBNA) and EB virus DNA by cytological hybridization in situ. Both EBNA and EB virus DNA were found in the tumour cells of three of the patients. The present findings suggest an association between EB virus and supraglottic carcinoma of the larynx.
The relative contents of Epstein-Barr virus (EBV)-associated nuclear antigen (EBNA) and average numbers of EBV genome equivalents per cell were determined (i) in Raji cells superinfected with P3HR-1 virus, (ii) in Raji cells induced by 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and sodium n-butyrate and (iii) in P3HR-1 cells induced by TPA and sodium n-butyrate. This treatment was followed by increases in the percentage of early antigen (in both Raji and P3HR-1 cells) and virus capsid antigen-positive cells (in P3HR-1 cells) and increases of approximately 20-fold in the average number of EBV DNA equivalents in superinfected Raji cells and in TPA- and sodium n-butyrate-induced P3HR-1 cells. However, the content of EBNA in these cells dropped. This was revealed by a decrease in both the complement-fixing antigen content and in the proportion of EBNA-positive cells as determined by anti-complement immunofluorescence. Thus, the positive correlation found previously between the content of EBNA and the number of EBV genome equivalents per cell in proliferating lymphoblastoid cultures does not seem to apply to the situation in either superinfected Raji cells or in P3HR-1 cells induced by TPA and sodium n-butyrate.
Lymphoblastoid cell line P3PFA was derived from P3HR-1 cells by long-term cultivation in the presence of phosphonoformate (PFA). Spontaneous production of Epstein-Barr virus antigens in PFA-selected subline was markedly reduced in comparison with the original cell line. Induction of early antigen (EA) and viral capsid antigen (VCA) syntheses with 5-iodo-2-deoxyuridine (IUDR), n-butyrate and 12-o-tetradecanoyl phorbol-13-acetate (TPA) was significantly less efficient in P3PFA than P3HR-1 cells. This was most likely associated with reduction in the number of viral genome copies per P3PFA cell that decreased more than one hundred times in comparison with P3HR-1 line. The synthesis of EA in both cell lines was not inhibited in the presence of PFA. On the other hand, PFA inhibited the synthesis of VCA by 95 and 30% in P3HR-1 and P3PFA, respectively. The relative resistance of VCA synthesis to PFA in P3PFA cells was not due to the presence of a drug-resistant virus-specific DNA-polymerase. It is noteworthy that the activity of virus-specific enzyme was markedly reduced, while cellular enzyme activity was significantly enhanced in induced P3PFA cells when compared to P3HR-1 cells treated in the same way.
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Sera from 48 tonsillar carcinoma (TC) patients, 48 matched controls and 16 recurrent exudative tonsillitis (RET) patients were examined for the presence of Epstein-Barr virus (EBV) associated nuclear antigen (EBNA), early antigen (EA) and virus capsid antigen (VCA). Higher prevalence and significantly higher antibody titres against all three EBV-associated antigens were observed in TC patients in comparison with controls and RET patients. Patients suffering from anaplastic TC had higher titres of antibodies against VCA and EA than TC patients with other histological diagnoses. Five out of 11 TC biopsies obtained from 9 patients were positive for EBV DNA at levels of 0.17, 4 to 5, 15 to 18 and in two cases 3 EBV genome equivalents per cellular genome. Among 16 RET patients, 4 were found positive at levels not exceeding 2.17 EBV genome equivalents per cellular genome. Higher titres of antibody against all EBV antigens were found in TC and RET patients with EBV DNA-positive tonsillar tissue than in those with EBV DNA-negative tonsillar tissue.
Binding of Epstein-Barr-virus-determined nuclear antigen (EBNA) to acid-fixed nuclei could be prevented by exposing EBNA to anti-EBNA antibodies. While EBNA is eluted from acid-fixed nuclei by 0.4 M NaCl, it was not eluted even by 1.5 M NaCl if EBNA bound to acid-fixed nuclei had been exposed to anti-EBNA antibody. In this case the reaction was abolished, and EBNA was at least partially eluted by 0.2 M glycine buffer, pH 2.8, or by 3 M NaSCN. EBNA not exposed to antibody was completely eluted from acid-fixed nuclei by treatment with a solution containing 200 microgram of DNA per 1 ml of isotonic buffer, pH 6.5; however, after treatment with antibody, EBNA was not eluted by DNA.
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Treatment of Epstein-Barr virus-determined nuclear antigen (EBNA) with DNA resulted in blocking of its ability to convert acid-fixed EBNA-negative cell nuclei to an EBNA-positive form. Epstein-Barr virus (EBV) DNA, herpes simplex virus type 2 (HSV-2) DNA and DNA isolated from three lymphoblastoid cell lines differed in their potency to block this reaction. EBV DNA was found to be about three times more effective than cellular DNAs in abolishing the ability of DNA-cellulose-purified EBNA to convert acid-fixed nuclei to the EBNA-positive form; the effect of HSV-2 DNA was of intermediate character. No difference was found between the blocking potency of DNAs isolated from EBV-genome-negative Ramos cells and EBV-genome-positive Raji and P3HR-1 cells.