Differentiation of adenovirus hexon epitopes with monoclonal antibodies by gel diffusion assays.

Nine monoclonal antibodies (MAbs) directed against crystallized human adenovirus type 1 (Ad h 1) hexon were tested with purified homologous and heterologous hexon preparations by gel diffusion. Six MAbs formed a single line with the homologous hexon in a 2-well pattern, and 3 MAbs formed lines only in biclonal combinations with an appropriate MAb. All of the 6 precipitating MAbs formed a continuous line of complete identity when tested simultaneously against homologous and different heterologous hexons. With Ad h 1 hexon a line of double partial identity (double spur) was formed when some pair combinations of 2 MAbs were placed in 2 juxtaposed wells. Other MAbs in the adjacent wells formed a line of identity. The MAbs could be divided into 2 antibody groups (groups A and B) based on this phenomenon. Members of antibody groups A and B apparently identified 2 sterically distinct epitopes: one of them is presumably the genus-specific epitope of the hexons (group A) and the other(s) should be intertype-specific epitope(s). Thus, the gel diffusion method can be used for selecting pairs of MAbs for their specificity to sterically independent epitopes. Mixtures of 2 MAbs belonging to the different antibody groups formed double lines with Ad h 1 hexons. Members of group A showed some helper effect to the members of group B for their precipitin line formation.

Multiple copies of identical epitopes on the adenovirus hexon.

A double monoclonal antibody (MAb) sandwich enzyme-linked immunosorbent assay (double MAb ELISA), which uses the same MAb as solid-phase immunosorbent (capture MAb) and as detector MAb (peroxidase-labeled), was developed to quantify the specific epitopes of adenovirus hexon. Four MAbs directed against crystallized adenovirus type 1 (Ad h 1) hexon were tested by this assay with homologous and different heterologous hexons. The lowest reacting concn with the homologous and heterologous hexon types both in direct and double MAb ELISA was determined and compared. At least two copies of four different epitopes were identified by the MAbs. Evidence is presented that more than one copy of identical or closely related epitopes exist on the homologous as well as on the heterologous hexon molecules. However, their presence could be detected only in higher concn of hexon preparations of subgenera A, B and D.

Taguchi optimisation of ELISA procedures.

We propose a new method in the field of ELISA optimization using an experimental design called the Taguchi method. This can be used to compare the net effects of different conditions which can be both qualitative and quantitative in nature. The method reduces the effects of the interactions of the optimized variables making it possible to access the optimum conditions even in cases where there are large interactions between the variables of the assay. Furthermore, the proposed special assignment of factors makes it possible to calculate the biochemical parameters of the ELISA procedure carried out under optimum conditions. Thus, the calibration curve, the sensitivity of the optimum assay, the intra-assay and inter-assay variability can be estimated. The method is fast, accessing the results in one step, compared to the traditional, time-consuming 'one-step-at-a-time' method. We exemplify the procedure with a method to optimize the detection of ScFv (single chain fragment of variable) phages by ELISA. All the necessary calculations can be carried out by a spreadsheet program without any special statistical knowledge.

Prospects for the control of AIDS patients by introducing defective-HIV harbouring leukocytes.

Introduction of leukocytes harbouring an artificially constructed defective HIV provirus into AIDS patients may result in inducing superinfection resistance against HIV and interfering with HIV receptors or replication of HIV. All these may slow down progression of the disease.

Recombinant DNA technology in adenovirus research.

A brief description of methods for cloning adenovirus genomic DNA and cDNA is presented, the use of recombinant clones in adenovirus research is illustrated by several examples, with particular emphasis on recent informations concerning the structure and function of adenovirus transforming genes.

Restriction site maps of the human adenovirus type 8 DNA.

Restriction site maps of human adenovirus 8 (Ad h 8) were constructed with BamHI, HindIII, PstI, SalI and KpnI endonucleases. The genome size was found to be 22.1 to 22.3 X 10(6) Mr. Comparison of the results with the data available on h Ad subgenera A, B, C showed that the SalI enzyme revealed subgenus-specific differences in the genomes. Similar patterns of the SalI fragments in both type 8 and 10 suggest that the differences were specific for the subgenus D of h Ad.

Parvovirus contamination of a human type 12 adenovirus strain.

A strain of human type 12 adenovirus was found contaminated with virions 27 nm in diameter. After separation by membrane filtration, adsorption on to and elution from erythrocytes and heat treatment, the contaminant was classified as a parvovirus based on its biological and physico-chemical properties and virion morphology. This parvovirus failed to produce interferon and did not affect the interferon-inducing ability of the adenovirus from which it had been separated.

A sensitive method for detection of polyclonal and monoclonal antibodies against the adenovirus hexon.

Chromic chloride and tannic acid methods were elaborated to bind purified adenovirus hexons to sheep red blood cells (SRBC). Either method gave very sensitive haemagglutination (HA) with adenovirus antisera, but treatment with tannic acid was more sensitive. Using this method, specific antibodies present in polyclonal immune sera against adenoviruses and adenovirus hexons, and the reactivity of monoclonal antibodies directed against adenovirus hexon type 1 were tested to 11 adenovirus types. With certain types, the high haemagglutination titres exceeded those obtained with ELISA, while with other types they remained slightly below the ELISA titres. The elaborated passive HA method with tannic acid is easily accomplished and may serve as an effective and useful tool in the study of both polyclonal and monoclonal antihexon antibodies.

Characterization of monoclonal antibodies to adenovirus type 35 hexon.

Twenty three monoclonal antibody-rich ascitic fluids (MIAFs) to human adenovirus (AV) type 35 hexon were studied by indirect ELISA using various tracer systems, passive haemagglutination (HA) as well as gel diffusion techniques. Eleven different human heterologous hexon types in addition to the homologous one, and two animal adenovirus (AV) hexons were used to determine the reactivity patterns (RPs) of the monoclonal antibodies (MoAbs). Based on the cross-reactivity with the different hexon types, the MoAbs exhibited genus, subgenus and type specificities; furthermore, a variety of intersubgenus and intertype specificities could be found. Fifteen of the MoAbs reacted in ELISA, but not in passive HA, suggesting that certain epitopes on the hexons bound to red blood cells were not available for the MoAbs in question. Four MoAbs were able to form a precipitin line with the hexon antigen in gel diffusion. Two of the four (MoAbs 35H10 and 35H51) formed with the homologous AV35 hexon a single confluent precipitin line only. In spite of the origin of these MoAbs from different hybrid cells (clones) their specificity was probably identical when recognizing the type-specific epitope of the AV35 hexon. The other two MoAbs (35H15 and 35H26) with a broad RPs were able to precipitate not only the homologous but also different heterologous hexon types.

Transfected lymphocyte extracts of patients with urological tumours: complement temperature-sensitive adenovirus mutants in vitro.

Patients with renal or bladder cancers exhibit a unique association with adenovirus (Ad) infections. About 60% of them contain antibodies to Ad early antigens. Both in their tumour cells and peripheral blood lymphocytes (PBL) they have detectable early Ad antigens known to be involved in malignant cell transformation. Transfection of tumour cell extracts resulted in complementing temperature-sensitive (ts) Ad mutants at nonpermissive temperatures (39 degrees C) indicating that some cells of the tumour mass possess active functions for Ad. Only 4 to 18% of control subjects were positive in these tests. Here we studied whether lymphocytes might be involved in tumourigenesis by Ad. PBL extracts of patients were transfected into HEp-2 culture cells, which were subsequently superinfected with Ad-5 ts18 and ts19 mutants at 39 degrees C. Titration of virus yields indicated complementation in 76% of patients with renal and bladder cancers in contrast to 20% of control individuals. Complementing ability of lymphocytes which had been prestimulated with phytohaemagglutinin (PHA) approached that of tumour extracts. It means that both specimens contain advanced functions in contrast to resting lymphocytes. Lymphocytes are nonpermissive for latently carried Ad infections. Expression, possible transfer of early Ad gene products via frequent contacts with tissue cells can result in removal of tumour suppressor gene products from complexes regulating cell cycle negatively. Further interaction with hormone-sensitive protooncogenes explains tissue, age and gender specificity of urological malignancies. These phenomena suggest an important cofactorial role for Ad in kidney and bladder tumours.

Study of antibodies to adenoviruses in patients with tumors of the urogenital system.

The presumable relationship between viruses and malignant disease has been studied. Approximately 4000 assays for antibodies to adenovirus were carried out with sera of patients with malignant and nonmalignant diseases of the urogenital organs and other organic diseases. The search was directed in the first place at antibodies to the early non-virion antigens of adenovirus-12 of oncogenic properties. On the evidence of the complement fixation reaction antibodies to adenoviruses were found to be less frequent in malignant tumors and in hypertrophy of the prostate than in the control group. Antibody to the non-virion antigen of adenoviruses was found in 53 per cent of patients with neoplastic diseases and prostatic hypertrophy, in 18 per cent of patients with urogenital diseases other than tumors and in 4 per cent of those with other organic diseases. The results point to a possible role of adenoviruses in tumors of the urogenital organs.

The endosomal epsilon-coatomer protein is involved in human adenovirus type 5 internalisation.

The effects of bafilomycin A1 and of the reduced level of endosomal epsilon-COP (coatomer protein) on the infectivity of human adenovirus type 5 were investigated in Coxsackie adenovirus receptor- (CAR-) transfected Chinese hamster ovary (CHO) cells. The endosomal proton pump inhibitor bafilomycin A1 was able to cause only partial inhibition. Using Id1F cells (an epsilon-COP thermosensitive mutant CHO cell line) the reduction of epsilon-COP level also had partial inhibitory effect. Based on these results and comparing them to existing models of the adenovirus entry, we propose a refined model in which there are two pathways of adenoviral entry: the first one involves the epsilon-COP as the downstream effector of the acidification and can be blocked by bafilomycin A1 and the second one is a pH-independent pathway.

Effect of an antibacterial varnish and amine-fluoride/stannous fluoride (AmF/SnF2) toothpaste on Streptococcus mutans counts in saliva and dental plaque of children.

The purpose of this study was to evaluate the effect of the simultaneous application of a chlorhexidine and thymol-containing varnish (Cervitec) and an amine fluoride/stannous fluoride containing toothpaste (Meridol) on Streptococcus mutans counts in saliva and dental plaque of school children 12-14 years of age, during a six-week period. The children were separated into group 1 (Cervitec varnish + fluoride-containing toothpaste), group 2 (Cervitec varnish + Meridol toothpaste), and group 3 (Meridol toothpaste alone). Over the six weeks the greatest improvement in salivary Streptococcus mutans count occurred in group 2. Overall, a statistically significant decrease in total microbiological count, and Streptococcus mutans was found in all three groups.

[Adenovirus vectors and their clinical application in gene therapy]. / Adenovírus vektorok és klinikai alkalmazásuk a génterápiában.

The potential therapeutic application of the gene transfer technology with adenovirus vectors seems to be enormous. Adenovirus vectors offer several advantages over other vectors, but several important limitations of adenovirus mediated gene transfer are also known. Great number of studies in inherited diseases and in different cancer therapy clinical trials have provided information of critical importance for design of efficient clinical protocols. Clinical trials have been extended to the treatment of many other diseases, too. There are about thirty currently active gene therapy protocols for the treatment only of HIV-1 infection in the USA. These programs aim to confer protective immunity against HIV-1 transmission to individuals who are in risk of infection, to develop preventive or therapeutic vaccines for patients with AIDS and other infectious diseases. Gene therapy represents one of the most important developments in oncology, however, before this can be realised as standard treatment the technical problems of gene delivery and higher safety must be overcome. The early--first and second generation--adenovirus vectors are now likely to be phased out for most diseases, and further experiments seem to be necessary. It might be change to the third generation or other, more modern vector application in clinical trials, as the helper dependent vectors. Almost all transcriptional unit is removed from the DNA of these vectors ("gutless vectors"), therefore they cannot reproduce, give higher gene expression and far less inflammatory. Despite the latest achievement reported in vector design it is not possible to predict yet to what extent and when gene therapy will be effective.

[Significance of recombinant adenoviruses in experimental gene therapy]. / A rekombináns adenovírusok jelentösége a kísérletes génterápiában.

In the past few years recombinant adenoviruses have emerged as promising technology in the gene therapy. They have been used for genetic modification of a variety of somatic cells in vitro and in vivo. They offer several advantages over other vectors. Replication defective vectors can be produced in very high titers (10(11) pfu/ml) thus allowing a substantially greater efficiency of direct gene transfer, they have the capacity to infect both replicating and nonreplicating cells, and the DNA functions in an extrachromosomal fashion, which eliminates the risk of insertional mutagenesis. Several important limitations of adenovirus-mediated gene transfer are also known, such as the relatively short term expression of foreign genes, the dose-related direct toxic effect, and that the host immune response to the viral proteins may substantially inhibit the effect of repeated infection with recombinant adenoviruses. Recombinant adenoviruses of types 2 and 5 are used most frequently as vectors for direct gene transfer. The author's experiments with monoclonal antibodies raised against the adenovirus hexon proteins revealed the existence of a remarkable homogeneity in the antigenic structure among the hexon types of subgenus C (types 1, 2, 5 and 6). Besides the genus and type specific epitopes the presence of at least 15 identical intertype specific epitopes were demonstrated on the surface of the hexons. The data suggest that recombinant adenoviruses constructed from the types of subgenus C cannot be replaced by each other to avoid the host immune response in repeated infections in gene therapy because of their close antigens relationships.

[New developments and trends in adenovirus research]. / Ujabb fejlemények és irányzatok az adenovírus-kutatásban.

Adenovirus infections have been associated with a variety of disorders of the respiratory, ocular, gastrointestinal and urogenital systems. In the last fifteen years, special attention has been given to the adenoviral infections in immunocompromised host, transplant recipients, cancer patients and AIDS patients. Adenoviruses in these patients can cause severe, often generalized illness, with high fatality rate. Data suggest a specific role for adenoviruses in AIDS pathogenesis. From AIDS patients many new and intermediate adenovirus serotypes could be isolated. Considerable effort should be devoted to this area of research in the coming years to understand the molecular mechanism of the interaction between AIDS and adenoviruses. In the last few years recombinant adenoviruses have been widely used as gene delivery vectors in experiments both with curative and preventive purposes. Adenovirus vectors have been used in the experimental gene therapy of genetic disorders, of immuno- and molecular therapy of a variety of cancers. The combination of recombinant adenovirus technology with chemotherapy (pro drug system) seems to be promising, as well as the specific destruction of tumor cells with modified, not recombinant adenoviruses. An the other hand, recombinant adenoviruses appear to be attractive candidates for vaccination against the infectious diseases, too. The current tendency is the construction of second-generation vectors which serve better the different purposes. Potential improvements could be the construction of targated vector by the modification of viral cell-tropism and the suppression of the immune response of the host organism directed against the vector and the vector virus infected cells.

[A microbiology clinic in outpatient gynecologic service]. / Klinikai mikrobiológiai ambulancia a nögyógyászati járóbeteg-ellátásban.

The authors report on their experiences gained at the sexually transmitted disease clinic they established at the First Department of Obstetrics and Gynecology of Semmelweis Medical University. A total of 456 patients presenting with signs and symptoms of lower genital tract infection have been examined in one year. The investigation of patients included aerobic and anaerobic culture of vaginal bacteria, vaginal smear and the identification of sexually transmitted Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasmatales and Gardnerella vaginalis. The authors conclude that a sexually transmitted disease clinic is appropriate to contribute to the prevention of horizontal and vertical spread of the sexually transmitted bacterial infections.

[Detection of the virus-specific antigens of adenoviruses and herpes simplex virus in patients with malignant genital diseases]. / Obnaruzhenie virusspetsificheskikh antigenov adenovirusov i virusa prostogo gerpesa u bol'nykh so zlokachestvennymi zabolevaniiami genitalii.

The possibility of detecting virus-specific antigens in women with various pathological changes of the genitalia (cancer and pre-cancer conditions) was demonstrated. Comparative examinations of specimens from patients with cancer diseases of the genitalia for the presence of structural and virus-induced early antigens of HSV-2 demonstrated immunofluorescence test (IFT) to be the diagnostic test for the detection of HSV antigens in patients with cervical carcinoma. The rate of virus-induced antigen findings in the group of patients with cervical carcinoma was 51.6%, in precancer conditions, 31.7%, in recurrent genital herpes, 78.5%, in the control group of healthy women, 5%, by the IFT. The detection of virus-specific adenovirus and herpes antigens in human tumors is of interest for further investigation of a possible role of these viruses in the generation of malignant neoplasias and for the development of methods for specific immunodiagnosis.

[Bacterial resistance of certain photopolymerizing filling materials and glass ionomer cements]. / Néhány fotopolimerizációs tömöanyag és üvegionomer cement baktériumállóságának vizsgálata.

In the course of the study the photopolimerizating filling materials (Visiomolar, Heliomolar, Heliosit, Silux, P-50, Prismatic) and the glass ionomer cements (Ketacfil, Ketac-silver) served as supposed sources of carbon for Ps. aeruginosa. All the examined materials have shown a suitable resistance to the studied bacterium species. If sunflower oil has been absorbed onto the surface of the examined materials, then a limited increase of the living germ counts has been observed. Ketac-silver was proved to be bactericidal in every case.