Complexity of the early genetic response to growth factors in mouse fibroblasts.

Genes whose expression is growth factor regulated are likely to be important components in the mechanisms controlling cell proliferation and differentiation. With the aim of identifying some of those genes, a lambda cDNA library was prepared with poly(A)+ RNA from quiescent NIH 3T3 cells stimulated with serum for 4 h in the presence of cycloheximide. Differential screening of approximately 200,000 recombinant phage plaques revealed 2,540 clones that cross hybridized preferentially with [32P]cDNA derived from RNA of stimulated cells rather than with cDNA derived from nonstimulated cells. Cross hybridization of these clones identified 82 independent sequences, including c-fos and c-myc. Seventy-one clones were further studied. Analysis of the changes in transcription and mRNA levels after serum stimulation demonstrated that the kinetics and extent of the induction vary dramatically between the different genes. Cycloheximide in all cases superinduced the mRNA levels by two mechanisms, inhibiting the shutoff of transcription and prolonging the half-lives of the mRNAs. Our results showed that induction of proliferation is accompanied by the onset of a complex genetic program.

Human serum induced opsonization of immunoglobulin G-coated polystyrene microspheres with complement components C3 and C4 as measured by flow cytometry.

Human IgG-coated polystyrene microspheres (IgG-ms) were incubated with human serum followed by biotinylated monoclonal anti-C3d or anti-C4d antibody, and phycoerythrin-streptavidin. The intensity of fluorescence was measured by flow cytometry and corresponds to the amount of deposited C3 and C4. Binding of C3 and C4 was dependent on the activation of the classical pathway of complement and on the amount of IgG adsorbed to the particles. No deposition was observed on control particles coated with bovine serum albumin or ovalbumin. Incubation of constant amounts of IgG-ms with increasing amounts of normal human serum (NHS) resulted in a dose-dependent increase in C3 deposition. The same result was found for C4 deposition at moderate NHS dilutions, but less C4 was detectable using a higher input of NHS. Half-maximum C3 and C4 deposition was observed at a mean serum dilution of 1/114 and 1/520, respectively (n = 26). No correlation was found between C4 or C3 deposition and either total C4 and C3 serum concentrations as measured by nephelometry or complement-mediated lysis of antibody-coated sheep red blood cells. Reduced or absent C4 or C3 deposition was found in the sera of patients with low amounts or deficiencies of components involved early in classical complement pathway activation whereas essentially normal C4 or C3 deposition was obtained with the sera of patients with deficiencies in components of the membrane attack complex. With this simple and specific functional assay using stable reagents an altered function of early components of the classical pathway of complement may be quickly and reliably detected in routine diagnostic laboratories. Moreover, such opsonized and well characterized particles may be useful in assays of phagocytic cell function.

Biochemistry and molecular biology of MCA.

The mucin-like carcinoma-associated antigen (MCA) is a mucin with a molecular weight of 350-500 kD. It circulates in the serum and its serum content can be determined with the Cobas Core MCA EIA test. Patients with breast cancer show elevated MCA serum levels. The molecule has a polypeptide backbone consisting of three parts: the C-terminus the N-terminus and the transmembrane sequences. The protein is heavily glucosylated with carbohydrate side chains that contain fucose, galactose and N-acetyl galactosamine. The antibody b-12 recognizes a repetitive epitope on the peptide portion of the MCA molecule. The epithelial mucin, which is coded by a unique gene, was cloned using PCR technology. Peptides corresponding to the N- and C-terminus were expressed in E. coli. Analysis of the purified peptides revealed molecular weights of 12 and 18 kD.

Radiographic measurements of alveolar bone loss in the rat.

A new method to evaluate alveolar bone loss in rodents is described. The palatal and lingual halves of maxillae and mandibles were radiographed. On enlarged positive prints, 5 vertical distances were drawn at defined sites from the cemento-enamel junction to points revealing fully intact bone structure. These were either located on the alveolar crest or at the depth of intrabony defects. These distances were recorded with a trace-reading pen coupled to a computer. Results were expressed in mm for each site separately and totals (left plus right values) for either maxillae or mandibles were calculated. This technique was compared to other methods for evaluating alveolar bone loss, using the jaws of rats subjected to a gnotobiotic regime in which the degree of bone loss was low. It was demonstrated that the measurement of vertical distances based on radiography by which also intrabony defects were defined was accurate, reproducible and more sensitive than other means of evaluating bone loss.

Amplification of DNA from whole blood.

A method is described for the amplification by PCR of human chromosomal DNA sequences from whole blood samples. Various amounts of blood samples, with either EDTA, citrate, or heparin used as the anticoagulant, have been used to determine optimal PCR conditions for each type of sample. Up to 80% (vol/vol) of whole blood sample is tolerated in PCR with Taq polymerase. Amplification from whole blood requires the optimization of salt (K+ and Mg++) according to sample volume and type of anticoagulant used. Pretreatment of fresh blood samples to lyse the leukocytes is required for EDTA-treated blood samples and is beneficent in PCR with heparin- and citrate-treated blood samples to obtain maximal amplicon amounts. A satisfactory method of pretreating samples is freeze/thawing. In addition, EDTA-treated blood samples require a heat treatment before PCR for maximal amplicon synthesis. It appears that purification of the DNA is not necessary for any of the whole blood samples analyzed by PCR. Results of amplification reactions from unpurified hepatitis B virus (HBV) sequences of whole sera samples are shown also.

Rat memory T lymphocytes. II. Differences in macrophage-dependent activation shown by Actinomyces viscosus antigens and by mitogens, using silica in vitro.

Protein-coated silica, a macrophage toxin, was used to assess the requirement for accessory cells in the induction of an in vitro proliferative response to (i) antigens from Actinomyces viscosus and (ii) the mitogens conconavalin A (Con A) and phytohaemagglutinin (PHA). T cells were obtained from RIC-Sprague-Dawley rats primed in vivo with A. viscosus Nyl by splenectomy and filtering the spleen cell suspensions through Degalan Ig-anti-IgG columns. In the presence of 100 microgram silica/ml during 4 days of culture, the proliferative response of T lymphocytes was not diminished. In contrast, when the T cells were precultured with silica for 24 h, washed, and subsequently cultured with the antigen fractions, antigen-induced proliferation was abolished. This procedure, however, had no influence on mitogen-induced proliferation was abolished. This procedure, however, had no influence on mitogen-induced T-cell activation. It is therefore concluded that the antigen-dependent anamnestic in vitro response (but not activation by mitogens) of rat T lymphocytes needs help from silica-sensitive macrophages.

Rat memory T lymphocytes: in vitro proliferation induced by antigens of Actinomyces viscosus.

Peripheral T lymphocytes from RIC-Sprague-Dawley rats primed in vivo several months earlier with Actinomyces viscosus Ny 1 reacted with a strong proliferative response upon antigenic challenge in vitro. Two different antigen preparations from A. viscosus Ny 1, a broken cell supernatant (BCS) and an extracellular fraction (EXC) gave maximum responses as measured by the uptake of [3H]thymidine at a concentration of 10 microgram at day 4 of culture. Phytohaemagglutinin (PHA) used as a control provoked a maximum proliferation in the same order of magnitude. T lymphocytes from unprimed, germfree and conventional animals showed a similar stimulation to PHA, but only a marginal reaction to BCS, and were not at all activated by EXC. Evidence is thus presented for efficient removal of cells responding non-specifically to B-cell mitogens.

Assessment of needs for plasma for fractionation in Europe.

In the early 1990s, a series of outbreaks of hepatitis C (HCV) infections clustering among recipients of certain lots of plasma-derived medicinal products (PDMP) alarmed regulatory authorities, manufacturers and the public alike. Also, a few episodes of Hepatitis A (HAV) infections occurred in haemophiliacs receiving solvent-detergent-treated factor VIII concentrates. Thus, several measures were brought into effect to reestablish the safety of the incriminated products and to further increase the margin of safety of PDMP in general. Therefore, intramuscular immunoglobulins had to be free of HCV RNA as shown by nucleic acid amplification technology (NAT) in the final products. Furthermore, the manufacturing process of PDMP had to be validated for both viral inactivation and elimination. Finally, HCV-NAT was to be standardised and implemented as a validated test of plasma pool samples. In 1994, a joint meeting of EPFA, EAPPI and Regulatory Authorities was held in Brussels to outline the state of the art and to delineate the actions to be taken. Five years later, in 1999, the incidence rates of HIV, HBV and HCV in unpaid blood donors have been minimized, especially in European countries. With probabilities for window period donations as low as 0.6 in 1 million for both HIV and HCV and 2.1 in 1 million for HBV in Switzerland, labile blood products have reached extreme, but not absolute safety. The introduction of HCV-NAT roughly doubles this safety resulting in a 1 in 3 million probability of a window donation.Concomittantly, extensive viral validation studies document effective inactivation and removal of viruses in PDMP. The demonstrated margins of safety, expressed as logarithmical reduction factors (LRF), range from 4 to over 20 log(10), depending on product, virus, and inactivation procedure used. Further progress to even safer PDMP shall be acomplished by consolidating the GMP processes, abandoning of obsolete requirements and harmonising national regulations within Europe. Before introducing new measures for additional agents such as HAV or Parvovirus B 19, gains and risks and even potential new threats have to be carefully assessed. Alternative efforts for the safeguard of patients, e.g. vaccination for HAV, need to be balanced against the risks of changing established and validated manufacturing procedures of PDMP with long-lasting safety records.

Interactions of antisera, sera, and oral fluid with glucosyltransferases.

Partially purified glucosyltransferases (GTF) isolated from Streptococcus mutans OMZ 176 and respective rabbit antisera were used to study enzyme-antibody interactions. A comparison between sensitive serological techniques and a functional inhibition test based on a radioenzyme assay demonstrated that the latter test system was the only one that discriminated between different antisera. Positive reactions in high dilutions in the former test systems were explained by the involvement of non-GTF contaminants and/or antibodies against enzyme regions distant to the catalytic site. The minute cross-reactions between two enzyme fractions and the respective antisera in the functional inhibition test indicated that the two immunogens contained mainly GTF that differed in the structure of their catalytic region. Control rabbit sera, rat oral fluid, and insoluble and soluble glucans considerably activated the GTF eluted with a 0.5 M phosphate buffer from hydroxapatite. It is suggested that these enzymes had additional binding sites for macromolecules inherent to rabbit sera and rat oral fluid, respectively, and that the observed increase in enzyme activity was due to a more stable enzyme conformation. Possibly the stimulation of GTF by the soluble glucan fraction was caused by a primer and/or acceptor function; however, this was not the case of the insoluble glucan. A stable complex was formed in the absence of the enzyme substrate, sucrose, the activity of which was not readily enhanced. It is concluded that the GTF of strain OMZ 176 are composed of multiple, multi-reactive molecules that enable these enzymes to act as cross-linking agents.

Characterization of the DNA methylase activity of the restriction enzyme from Escherichia coli K.

The restriction endonuclease from Escherichia coli K is a multifunctional protein which efficiently methylates heteroduplex DNA (one strand modified and one strand unmodified) in the presence of S-adenosylmethionine (AdoMet), ATP, and Mg2+. The methylase activity is catalytic, and seems to modify different heteroduplex host specificity sites for E. coli K with equal efficiency. In the methylase reaction, both AdoMet and ATP (or its imido analog) act as allosteric effectors, but AdoMet also serves as a methyl donor. Preincubation of the enzyme with AdoMet eliminates the lag period observed in DNA methylation. The rate of enzyme activation was determined using the AdoMet analog Sinefungin. The result are consistent with the hypothesis that the early steps of AdoMet binding and enzyme activation are common to both restriction and modification reactions.

Persistence of the competent state in mouse fibroblasts is independent of c-fos and c-myc expression.

Induction of competence in quiescent fibroblasts by platelet-derived growth factor (PDGF) is accompanied by a dramatic increase in the expression of c-fos and c-myc genes. However, the maintenance of the competent state and progression through G1 does not require high expression of these proto-oncogenes. These results suggest that the induction of c-fos and c-myc by growth factors in quiescent fibroblasts may be required to render the cells competent for progression.

Cyclosporin A: in vivo and in vitro suppression of rat T-lymphocyte function.

The immunosuppressive effect of cyclosporin A (CS-A) was investigated in RIC-Sprague-Dawley rats. In vivo, CS-A totally abolished the formation of antibodies to the hapten dinitrophenyl (DNP) in rats immunized with DNP-keyhole limpet haemocyanin. In vitro, the effect of CS-A was investigated in spleen cell cultures stimulated by concanavalin A, phytohaemagglutinin or lipopolysaccharide. The suppression due to CS-A was more pronounced in cultures set up with cells from rats fed the drug than in spleen cell cultures from control animals supplemented with serum containing CS-A. Purified by filtration through Degalan-rat Ig-anti IgG columns, T lymphocytes from CS-A treated rats were no longer suppressed by CS-A serum in contrast to purified T cells obtained from control rats. Thus, CS-A seems to interfere with the mitogenic triggering of a subpopulation of T lymphocytes resulting in a functional clonal deletion.

Prevention of hepatitis C infection in haemodialysis units. A prospective study.

A prospective study was begun in our haemodialysis unit after four previously negative patients were found to be anti-HCV positive. A dedicated area and dedicated dialysis equipment (but not a separate room) were assigned to anti-HCV-positive patients and testing for HCV antibodies was performed every 3 months. A total of 131 patients were treated during the study period of 18 months. Of these, 50 patients were dialysed during the entire 18 months, and 21 were available to be tested six or more months after having left the centre. During the first 6 weeks after implementing the precautions two more anti-HCV-positive patients were detected. However, during the rest of the study period no further newly infected patients were found. It is concluded that the spread of HCV infection in a haemodialysis environment can be prevented by limited isolation procedures.

Detection of labelled RNA species by contact hybridization.

An improved contact hybridization technique for the analysis of labelled RNA species is presented. The method combines high sensitivity of detection with the high resolution of polyacrylamide gel electrophoresis and should be especially useful for the characterization of transient RNA precursor molecules. Its application to gene mapping is illustrated.

DNA translocation by the restriction enzyme from E. coli K.

The restriction endonuclease Eco K binds to a host specificity site and then proceeds to cleave the DNA at sites that may to several thousand bases away. It does this by translocating the DNA past the enzyme in an ATP-dependent reaction that results in the formation of highly twisted loop intermediates. DNA cleavage can occur on either side of the host specificity site.