Measurement of hematological, clinical chemistry, and infection parameters from hirudinized blood collected in universal blood sampling tubes.

Hirudin, the anticoagulatory polypeptide of the leech Hirudo medicinalis, strongly inhibits thrombus formation by specifically interacting with thrombin. For diagnostic purposes, hirudin should be superior to other anticlotting compounds because it only minimally alters the mineral, protein, and cellular blood constituents. To test this hypothesis, hirudinized and routinely processed venous blood from 80 healthy volunteers and patients was subjected to a variety of automated blood tests. A strong correlation was found between the results of automated complete blood counts obtained from K(2)-ethylenediaminetetraacetic acid (EDTA) anticoagulated and hirudinized blood (1000 antithrombin units [ATU] hirudin/ml). In addition, clinical chemistry and serological infection parameters (asparlat amintransferase [ASAT], lactate dehydrogenase [LDH], sodium, and so on, and antibodies against hepatitis B and C and human immunodeficiency virus [HIV]1/2, respectively) correlated well when measured in serum as compared with hirudinized plasma. Contrary to single clotting factors, global coagulation parameters (activated partial thromboplastin time [aPTT], prothrombin time [PT]) could not be measured in hirudinized blood. Recombinant hirudin neither interfered with immunophenotyping of mononuclear cells using FACScan analysis, nor did it alter the detection of Wilms' tumor gene expression by RT-PCR technology even at high doses (5000 ATU hirudin). Thus, a hirudin-containing blood sampling tube can be designed as a universal blood sampling tube (UBT) for testing the majority of diagnostic blood parameters.

Exogenous hepatitis B surface antigen particles processed by dendritic cells or macrophages prime murine MHC class I-restricted cytotoxic T lymphocytes in vivo.

Injection of low doses of particulate hepatitis B surface Ag (HBsAg) into H-2d mice without adjuvants primes an Ld-restricted, S28-39-specific T cell response. This study indicates that dendritic cells (DC) and macrophages (M phi) both serve as APCs that support priming of CD8+ CTL precursors in vivo to exogenous HBsAg particles. After transfer into a syngeneic, naive host, HBsAg particle-pulsed DC, either freshly purified from skin or derived from a cloned DC line, efficiently primed class I-restricted, HBsAg-specific CTL precursors. M phi, either harvested from the peritoneal cavity or generated in macrophage-CSF-stimulated bone marrow cell cultures in vitro or derived from established, cloned M phi lines (PU5-1.8, J774A.1), pulsed with HBsAg particles in vivo or in vitro, elicited a class I-restricted, HBsAg-specific CTL response after adoptive transfer into naive hosts. The class I-restricted CTL response induced by HBsAg particle immunization was suppressed in carrageenan-treated mice, but was restored when carrageenan-treated mice were immunized with syngeneic, HBsAg-pulsed M phi. Selective elimination of M phi by liposome-incorporated dichloromethylene-diphosphonat did not suppress the induction of a CTL response of H-2d mice by HBsAg particle immunization. HBsAg-pulsed, freshly prepared DC are more potent than pulsed M phi in priming class I-restricted CTL in vivo. The relative importance of both types of APC in priming CTL remains to be resolved.

Priming of class I-restricted cytotoxic T lymphocytes by vaccination with recombinant protein antigens.

We investigated the specific priming of MHC class I-restricted cytotoxic T lymphocytes (CTL) by different protein antigen preparations in mice. The recombinant viral protein antigens tested are of potential relevance for the design of subunit vaccines. They include the hepatitis B virus (HBV) surface antigen (S-antigen), the HIV-1 gp160 envelope protein, and a chimeric HIV-1 Pr55-gag/V3-3 retrovirus-like particle. In addition, ovalbumin (OVA) was tested. The native or denatured particulate (multimeric) or monomeric form of these protein antigens was injected by various routes into mice. Class I-restricted CTL were efficiently primed by a single low-dose injection of HBV S-antigen particles or the chimeric HIV-1 Pr55-gag/V3-3 particles. After SDS-denaturation, gel-purified monomeric S-antigen and monomeric Pr55-gag/V3-3 fusion protein were still very efficient in priming CTL. CTL sensitization was not detected in a (primary or boosted) response to even high doses of native OVA or native HIV-1 gp160. Denaturation of these two antigens by detergent strikingly increased their immunogenicity for CTL. Immunization of mice with non-treated or SDS-denatured antigenic peptides representing the relevant CTL-defined epitopes of the tested protein antigens did not prime CTL. These data indicate that native, particulate and denatured, monomeric protein antigens efficiently stimulate a class I-restricted CTL response.

Hepatitis B virus small surface antigen particles are processed in a novel endosomal pathway for major histocompatibility complex class I-restricted epitope presentation.

We investigated the major histocompatibility complex (MHC) class I-restricted presentation of an epitope of the hepatitis B virus small surface (S) antigen particle to cloned murine cytotoxic T lymphocytes (CTL). Efficient Ld-restricted presentation of the S28-39 epitope to CTL is observed in cells of different tissue origin pulsed in vitro, either with the antigenic S28-39 12-mer S-peptide, or with particulate S-antigen. The kinetics of epitope presentation differ in S-peptide-pulsed and in S-particle-pulsed cells: while a 15-min pulse with the antigenic peptide sensitizes targets for class I-restricted CTL lysis, presentation of S-particles requires 30-60 min to sensitize cells for CTL lysis. Uptake of antigenic material and active metabolism of the presenting cell are required for processing of S-particles, but not for sensitizing targets with S-peptides. Intracellular processing and presentation of S-particles is blocked in cells treated with chloroquine, NH4Cl, primaquine, or leupeptin, but not by treatment with cycloheximide or brefeldin A. This processing pathway operates efficiently in peptide-transporter-deficient, Ld-transfected T2 cells, revealing a novel endosomal/lysosomal processing pathway for class I-restricted presentation of peptides derived from exogenous S-particles.

Selective stimulation of murine cytotoxic T cell and antibody responses by particulate or monomeric hepatitis B virus surface (S) antigen.

In the murine system, we tested in vivo the immunogenicity of different preparations of the yeast-derived surface antigen (S-antigen or S-protein) of hepatitis B virus (HBV). Native S-protein molecules self-assemble into stable 22-nm particles. BALB/c mice immunized with low doses of native S-particles without adjuvants efficiently generated an H-2 class I-restricted CD8+ cytotoxic T lymphocyte (CTL) response, and developed easily detectable serum antibody titers against conformational determinants of the native S-particle or linear epitopes of the denatured S-protein. Disruption of S-particles with sodium dodecyl sulfate and beta-2-mercaptoethanol generated p24 S-monomers. Injection of an equal dose of S-monomers into mice efficiently primed CTL, but did not stimulate an antibody response against conformational or linear epitopes of the native or denatured S-protein. In vivo priming of CTL by S-particles or S-monomers required "endogenous" processing of the antigen because the injection of an equimolar (or higher) dose of an antigenic, S-derived 12-mer peptide into mice did not prime CTL. Native (particulate) or denatured (monomeric) S-antigen injected with mineral oil (incomplete Freund's adjuvant) or aluminum hydroxide failed to stimulate a CTL response. Hence, different preparations can be produced from a small protein antigen which specifically stimulate selected compartments of the immune system.

Immunization with soluble hepatitis B virus surface protein elicits murine H-2 class I-restricted CD8+ cytotoxic T lymphocyte responses in vivo.

Immunization with soluble proteins only rarely induces a specific response of CD8+ CTL. We describe experiments that demonstrate the efficient and specific in vivo priming of CTL in BALB/c mice immunized with soluble hepatitis B virus (HBV)-derived surface (S) protein. A single (s.c., i.p. or i.v.) injection of a low dose (30 ng to 3 micrograms per mouse) of recombinant S protein particles without adjuvants induced a CTL response. This specific cytotoxic response was read out against a panel of different S protein-expressing transfected mouse cell lines. Effector cells of this response were Ld-restricted, CD3+ CD4- CD8+ CTL. H-2d/Ld+ (BALB/c, C.B-17) mice were responders; H-2d/Ld- (dm2) mutant mice and H-2b (C57BL/6) mice were nonresponders. Injections of various dosages of a S protein-derived, immunogenic, synthetic peptide into BALB/c mice by various routes did not prime CTL. After incorporation of S protein particles into IFA or aluminum hydroxide, these protein Ag lost their ability to specifically stimulate CTL in vivo. After priming of mice with S protein emulsified in IFA or adsorbed to aluminum hydroxide boost injections with native S protein particles were inefficient in stimulating a specific CTL response. These findings are of relevance for the design of synthetic subunit vaccines for which specific stimulation of CD8+ T effector functions is desired.

Heterologous protein production in yeast.

The exploitation of recombinant DNA technology to engineer expression systems for heterologous proteins represented a major task within the field of biotechnology during the last decade. Yeasts attracted the attention of molecular biologists because of properties most favourable for their use as hosts in heterologous protein production. Yeasts follow the general eukaryotic posttranslational modification pattern of expressed polypeptides, exhibit the ability to secrete heterologous proteins and benefit from an established fermentation technology. Aside from the baker's yeast Saccharomyces cerevisiae, an increasing number of alternative non-Saccharomyces yeast species are used as expression systems in basic research and for an industrial application. In the following review a selection from the different yeast systems is described and compared.

High-level expression of foreign genes in Hansenula polymorpha.

The methylotrophic yeast Hansenula polymorpha belongs to a limited number of non-Saccharomyces yeast species used as hosts for heterologous gene expression. It has successfully been applied for the production of hormones, antigens and enzymes. The system excells by mitotically stable recombinant strains, high productivity and faithful processing of the produced polypeptides. The favourable characteristics of this microorganism for protein production at an industrial scale are described in the following article focusing on some recent representative examples.

Constitutive expression of multiple growth factor genes by melanoma cells but not normal melanocytes.

In a panel of metastatic melanoma cell lines we found steady-state mRNA transcripts for multiple growth factors including basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF)-A, PDGF-B, transforming growth factor (TGF)- beta 1, TGF- alpha, melanoma growth-stimulating activity (MGSA), interleukin (IL)-1 alpha, and IL-1 beta but not insulin-like growth factor (IGF)-1 or IGF-2. Expression of growth factor genes was constitutive because prior to RNA extraction melanoma cells were maintained in a chemically defined culture medium free of exogenous growth factors. Each of four cell lines had an individual pattern of expression of either two, four, five, or seven growth factors; however, all cell lines shared expression of the bFGF gene. Two strains of normal melanocytes expressed TGF- beta 1 but not bFGF, PDGF, TGF- alpha , or MGSA mRNA at detectable levels. We tested growth-modulatory effects of the growth factors most frequently expressed by melanoma cells (bFGF, TGF- alpha, TGF- beta, PDGF). None of these stimulated melanoma cell growth consistently, whereas exogenous, acid-activated TGF- beta inhibited melanoma growth at concentrations greater than 10 ng/ml, suggesting that bioactive TGF- beta may represent a physiologic growth inhibitor. Neither neutralizing antisera to PDGF or TGF- alpha nor a monoclonal antibody to the epidermal growth factor (EGF)-receptor inhibited melanoma cell growth. Our results indicate that multiple growth factors are expressed simultaneously and constitutively by melanoma cells but not normal melanocytes in culture. Expression of bFGF is a common feature underscoring the significance of bFGF as an autocrine factor for melanoma cells as described earlier. Secreted PDGF and TGF- alpha are apparently not involved in or not essential for autocrine growth stimulation of melanoma cells.

SV40-transfected human melanocyte sensitivity to growth inhibition by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate.

Normal human melanocytes, which require the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) for growth in culture, were transfected with a SV40 T-antigen-containing plasmid by using the technique of electroporation, and were scored for colonies of morphologically altered cells. The frequency of transformed colonies was higher when selection was done in the absence rather than the presence of TPA in the medium. Three cell lines derived from transformed colonies were characterized. All show an enhanced growth rate compared to parental cells, anchorage independence, loss of dependence on medium supplements for growth, chromosomal abnormalities, an extended life span, and growth inhibition by TPA. They express nerve growth factor receptor and Mr 97,000 protein, melanotransferrin, two antigens usually associated with melanocytic cells, but the transformed cells are not pigmented. The three cell lines underwent crisis at about passage 10 posttransfection; one cell line recovered and appears to have unlimited growth potential. None of the cell lines is tumorigenic. They should be interesting models for studying multistage carcinogenesis in human cells and transcriptional activation by TPA.

Tumor promoters induce a transient expression of tumor-associated genes in both basal and differentiated cells of the mouse epidermis.

The effect of tumor promoters on the in vivo expression of tumor-associated, overexpressed genes was studied. Two of the tumor-associated genes, mal 1 and mal 2 were overexpressed already in the benign papilloma stage of mouse skin carcinogenesis. Overexpression of the other two genes, mal 4 and transin, was specific for the malignant state. Treatment of the normal adult epidermis with the complete tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and the incomplete, second-stage promoter 12-O-retinylphorbol-13-acetate (RPA) enhanced transiently the expression of the mal sequences and transin. Fractionation of the adult epidermis on Percoll gradients into basal cells and differentiated, suprabasal cells showed that expression of the mal sequences was enhanced by TPA in both basal and differentiated cells. In contrast, transin expression, which was undetectable in cells of the normal epidermis, was enhanced in only the basal cells of the TPA-treated epidermis. The non-tumor-promoting hyperplastic agent, ethylphenyl propiolate (EPP), applied to the skin at a hyperplastic dose level did not enhance the expression of the mal 4 or transin sequences in the epidermis and had only a slight enhancing effect on the levels of mal 1 and mal 2 transcripts in the epidermis. Our results suggest that the observed stimulated expression of mal 1 and mal 2 is related to proliferative processes, whereas stimulated expression of mal 4 and transin reflects tumor-promoter-specific responses.

Molecular cloning of sequences activated during multi-stage carcinogenesis in mouse skin.

Recombinant DNA techniques were employed to isolate sequences that were activated during multi-stage carcinogenesis in the skin of NMRI mice. Differential screening of 5000 cDNA clones made from poly(A)+ RNA of squamous cell carcinomas induced by 7,12-dimethylbenz[a]anthracene (DMBA) and the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) resulted in the identification of 35 cDNA clones displaying a different hybridization signal. Eight cDNA clones, three of which proved to be identical, were used as probes in transfer hybridization experiments. These cDNA clones, designated pmal-1 to pmal-6, showed a strong signal with carcinoma RNA but either a weak or no signal with RNA from normal epidermis. Clone pmal-2 contained repetitive sequences as shown by Southern analysis resulting in a smeared RNA hybridization signal in RNA blots whereas the other five clones corresponded to discrete size classes of mRNA. Studies on the expression pattern of mal-1,-2 and -3 related sequences revealed transcriptional activation already in the benign papilloma stage of multi-step carcinogenesis.

DNA and RNA virus species are inhibited by xanthates, a class of antiviral compounds with unique properties.

Various DNA and RNA virus species are inhibited by xanthate compounds at concentrations that leave the mitotic activity of uninfected cells unimpaired. The concentration of tricyclodecan -9-yl- xanthogenate that reduces the yield of herpes simplex virus types 1 and 2 by 50% is between 4.5 and 33 microM. The replication of DNA viruses such as simian virus 40 can be blocked at the DNA and RNA level both early and late after infection. The xanthates are not incorporated into nucleic acids. Episomal bovine papilloma virus DNA replication and transcription are also inhibited in transformed cells. The treated cells revert to the normal phenotype by acquisition of contact inhibition and a flat morphology.