Karel Raška

Chimerasome-mediated gene transfer in vitro and in vivo

Proteoliposome delivery vesicles can be prepared by the protein-cochleate method [Gould-Fogerite and Mannino, Anal. Biochem. 148 (1985) 15-25; Mannino and Gould-Fogerite, Biotechniques 6 (1988) 682-690]. Proteins which mediate the entry of enveloped viruses into cells are integrated in the lipid bilayer, and materials are encapsulated at high efficiency within the aqueous interior of these vesicles. We describe proteoliposome-mediated delivery of proteins and drugs into entire populations of cells in culture. Material can be delivered gradually by Sendai-virus-glycoprotein-containing proteoliposomes. Alternatively, synchronous delivery to a population can be achieved by exposing cell-bound influenza glycoprotein vesicles briefly to low pH buffer. When DNA is encapsulated, chimeric proteoliposome gene-transfer vesicles (chimerasomes), which mediate high-efficiency gene transfer in vitro and in vivo, are produced. Stable expression of a bovine papilloma virus-based plasmid in tissue-cultured cells, at 100,000 times greater efficiency than Ca.phosphate precipitation of DNA, with respect to the quantity of DNA used, has been achieved. Stable gene transfer and expression in mice has been obtained by subcutaneous injection of chimerasomes containing a plasmid expressing the early region of polyoma virus. In one experimental group, 50% of the mice developed tumors which were shown to express polyoma virus early proteins and contain the transferred DNA. This is the first report of stable gene transfer in animals mediated by a liposome- or proteoliposome-based system.

IMMUNE DEFICIENCY IN UREMIA : INTERLEUKIN-2 PRODUCTION AND RESPONSIVENESS AND INTERLEUKIN-2 RECEPTOR EXPRESSION AND RELEASE

We have studied the role of interleukin-2 (IL-2) and its receptors in the impaired in vitro lymphocyte response characteristic of hemodialysis patients treated by means of cuprophane membranes. The proliferative response of T lymphocytes as well as T-cell-dependent B cell proliferation after stimulation with mitogens was significantly reduced in hemodialysis patients. The in vitro production of IL-2 after such stimulation in parallel cultures was found to be similar in patients and in controls. The expression of IL-2 receptor on the lymphocyte cellular membrane in the hemodialysis group was also similar to controls. The in vitro proliferative response of uremic lymphocytes to exogenous IL-2, however, was significantly depressed suggesting a reduced availability of biologically active IL-2 receptor. The release of soluble IL-2 receptor by lectin-stimulated lymphocytes in culture was also significantly lower in the patient group; yet, hemodialysis patients has a strikingly elevated level of plasma soluble IL-2 receptor, and similar high levels were also found in three other groups of end-stage renal disease patients dialyzed by means of cellulose acetate, polysulfone and polyacrylonitrile membranes, as well as in a group of uremic patients on conservative treatment. In the hemodialysis patient group a significant positive correlation between levels of soluble IL-2 receptor and the duration of hemodialysis was found. Since soluble IL-2 receptor has been reported to down-regulate lymphocyte responses, the elevation in plasma levels of soluble IL-2 receptor in hemodialysis patients may be a pathogenetic factor in the progressive development of impaired immunity associated with end-stage renal disease.

Soluble and cellular markers of immune activation in patients with systemic sclerosis

The peripheral blood lymphocyte pattern, the lymphocyte responses in vitro, as well as the soluble markers of immune activation were studied in 24 patients with systemic sclerosis (SSc patients). The proportions of total T cells (CD3), their CD4 subset, as well as B lymphocytes were within the normal range. The relative proportion of CD8 lymphocytes, however, was significantly reduced. Patients with SSc had a slightly lower percentage of CD4/4B4+ cells, whereas their proportion of CD4/2H4+ cells was elevated as compared to healthy controls. The proportion of lymphocytes expressing the interleukin-2 receptor (IL-2R) was significantly higher in SSc patients. The proliferative responses of peripheral blood mononuclear cells to PHA stimulation were reduced in the patient group, while expression of IL-2R on lymphocytes after such in vitro stimulation was comparable to that of controls. Expression of IL-2R on patient but not control lymphocytes was increased after in vitro exposure to laminin. Such exposure failed to induce IL-2 production or cell proliferative responses. Soluble plasma IL-2R level (sIL-2R) and soluble CD8 (sCD8) molecule levels in SSc patients were significantly elevated. These results indicate the presence of an ongoing lymphocyte activation in this disease process.

Effects of cyclosporine and rapamycin on immunoglobulin production by preactivated human B cells

In order to assess the direct effects of cyclosporine A (CsA) and rapamycin on B cells, we utilized a two‐segment culture system of highly purified B lymphocytes consisting of induction (activation) in the presence of the formalinized Staphylococcus aureus bacteria and IL‐2. and differentiation, respectively, in the presence of various combinations of cytokines. Results show that rapamycin strongly inhibited production of both IgM and IgG measured at the end of the secondary culture supported by IL‐2/IL‐6, whereas CsA up‐regulated the immunoglobulin production. The stimulatory effect of CsA was also observed when preactivated B cells were recultured in absence of any cytokines. These results show that rapamycin and CsA have dearly distinct effects on human B lymphocyte responses in vitro. Rapamycin is a more potent in vitro immunosuppressant of B lymphocytes than CsA. It is effective at significantly lower concentrations, and it does not stimulate either the proliferation or antibody production by preactivated B cells.

Ultrastructural immunolocalization of cyclin/PCNA in synchronized 3T3 cells.

The immunolocalization of cyclin/PCNA in synchronized 3T3 cells was performed with human autoantibodies using an immunogold technique performed on thin cryosections. Previous immunofluorescent studies demonstrated that the DNA replication sites correspond to the localization of bound cyclin. We have found that in the early periods of S phase, the DNA replication sites (or sites potentially ready for the replication during the hydroxyurea DNA synthesis block) are situated in the perichromatin region and correspond to clustered gold particles present frequently over a morphologically distinct small nuclear area. Heavily labeled chromocenters, including perinucleolar condensed chromatin, exhibiting several such distinct areas were found in later periods of S phase.

Normal T Lymphocyte Function in Patients with End-Stage Renal Disease Hemodialyzed with ‘High-Flux’ Polysulfone Membranes

T lymphocyte function was analyzed in patients hemodialyzed with high-flux polysulfone membranes, which have been reported to improve the patients overall clinical condition and well-being. For comparison purposes, patients treated by the use of low-flux cuprophane membranes were also studied. Peripheral blood white cell counts, numbers of lymphocytes as well as the numbers of T cells and their CD4 and CD8 subsets were within normal range in both patient groups. The absolute number of B cells was slightly decreased in cuprophane-membrane- but not polysulfone-membrane-treated patients. The proliferative response of T lymphocytes after stimulation with optimal concentration of phytohemagglutinin (PHA) was normal in patients treated with high-flux membrane dialysis but significantly reduced in those treated with cuprophane membranes. The generation of interleukin-2 (IL-2) receptor on T lymphocytes after PHA stimulation was normal in the polysulfone-membrane-treated group and slightly impaired in the cuprophane-membrane-dialyzed patients. Production of both IL-2 and interleukin-1, as well as the natural killer cell activity, in patients treated by high-flux membrane dialysis were also comparable to controls. The levels of serum beta 2-microglobulin were significantly elevated in patients-maintained on high-flux dialysis membranes but did not reach the levels seen in patients dialyzed by cuprophane membranes. The beta 2-microglobulin at levels seen in patients on cuprophane dialysis had no effects on activation and proliferation of control lymphocytes in vitro. These results suggest that impaired functional responses of T lymphocytes seen in end-stage disease patients on prolonged hemodialysis with cuprophane membranes are not seen in similar patients hemodialyzed with polysulfone membranes.

Product of adenovirus type 2 early gene block E1 in transformed cells elicits cytolytic response in syngeneic rats

Abstract The cytotoxic immune response to three well-characterized adenovirus type 2 (Ad2)-transformed cell lines (A2T8, 50A, and T2C4) was studied in syngeneic LIS rats. Antibodies against the three virus-transformed lines were raised by hyperimmunization. Cytolytic T cells were generated by in vivo immunization and in vitro education with A2T8 and 50A cells. The complement-dependent antibody-mediated cytotoxicity with hyperimmune sera, T-cell cytotoxicity, and antibody-blocking effects in T-cell cytotoxicity assays indicate that these three Ad2-transformed cell lines share specific antigen. This observation was further confirmed by indirect immunofluorescence staining of the cell surface. Because it is absent in syngeneic LIS embryo fibroblasts and in syngeneic LIS cells transformed with EcoRI-C fragment of Ad12 DNA, this antigen appears to be Ad2 specific. Since the only Ad2 function shared and expressed by all three of these cell lines is the early gene block E1, it is concluded that its product or products elicit both humoral and T-cell cytolytic responses.

Adenovirus type 5 and adenovirus type 12 recombinant viruses containing heterologous E1 Genes are viable, transform rat cells, but are not tumorigenic in rats

Two sets of adenovirus type 5 (Ad5)-adenovirus type 12 (Ad12) recombinant viruses were constructed and analyzed. In one case the Ad12 E1A, E1B, or E1A plus E1B genes were substituted for the corresponding Ad5 genes in the Ad5 chromosome. The second set contained the Ad5 E1A, E1B, or E1A plus E1B genes in place of the cognate Ad12 genes in the Ad12 chromosome. The hybrid viruses were all viable and expressed the appropriate E1 antigens. They were able to transform secondary rat fibroblasts, but at reduced efficiency as compared to either parental virus. Fibroblasts transformed with the recombinant Ad5 virus carrying the Ad12 E1A plus E1B genes were tumorigenic in newborn, syngeneic rats. Some of the cell lines transformed with the Ad5 virus containing the Ad12 E1A gene were tumorigenic but none of the recombinants with the Ad12 E1B gene was able to induce tumors in this assay. Although Ad12 was tumorigenic, none of the Ad5 or Ad12 recombinant viruses induced tumors in newborn rats injected either intracerebrally or subcutaneously with virus particles.

The response of BHK21 cells to infection with type 12 adenovirus: VI. Synthesis of virus-specific RNA

Abstract When G1-arrested BHK21 cells are infected with type 12 adenovirus (Ad12), some of the newly synthesized RNA is virus specific. RNA-DNA hybridization-competition experiments have demonstrated that this RNA corresponds to up to 60% of those “early” mRNA sequences which are transcribed in productively infected human embryonic kidney (HEK) cells. No “late” Ad12 mRNA was detected in BHK21 cells. Comparison of Ad12-specific RNA sequences transcribed in Ad12-infected BHK21 cells and in Ad12-induced hamster tumor cells (line HT 2 ) by hybridization-competition reaction indicated that no virus-specific sequences are transcribed in HT 2 cells which are not also present in BHK21 cells abortively infected with Ad12. Attempts to detect any Ad12 DNA synthesis in BHK21 cells by DNA-DNA hybridization, under conditions of minimal background cellular DNA synthesis, were unsuccessful. Correspondingly, immunofluorescent staining experiments failed to detect Ad12 capsid protein synthesis in over 10 4 infected cells.

Effects of arginine starvation on macromolecular synthesis in infection with type 2 adenovirus: II. Synthesis of virus-specific RNA and DNA☆

Abstract The effects of arginine starvation on DNA and RNA synthesis and on polyribosomes in KB cells infected with type 2 adenovirus were studied. Synthesis of viral DNA in arginine-starved (Arg−) cells was demonstrated by CsCl equilibrium density gradient centrifugation, zonal velocity sedimentation in alkaline sucrose gradients, and DNA-DNA hybridization. However, the amounts of cellular and especially viral DNA in Arg− cells were significantly lower than in cells maintained in complete medium (Arg+). No evidence was obtained to suggest that this quantitative difference was due to degradation of newly synthesized DNA in Arg− cells. Up to 12 hr after infection, no quantitative or qualitative difference was found between Arg+ and Arg− cells with regard to viral mRNA transcribed. Thereafter (to 17 hr p.i.), the rate of viral RNA synthesis decreased but the same species continued to be synthesized. At 17.5 hr p.i., the lowering of the rate of RNA synthesis was reflected both in the nucleus and in the cytoplasm of Arg− cells and affected cellular as well as viral RNA. However, the relative distribution of viral mRNA between cytoplasmic fractions (polyribosomal and nonpolyribosomal) was unaffected by arginine deprivation. Polyribosomes decreased progressively after infection in Arg− cells while they increased in Arg+ cells. The change in polyribosomal profile was not dependent on Ad2 infection, but also occurred in uninfected Arg− cells. Under both conditions, the changes induced by arginine deprivation were reversible by arginine restoration as late as 20 hr after infection and/or arginine deprivation. It is postulated that failure of virion assembly to occur in Arg− cells is not due to failure of a major structural viral constituent to be synthesized ( see also Rouse and Schlesinger, 1972 ); rather it must involve lack of an exquisitely arginine-dependent factor (or factors) needed for normal DNA synthesis and, especially, for enabling the DNA that is made to associate with the structural proteins that are also synthesized. This conclusion is supported by the earlier finding that the DNA preformed in Arg− cells does, but the preformed viral proteins do not, participate in virion assembly which occurs upon arginine restoration.