Two-step chromatographic purification of recombinant Plasmodium falciparum circumsporozoite protein from Escherichia coli.

The Plasmodium falciparum circumsporozoite (PfCS) protein (aa 19-405) has been cloned and expressed in E. coli. The protein was purified in a two-step process that was rapid and reproducible. E. coli cells were grown to a high density before induction for 1 h. Cells were disrupted by high pressure microfluidization and the total bacterial protein solubilized in 6 M Gu-HCl. The protein was refolded while bound to Ni-NTA agarose by exchange of 6 M Gu-HCl for 8 M urea and then slow removal of the urea. The eluted protein was further purified on Q Sepharose Fast Flow using conditions developed to remove E. coli proteins and reduce endotoxin (to 10 EU/50 microg). Yield was 20 mg of PfCS protein from 10 g of wet cell paste. The final protein product bound to HepG2 liver cells in culture and inhibited the invasion of those cells by sporozoites in an ISI assay greater than 80% over control cultures when used at 10 microg/ml.

[Expression of a cloned hemagglutinin-neuraminidase gene from Newcastle disease virus in murine myeloma cells]. / Ekspressiia klonirovannogo gena gemaggliutinin-neiraminidazy virusa bolezni N'iukasla v kletkakh myshinoi mielomy.

Vaccination with autologous cancer cells expressing a potent foreign antigen is promising for immunotherapy of tumors. A construct was obtained to transfect cancer cells with the hemagglutinin-neuraminidase (HN) gene of the Newcastle disease virus (NDV). Specific primers were designed, and the HN cDNA was amplified from RNA isolated from the allantoid fluid of NDV-infected embryonated chicken eggs. The amplified fragment was cloned in pCR2.1, sequenced, and recloned in expression vector pCDNA3.1/Zeo(+). The resulting construct was used to transfect mouse myeloma cells SP2/0. Production of HN was checked by ELISA and by a neuraminidase activity assay. Cell agglutination on ice was proposed as a test for surface HN.

Inhibitory and blocking monoclonal antibody epitopes on merozoite surface protein 1 of the malaria parasite Plasmodium falciparum.

Merozoite surface protein 1 (MSP-1) is a precursor to major antigens on the surface of Plasmodium spp. merozoites, which are involved in erythrocyte binding and invasion. MSP-1 is initially processed into smaller fragments; and at the time of erythrocyte invasion one of these of 42 kDa (MSP-1(42)) is subjected to a second processing, producing 33 kDa and 19 kDa fragments (MSP-1(33) and MSP-1(19)). Certain MSP-1-specific monoclonal antibodies (mAbs) react with conformational epitopes contained within the two epidermal growth factor domains that comprise MSP-1(19), and are classified as either inhibitory (inhibit processing of MSP-1(42) and erythrocyte invasion), blocking (block the binding and function of the inhibitory mAb), or neutral (neither inhibitory nor blocking). We have mapped the epitopes for inhibitory mAbs 12.8 and 12.10, and blocking mAbs such as 1E1 and 7.5 by using site-directed mutagenesis to change specific amino acid residues in MSP-1(19) and abolish antibody binding, and by using PEPSCAN to measure the reaction of the antibodies with every octapeptide within MSP-1(42). Twenty-six individual amino acid residue changes were made and the effect of each on the binding of mAbs was assessed by Western blotting and BIAcore analysis. Individual changes had either no effect, or reduced, or completely abolished the binding of individual mAbs. No two antibodies had an identical pattern of reactivity with the modified proteins. Using PEPSCAN each mAb reacted with a number of octapeptides, most of which were derived from within the first epidermal growth factor domain, although 1E1 also reacted with peptides spanning the processing site. When the single amino acid changes and the reactive peptides were mapped onto the three-dimensional structure of MSP-1(19), it was apparent that the epitopes for the mAbs could be defined more fully by using a combination of both mutagenesis and PEPSCAN than by either method alone, and differences in the fine specificity of binding for all the different antibodies could be distinguished. The incorporation of several specific amino acid changes enabled the design of proteins that bound inhibitory but not blocking antibodies. These may be suitable for the development of MSP-1-based vaccines against malaria.

Removal of the circumsporozoite protein (CSP) glycosylphosphatidylinositol signal sequence from a CSP DNA vaccine enhances induction of CSP-specific Th2 type immune responses and improvesprotection against malaria infection.

The C terminus of the circumsporozoite protein (CSP) is anchored to the parasite cell membrane by a glycosylphosphatidylinositol (GPI) glycolipid. This GPI signal sequence functions poorly in heterologous eukaryotic cells, causing CSP retention within internal cell organelles during genetic immunization. Cellular location of antigen has quantitative and qualitative effects on immune responses induced by genetic immunization. Removal of the GPI signal sequence had a profound effect on induction and efficacy of CSP-specific immune response after genetic immunization of BALB/c mice with a gene gun. The CSP produced from the plasmid lacking the GPI anchor signal sequence (CSP-A) was secreted and soluble, but that produced by the CSP+A plasmid was not. The CSP-A plasmid induced a highly polarized Th2 type response, in which the CSP-specific IgG antibody titer was three- to fourfold higher, and the protective effect was significantly greater than that induced by the CSP+A plasmid. Thus, these two physical forms of CSP induced quantitatively and qualitatively different immune responses that also differed in protective efficacy. Engineering plasmid constructs for proper cellular localization of gene products is a primary consideration for the preparation of optimally efficacious DNA vaccines.

Induction of monocyte- and T-cell-attracting chemokines in the lung during the generation of idiopathic pneumonia syndrome following allogeneic murine bone marrow transplantation.

Idiopathic pneumonia syndrome (IPS) is a significant complication following bone marrow transplantation (BMT). We have developed a murine model in which severe IPS is induced by pre-BMT conditioning and allogeneic T cells and is characterized by the recruitment of host monocytes and donor T cells into the lung by day 7 post-BMT. Chemokines regulate cellular recruitment and the migration of cells into inflammatory lesions. In this study, we examined the profiles of chemokines produced locally in the lung (parenchyma and bronchoalveolar lavage fluid) and systemically (serum) during the generation of IPS in the peri-BMT period. Protein and messenger RNA (mRNA) levels of CC chemokines (monocyte/lymphocyte attractants), especially monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1alpha, RANTES (regulated upon activation normal T-cell expressed and secreted), and C10, were preferentially induced in the lung by day 7 postallogeneic BMT. In addition, there was an increase in mRNA for IP-10 (a monocyte and Th1-cell chemoattractant). The CXC chemokines MIP-2 and KC, known neutrophil attractants, were moderately elevated. For the most part, these increases in chemokines were dependent on the coinfusion of allogeneic T cells with the BM inoculum. Ribonuclease protection assay and in situ hybridization analyses post-BMT showed that the lung was a major producer of MCP-1, a potent inducer of monocyte chemotaxis. Increases in MCP-1 levels in the lung preceded host APC influx whereas MIP-1alpha levels accompanied donor T-cell infiltration. In summary, we have shown that monocyte- and T-cell-attracting chemokines are associated with monocyte and T-cell recruitment during IPS.

Expression of IL-8 gene in human monocytes and lymphocytes: differential regulation by TNF and IL-1.

TNF-alpha and IL-1 were reported to be the most powerful inducers of IL-8 in a multitude of cells, including leukocytes. In this study, we investigated TNF-alpha- and IL-1-mediated regulation of IL-8 gene expression in non-fractionated PBMC, and purified monocyte (MO) and lymphocyte (LY) fractions. Our analysis revealed that purified human MO did not respond to exogenous TNF-alpha with the induction of IL-8 mRNA or protein, nor require endogenous TNF-alpha for IL-8 expression. In contrast, in the presence of exogenous IL-1alpha and IL-1beta a substantial enhancement of IL-8 mRNA and protein expression in MO was observed. Nevertheless, antibodies to IL-1alpha and IL-1beta were unable to downregulate the expression of IL-8 in resting adherent or Staphylococcus aureus Cowan 1 (SAC)-stimulated MO. In contrast with MO, purified LY and non-fractionated PBMC expressed IL-8 in response to exogenous TNF-alpha, similar to exogenous IL-1alpha and IL-1beta. As was seen with MO, antibodies to TNF-alpha, IL-1alpha and IL-1beta did not inhibit the expression of IL-8 in purified LY and non-fractionated PBMC stimulated with SAC and LPS. Taken together, our data demonstrate major differences in responsiveness of MO and LY to exogenous TNF-alpha and IL-1, and suggest relative autonomy of IL-8 gene expression in these cells that does not require accessory cytokines but can be induced directly by exogenous stimuli.

Resistance of Staphylococcal enterotoxin B- induced proliferation and apoptosis to the effects of dexamethasone in mouse lymphocyte cultures.

Staphylococcal enterotoxin B (SEB) is a superantigen causing lymphocyte proliferation and apoptosis. Glucocorticoids are immunosuppressants and are released immediately following SEB intoxication in mice. Whether glucocorticoids affect lymphocyte proliferation and apoptosis in SEB-intoxicated mice is still unknown. To study this question, we examined the effects of dexamethasone (DEX), a synthetic glucocorticoid, on SEB-stimulated lymphocyte cultures from mouse thymus and peripheral lymphoid tissues (PLT). SEB, as well as concanavalin A (Con A), induced lymphocyte proliferation which peaked on day 4 and declined significantly on day 7. As expected, in Con A-stimulated cultures, DEX completely suppressed the proliferation of lymphocytes from both the thymus and PLT. However, in SEB-stimulated cultures, while DEX completely suppressed thymocyte proliferation, it did not suppress PLT cell proliferation even at a high concentration of 10(-7) M. The proliferating cells were Vbeta8(+) T cells of both the CD4(+) and CD8(+) subsets. DEX caused apoptosis. SEB also caused apoptosis, which was manifested by a maximal DNA subdiploidy on day 4 and by a maximal DNA fragmentation on day 7. Both events appeared not to be affected by DEX. The failure of DEX to affect the proliferation and apoptosis was consistent with high levels of cytokines (IL-1alpha, IL-2, IL-4, IL-6 and IFN-gamma) produced in the SEB-stimulated cultures, suggesting that the cytokines act in concert to circumvent the effects of DEX.

[Design of constructions for the expression of proteolytic antibody light chain]. / Dizain konstruktsii dlia ékspressii legkoi tsepi proteoliticheskogo antitela.

A new system for the expression of a catalytic light chain antibody to the vasoactive intestinal peptide is described. The system made possible the isolation the large amounts of a homogeneous protein without any additional peptide domains. The preparation obtained can be used in further experiments on light chain crystallization and in X-ray-structural analysis of its catalytic center.

[Participation of 2-C-methyl-D-erythritol-2,4-cyclopyrophosphate in reactions of bacteria on oxidative stress and their persistence in macrophages]. / Uchstie 2-C-metil-D-éritritol-2,4-tsiklopirofosfata v reaktsii bakterii na okislitel'nyi stress i v ikh persistentsii v makrofagakh.

In aerated medium, Corynebacterium ammoniagenes cells accumulate 2-C-methyl-D-erythritol-2,4-cyclopyrophosphate (MEC) during heat shock and in the presence of O2--generating compounds or ozone. The ability to accumulate MEC was genetically transformed from C. ammoniagenes to E. coli XL-1; transformed E. coli (2-31 clone) accumulates MEC in the presence of glucose and glucose oxidase (generation of H2O2) or benzylviologen (generation of O2-); the viability of transformed bacteria inside the murine peritoneal macrophages also significantly increases. However, model conditions of phagosomes of warm-blooded animals (NO + H2O2 + O2-) did not cause MEC accumulation by C. ammoniagenes but increased the formation of polyphosphate which can be due to selective oxidative aberration of biosynthetic processes. Growth rate of Acanthamoeba castellanii on solid medium with bacterial lawn was not significantly different in C. ammoniagenes, C. ammoniagenes with preaccumulated MEC, E. coli XL-1, and E. coli 2-31 and did not depend on the accumulation of MEC by bacteria. Unlike the recipient E. coli strain, the transformed 2-31 clone synthesizes two nonpolar lipids (Rf = = 0.85 and 0.75; TCL on Silufol in hexane) and carotinoid pigments; this can be due to changes in metabolic pathways of isopentenylpyrophosphate that can be a precursor of MEC biosynthesis. Thus, MEC is involved in bacterial responses to certain components of oxidative stress and in bacterial persistence inside the macrophages.

The ability of a recombinant Escherichia coli strain to synthesize 2-C-methyl-D-erythritol-2,4-cyclopyrophosphate correlates with its tolerance to in vitro induced oxidative stress and to the bactericidal action of murine peritoneal macrophages.

A number of bacteria are able to synthesize 2-C-methyl-D-erythritol-2,4-cyclopyrophosphate (BOSS) in response to oxidative stress. Here we show that the ability to synthesize BOSS can be genetically transferred from Corynebacterium ammoniagenes to Escherichia coli. A total DNA library from C. ammoniagenes ATCC 6872 established in the pBluescript SKII+vector backbone was transfected into E. coli XL-1 blue. Recombinant clone 2-31, which was resistant to redox-cycling agents, was selected. NMR studies showed that this clone was able to synthesize BOSS. We also studied the resistance of clone 2-31 to the bactericidal action of macrophages. Clone 2-31 cells had better survival within murine peritoneal macrophages than parental E. coli XL-1-blue cells. Since the ability to synthesize BOSS correlates with increased survival of bacteria within macrophages, we suggest that the pathogenicity of Corynebacteria could be mediated through the synthesis of BOSS.

Differential action of cycloheximide and activation stimuli on transcription of tumor necrosis factor-alpha, IL-1 beta, IL-8, and P53 genes in human monocytes.

In the present study we have analyzed superinduction of TNF-alpha mRNA and enhancement of TNF-alpha gene transcription by cycloheximide (Chx) in human blood monocytes isolated by continuous Percoll gradient and activated in vitro. In the same monocyte cultures, we have compared the rate of gene transcription of TNF-alpha, IL-1 beta, IL-8, and the P53-antioncogene under the influence of plastic adherence, Staphylococcus aureus Cowan 1 (SAC), and Chx added at different times of monocyte culture. It was shown that the cytokine genes have low or negligible transcriptional activity in freshly isolated monocytes, whereas P53 gene transcription was constant in freshly isolated and in vitro-stimulated cells. Transcription of the IL-1 beta and IL-8 genes was induced by adherence and was not more enhanced by SAC. Transcription of the TNF-alpha gene was not induced by adherence. Chx added at the beginning of the monocyte culture did not block TNF-alpha or IL-1 beta gene transcription. IL-8 gene transcription, however, was abrogated by Chx. Addition of SAC to monocyte culture containing Chx caused significant enhancement of TNF-alpha gene transcription. Addition of Chx after 2.5 or 4 h of SAC activation caused "superinduction" of TNF-alpha mRNA and enhancement of TNF-alpha gene transcription. The data imply that TNF-alpha gene transcription in activated human monocytes might be regulated by both positive and negative regulatory factors that differ in their stability and protein synthesis dependence. In addition, results demonstrate that TNF-alpha, IL-1 beta, IL-8, and p53 genes in human monocytes are differently regulated.