Cloning and recombinant expression of a 822 bp region of a Pf403 Plasmodium falciparum gene.

Thesis (M.Sc.) - University of Natal, Pietermaritzburg, 2003.Malaria is a devastating parasitic disease in humans caused by species in the genus Plasmodium. With over 100 million cases and at least 1.5 million fatalities each year, the disease accounts for 4-5% of all fatalities in the world. A recent increase in the number of malaria cases in South Africa has imposed severe costs on the economy and public health. Immunity to malaria is a multi-component system involving both B and T celllymphocytes. Pc96 is a 96 kDa antigen identified in the mouse malaria model Plasmodium chabaudi adami. It is known to be associated with the outer membrane of mouse erythrocytes infected with the parasite and has shown protective roles in mice challenged with P. chabaudi adami. A specific T cell clone has been identified that adoptively provides protection to athymic mice infected with P. chabaudi adami. Antibodies raised against Pc96 identified proteins that induced the proliferation of the protective T cell clones. At least four other antigens of different species of. malaria share at least one cross-reactive epitope. In an attempt to identify a Plasmodiumfalciparum homologue ofPc96, the amino-acid sequence was used in a BLAST search of the P. falciparum genome database, identifying a 403 kDa protein with a high degree of homology to Pc96. Sequence alignments indicated a region spanning 90 amino acids in Pf403 that overlaps the Pc96 amino acid sequence. A 178 kDa protein in P. yoelii yoelii (Pyy178) was shown to be highly similar to Pc96. Tvcell epitope prediction programs identified putative T cell epitopes in Pc96 which appear to be conserved in Pf403 and Pyy178. A casein kinase IT phosphorylation site was also identified in this region and is conserved in both sequences. PCR primers were designed to amplify regions of the MAL3P6.11 gene coding for Pf403 from P.falciparum genomic DNA. An 817 bp region in the MAL3P6.11 gene was amplified. This codes for the region ofPf403 that shows high homology to Pc96 and contains the conserved T cell epitopes and casein kinase phophorylation site. A BamHI site was incorporated into the forward primer to facilitate in-frame ligation with cloning vectors. The PCRproduct obtained was verified by restriction analysis using HindIII and EcoRI sites within the fragment. The 817 bp peR product was cloned into the pMOSBlue vector using a blunt-endedPCR cloning kit, and transformed into MOSBlue competent cells. Recombinants were identified using the uIV complementation system, and verified by PCR, plasmid DNA isolation, and restriction digestion analysis. The insertDNA in pMOSBlue was cut out with BamHI and sub-cloned into the BamHI site in the pMAL-C2x expression vector. Sequencing ofthe construct confirmed the identity of the cloned insert and showed the sequence to be in frame with the malE gene coding for maltose binding protein (MBP). The fusion protein, MBP-Pf32 .5, was induced and expressed as a 75 kDa protein comprising ofthe 32.5 kDa region ofPf403, and MBP (42.5 kDa) and was detected by anti-MBP antibodies, by western blotting. This recombinant protein has many applications for further studies involving the characterisation of the Pf403 protein, and the determination of possible roles that the protein may have in stimulating an immune response during human malaria infections

Bruton's tyrosine kinase regulates TLR7/8-induced TNF transcription via nuclear factor-κB recruitment

Tumour necrosis factor (TNF) is produced by primary human macrophages in response to stimulation by exogenous pathogen-associated molecular patterns (PAMPs) and endogenous damage-associated molecular patterns (DAMPs) via Toll-like receptor (TLR) signalling. However, uncontrolled TNF production can be deleterious and hence it is tightly controlled at multiple stages. We have previously shown that Bruton's tyrosine kinase (Btk) regulates TLR4-induced TNF production via p38 MAP Kinase by stabilising TNF messenger RNA. Using both gene over-expression and siRNA-mediated knockdown we have examined the role of Btk in TLR7/8 mediated TNF production. Our data shows that Btk acts in the TLR7/8 pathway and mediates Ser-536 phosphorylation of p65 RelA and subsequent nuclear entry in primary human macrophages. These data show an important role for Btk in TLR7/8 mediated TNF production and reveal distinct differences for Btk in TLR4 versus TLR7/8 signalling

Cytokines and inflammatory mediators: 25. Certolizumab Pegol has a Different Profile from the other Anti-TNFS, Including Golimumab, in a Variety of in Vitro Assays

Background: Activities of the anti-TNFs, certolizumab pegol (CZP), etanercept (ETA), infliximab (IFX) and adalimumab (ADA), have been compared in a range of in vitro assays. CZP is the only licensed PEGylated Fab' anti-TNF; ETA is a fusion protein with an IgG1 Fc, and IFX and ADA are both antibodies with an IgG1 Fc. Golimumab (GLM) is a monoclonal IgG1 TNF inhibitor recently approved for a number of indications; it is thus of interest to assess the in vitro activity of GLM. In vitro assays previously used were neutralisation of TNF in the L929 bioassay, inhibition of LPS-driven cytokine production by monocytes, induction of apoptosis in activated lymphocytes and monocytes, and induction of neutrophil necrosis. Methods: Neutralisation of human TNF was assessed in the L929 bioassay using a range of concentrations of the anti-TNFs and a fixed concentration of TNF (100 pg/mL). Activity of the anti-TNFs at inhibiting LPS-driven IL-1β secretion by monocytes was assessed by incubating peripheral blood monocytes with various concentrations of the anti-TNF for 1 hour (hr) and then washing the cells. LPS was added for 4 hrs, the supernatants collected and the IL-1β level measured by ELISA. To assess induction of apoptosis, peripheral blood lymphocytes were activated for 2 days with 2 μg/mL CD3/CD28 and monocytes with 300 U/mL IL-4 and GMCSF for 3 days. The effect of the anti-TNFs on apoptosis was assessed by Annexin V staining using flow cytometry 24 hrs later. The effect of the anti-TNFs on neutrophil necrosis was determined by measuring myeloperoxidase release after 12 hrs. An isotype-matched control was used in all assays except the L929 bioassay. Results: IC90 neutralisation activity of the anti-TNFs in the L929 bioassay was 0.3 ng/mL for ETA, 4 ng/mL for GLM, 15 ng/mL for ADA, and 20 ng/mL for IFX, compared with 2.5 ng/mL for CZP. CZP was the most potent inhibitor of LPS-driven IL-1β secretion (IC50 ∼0.1 ng/mL), followed by GLM (20 ng/mL) and IFX (50 ng/mL). GLM, ADA, IFX and ETA induced apoptosis of monocytes and lymphocytes to a similar degree reaching a level of 23% and ∼40% at 100 μg/mL, respectively. CZP caused no increase in apoptosis above the levels seen with the isotype-matched control. In the neutrophil necrosis assay, ADA,IFX and GLM caused ∼70% necrosis at 100 μg/mL, and ETA 48%. CZP did not increase the level of necrosis above the level of the control. Conclusions: Bioactivity of the IgG1 molecules GLM, IFX and ADA in neutralising human TNF was inferior to that of CZP and ETA. CZP, the only PEGylated anti-TNF, had a different profile to the other anti-TNFs as it was the most potent at inhibiting LPS-driven IL-1β production by monocytes, did not induce apoptosis of activated monocytes and lymphocytes, and did not cause neutrophil necrosis. The clinical relevance of these in vitro effects is unknown. Nevertheless, these assays show interesting in vitro differences between the anti-TNFs. Disclosure statement: G.F. and A.N. are employees of UC

IL-10 mediated control of TNF gene regulation in human macrophages

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Expression of vFLIP in a Lentiviral Vaccine Vector Activates NF-κB, Matures Dendritic Cells, and Increases CD8+ T-Cell Responses▿

Lentiviral vectors deliver antigens to dendritic cells (DCs) in vivo, but they do not trigger DC maturation. We therefore expressed a viral protein that constitutively activates NF-κB, vFLIP from Kaposi's sarcoma-associated herpesvirus (KSHV), in a lentivector to mature DCs. vFLIP activated NF-κB in mouse bone marrow-derived DCs in vitro and matured these DCs to a similar extent as lipopolysaccharide; costimulatory markers CD80, CD86, CD40, and ICAM-1 were upregulated and tumor necrosis factor alpha and interleukin-12 secreted. The vFLIP-expressing lentivector also matured DCs in vivo. When we coexpressed vFLIP in a lentivector with ovalbumin (Ova), we found an increased immune response to Ova; up to 10 times more Ova-specific CD8+ T cells secreting gamma interferon were detected in the spleens of vFLIP_Ova-immunized mice than in the spleens of mice immunized with GFP_Ova. Furthermore, this increased CD8+ T-cell response correlated with improved tumor-free survival in a tumor therapy model. A single immunization with vFLIP_Ova also reduced the parasite load when mice were challenged with OVA-Leishmania donovani. In conclusion, vFLIP from KSHV is a DC activator, maturing DCs in vitro and in vivo. This demonstrates that NF-κB activation is sufficient to induce many aspects of DC maturation and that expression of a constitutive NF-κB activator can improve the efficacy of a vaccine vector

Knowing me, knowing you : own orientation and information about the

The purpose of this study was to examine the effects of motivational orientations on negotiation outcomes in unstable negotiation contexts. Instability was created by pitting individualists against cooperators (mixed dyads), and by giving only one of the parties information about the other party’s orientation. A total of 162 subjects participated in negotiation simulations, where orientation and information were manipulated through instructions from management. The cooperative dyads got better outcomes than did the individualistic dyads. The mixed dyads did as well as the cooperative dyads when the cooperators had information, but did as badly as the individualistic dyads when the individualists had information. The process analyses indicated that the dyads with high outcomes achieved their results because the integrative activities increased over time. In the mixed dyads with informed individualists, the individualists reached higher individual outcome than their cooperative (uninformed) opponents. Thus, naive cooperators can easily be exploited