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Coccidiosis is caused by infection with Eimeria spp. The disease is responsible for major economic loss to the poultry industry unless infections are controlled by anticoccidial drugs. John Ellis and Fiona Tomley discuss recent research on the characterization and cloning of antigens from Eimeria spp and advances towards the development of genetically engineered vaccines against poultry coccidiosis.
Articular cartilage injuries are commonly encountered problems in sports medicine and orthopaedics. The treatment of chondral and osteochondral lesions, which possess only a very limited potential for healing, still represents a great challenge to clinicians and to scientists. Experimental investigations reported over the last 20 years have shown that a variety of methods, including implantation of periosteum, perichondrium, artificial matrices, growth factors, and transplanted cells, can stimulate formation of new cartilage. Genetic engineering--a combination of gene transfer techniques and tissue engineering--will facilitate new approaches to the treatment of articular cartilage injuries.
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Metal concentrations in soils are locally quite high, and are still increasing due to many human activities, leading to elevated risk for health and the environment. Phytoremediation may offer a viable solution to this problem, and the approach is gaining increasing interest. Improvement of plants by genetic engineering, i.e. by modifying characteristics like metal uptake, transport and accumulation as well as metal tolerance, opens up new possibilities for phytoremediation. So far, only a few cases have been reported where one or more of these characteristics have been successfully altered; e.g. mercuric ion reduction causing improved resistance and phytoextraction, and metallothionein causing enhanced cadmium tolerance. These, together with other approaches and potentially promising genes for transformation of target plants are discussed.
Previously, we have shown that some rheumatoid factors (RFs) produced by Epstein-Barr virus (EBV)-transformed B cells from patients with rheumatoid arthritis (EBV-RA-RF) appear to be disease-specific autoantibodies that bind differently to defined epitopes on genetically engineered IgG antibodies, compared with RFs expressed by patients with Waldenstrom's macroglobulinaemia (Wmac-RFs) and healthy immunized donors (HID-RFs). To exclude the possibility that EBV transformation is responsible for these differences, we have now studied 15 other monoclonal IgM RFs from patients with RA that were produced by heterohybridoma-B-cell fusion (HRA-RFs). These HRA-RFs show the same gross specificity profiles for IgG as do their EBV-RA-RF counterparts. However, when the specificities of the HRA-RF and EBV-RA-RF panels were combined and compared with those RFs from patients with Wmac or HID, significant differences in binding specificity were again observed. Hybrid IgG3/4 antibodies made by exon shuffles between the IgG3 and IgG4 wild-type genes, and families of IgG variant antibodies made by site-directed mutagenesis, were used to map the fine specificity of HRA-RFs. The fine specificity of HRA-RFs were also similar to those of EBV-RA-RFs. These studies demonstrate that the method used for immortalizing IgM, RF-producing B cells from RA patients does not influence the specificity of the RFs obtained. Furthermore, some RFs expressed in RA have distinct and unique specificities, and may therefore represent disease-specific autoantibodies.
Polarization of an immune response toward tolerance or immunity is dictated by the interactions between T cells and dendritic cells (DC), which in turn are modulated by the expression of distinct cell surface molecules, and the cytokine milieu in which these interactions are taking place. Genetic modification of DC with genes coding for specific immunoregulatory cell surface molecules and cytokines offers the potential of inhibiting immune responses by selectively targeting Ag-specific T cells. In this study, the immunomodulatory effects of transfecting murine bone marrow-derived DC with Fas ligand (FasL) were investigated. In this study, we show that FasL transfection of DC markedly augmented their capacity to induce apoptosis of Fas+ cells. FasL-transfected DC inhibited allogeneic MLR in vitro, and induced hyporesponsiveness to alloantigen in vivo. The induction of hyporesponsiveness was Ag specific and was dependent on the interaction between FasL on DC and Fas on T cells. Finally, we show that transfusion of FasL-DC significantly prolonged the survival of fully MHC-mismatched vascularized cardiac allografts. Our findings suggest that DC transduced with FasL may facilitate the development of Ag-specific unresponsiveness for the prevention of organ rejection. Moreover, they highlight the potential of genetically engineering DC to express other genes that affect immune responses.
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Interleukin-12 (IL-12) mediates significant antitumor effects in animal models but associated with dose-dependent toxicity in human. To achieve local expression of IL-12 at the tumor site without systemic toxicity, we performed intra-arterial administration of fibroblasts genetically engineered to produce IL-12 protein with or without retrovirus (CRIP- IL-12 or 3T3-IL-12) in liver metastasis model. Rat breast cancer cells ( MADB - 106) were injected into the portal vein of syngeneic Fisher rats on day 0, and fibroblasts were injected into the hepatic artery on day 7. On day 21, liver weight and number of liver tumors were examined. As controls, CRIP cells expressing retrovirus carrying lacZ marker gene (CRIP-lacZ) or saline (Hanks balanced salt solution, HBSS) were injected. Administration of CRIP-IL-12 significantly reduced tumor metastasis in liver measured by number of foci (CRIP- IL-12: 45.2 +/- 36.7, CRIP-lacZ: >250, HBSS: >250, P<.05) and by liver weight (CRIP-IL-12: 13.0+/-2.5 g, CRIP-lacZ: 30.4+/-8.5 g, HBSS: 26.0+/-7.6 g, P<.05). 3T3-IL-12, which produced only IL-12 protein but not IL-12 retrovirus, also had significant antitumor effects equivalent to CRIP-IL-12. Intra-arterial injection of IL-12--producing fibroblasts into the liver may be an effective therapy for liver tumors reducing systemic toxicity, and could be developed for clinical application.
The possibility of a disease-free world seems to be at the fingertips of the Human Genome Project, but not yet fully within its grasp. Consequently, the advent of the Human Genome Project has ushered in the necessity for a clear delineation of the definition of "disease." This very concept of disease initially must be defined before genetic treatment can be addressed, ensuring that only true disease, not every anomaly of the human condition, are made eligible for genetic alterations. The benefits of such a precautionary measure are innumerable, since a regulation standard could be established and evolutionary implications could be taken into consideration.
The prokaryotic Cre-loxP recombination system is a powerful tool that enables in vitro and in vivo site-specific manipulations of the genome of eukaryotic cells as well as of DNA viruses and their derived vectors. This system, however, has not yet been exploited in the context of herpes simplex virus type 1 (HSV-1) infected cells, perhaps because this virus encodes several functions that induce a strong shutoff of cellular protein synthesis, a fact that could preclude expression of cellular-encoded Cre recombinase. In the present study, we show that efficient site-specific recombination can take place in cell lines expressing Cre, even in the context of HSV-1 infection, as evidenced by the engineering of an HSV-1 recombinant virus and several viral vectors carrying one or two loxP sequences. More precisely, we have used this system to induce an irreversible switch in the expression of a viral complex transcription unit encoding two different open reading frames and allowing consecutive expression of two reporter genes. Furthermore Cre recombinations were also used to induce the decatenation of the genomic concatemers harbored by amplicon particles upon infection of cells under nonreplicative conditions, thus enabling the rescue of many independent plasmids corresponding to the original amplicon plasmid used to generate the vectors. Thus the Cre-loxP recombination system can successfully be used for engineering the genome of HSV-1 or HSV-1-based vectors in cultured cells.
Transplantation of an excessive number of islets of Langerhans (two to four pancreata per recipient) into patients with type I diabetes is required to restore euglycemia. Hypoxia, nutrient deprivation, local inflammation, and the beta cell inflammatory response (up-regulation of NF-kappaB-dependent genes such as inos) result in beta cell destruction in the early post-transplantation period. Genetic engineering of islets with anti-inflammatory and antiapoptotic genes may prevent beta cell loss and primary nonfunction. We have shown in vitro that A20 inhibits NF-kappaB activation in islets and protects from cytokine- and death receptor-mediated apoptosis. In vivo, protection of newly transplanted islets would reduce the number of islets required for successful transplantation. Transplantation of 500 B6/AF(1) mouse islets into syngeneic, diabetic recipients resulted in a cure rate of 100% within 5 days. Transplantation of 250 islets resulted in a cure rate of only 20%. Transplantation of 250 islets overexpressing A20 resulted in a cure rate of 75% with a mean time to cure of 5.2 days, comparable to that achieved with 500 islets. A20-expressing islets preserve functional beta cell mass and are protected from cell death. These data demonstrate that A20 is an ideal cytoprotective gene therapy candidate for islet transplantation.
Intrinsic tyrosines, as monitored by fluorescence spectroscopy, are sensitive reporters of local, Ca(2+)-induced conformational changes in troponin C (TnC). Rabbit skeletal TnC contains two tyrosines (Y10 in the N-helix, and Y109 in site 3 in the C-terminal domain) in distinct microenvironments: their individual contributions to total fluorescence intensity are elucidated here utilizing bacterially synthesized rabbit skeletal TnC (sTnC4) and a genetically engineered variant, termed 109YF, lacking one of the tyrosines (Y109 replaced with F109). The steady-state fluorescence emission spectra following excitation at 280 nm were recorded in EGTA (Ca(2+)-free) and Ca(2+)-saturated (pCa4) solutions. For the wild-type sTnC4, pCa4 causes a significant (46%) increase in the peak fluorescence intensity over the value in EGTA. For the mutant 109YF, the EGTA fluorescence is only marginally affected (74% of the wild-type FEGTA), but interestingly the Ca2+ effect is completely suppressed (delta F = FpCa4-FEGTA = 2% of the wild-type value). These results indicate that the two tyrosines make disparate contributions to the fluorescence spectrum of wild-type sTnC, both in the presence and absence of Ca2+; whereas Y10 in the N-helix is dominant in Ca(2+)-free solution, Y109 is the sole contributor to the Ca2+ effect. Furthermore, to explain the biphasic fluorescence response of Y109 obtained during Ca2+ titrations, the findings yield the most unequivocal evidence that Ca(2+)-induced conformational changes in the trigger sites operating the contractile switch modify properties of the C-terminal sites in TnC pari passu.
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Activation tagging is a new method for isolation and functional identification. It can generate dominant gain-of-function mutants by overexpression of a particular endogenous gene. Due to this special characteristics of activation tagging,this method has been a powerful tool for new gene discovery and gene functional analysis. This paper reviewed the principle and study conditions of activation tagging,as well as its use in plant genetic engineering.
Regulated-on-activation-normal-T-cell-expressed-and-secreted (RANTES), a CC-chemokine, enhances antigen-specific T helper (Th) type-1 responses against HIV-1. To evaluate the adjuvant effects of RANTES against HIV vaccine candidate in SHIV-macaque models, we genetically engineered a live-attenuated SHIV to express the RANTES gene (SHIV-RANTES) and characterized the virus's properties in vivo. After the vaccination, the plasma viral loads were same in the SHIV-RANTES-inoculated monkeys and the parental nef-deleted SHIV (SHIV-NI)-inoculated monkeys. SHIV-RANTES provided some immunity in monkeys by remarkably increasing the antigen-specific CD4+ Th cell-proliferative response and by inducing an antigen-specific IFN-gamma ELISpot response. The magnitude of the immunity in SHIV-RANTES-immunized animals, however, failed to afford greater protection against a heterologous pathogenic SHIV (SHIV-C2/1) challenge compared to control SHIV-NI-immunized animals. SHIV-RANTES immunized monkeys, elicited robust cellular CD4+ Th responses and IFN-gamma ELISpot responses after SHIV-C2/1 challenge. These findings suggest that the chemokine RANTES can augment vaccine-elicited, HIV-specific CD4+ T cell responses.
Alpha-hemolysin is an Escherichia coli exotoxin that enhances bacterial virulence, has profound effects on leukocytes in vitro, and induces the release of interleukin-1 (IL-1) but not tumor necrosis factor (TNF) from human monocytes in vitro. The purpose of this study was to examine alpha-hemolysin's influence on virulence and TNF and IL-1 production in vivo. Two genetically engineered, isogeneic strains of E. coli were used; one variant produces alpha-hemolysin, and the other does not. Male BALB/c mice were injected with either of the two variants and serum TNF and IL-1 were assayed. These results were compared with those obtained from the injection of either of two serotypes of lipopolysaccharide (LPS). The nonhemolytic E. coli strain produced no mortality and no significant elevation of serum TNF or IL-1 levels. In contrast, equal inocula of the hemolytic E. coli strain produced significant mortality and elevation of serum IL-1 levels. No significant elevation of TNF levels was detected in this group despite high-level mortality. A pattern of induction of mortality and elevation of serum IL-1 levels without elevation of serum TNF levels is distinct from the pattern typical of LPS. In these experiments, both serotypes of LPS caused elevations of TNF and IL-1 levels whether or not mortality was induced. Thus, alpha-hemolysin produces a cytokine response in vivo that is similar to that previously demonstrated in vitro by Bhakdi et al. (S. Bhakdi, M. Muhly, S. Korom, and G. Schmidt, J. Clin. Invest. 85:1746-1753, 1990) and appears to induce mortality independently of serum TNF.