Lentivectors encoding immunosuppressive proteins genetically engineer pancreatic beta-cells to correct diabetes in allogeneic mice.

The effectiveness of genetic engineering with lentivectors to protect transplanted cells from allogeneic rejection was examined using, as a model, type 1 diabetes treatment with beta-cell transplantation, whose widespread use has been limited by the requirement for sustained immunosuppressive treatment to prevent graft rejection. We examined whether lentivectors expressing select immunosuppressive proteins encoded by the adenoviral genome early region 3 (AdE3) would protect transplanted beta-cells from an alloimmune attack. The insulin-producing beta-cell line beta TC-tet (C3HeB/FeJ-derived) was transduced with lentiviruses encoding the AdE3 proteins gp19K and RID alpha/beta. The efficiency of lentiviral transduction of beta TC-tet cells exceeded 85%. Lentivector expression of gp19K decreased surface class I major histocompatibility complex expression by over 90%, whereas RID alpha/beta expression inhibited cytokine-induced Fas upregulation by over 75%. beta TC-tet cells transduced with gp19K and RID alpha/beta lentivectors, but not with a control lentivector, provided prolonged correction of hyperglycemia after transplantation into diabetic BALB/c severe combined immunodeficient mice reconstituted with allogeneic immune effector cells or into diabetic allogeneic BALB/c mice. Thus, genetic engineering of beta-cells using gp19K- and RID alpha/beta-expressing lentiviral vectors may provide an alternative that has the potential to eliminate or reduce treatment with the potent immunosuppressive agents necessary at present for prolonged engraftment with transplanted islets.

Mechanisms of E3 modulation of immune and inflammatory responses.

Adenoviruses contain genes that have evolved to control the host immune and inflammatory responses; however, it is not clear whether these genes function primarily to facilitate survival of the virus during acute infection or during its persistent phase. These issues have assumed greater importance as the use of adenoviruses as vectors for gene therapy has been expanded. This review will focus on the mechanism of immune evasion mediated by the proteins encoded within the early region 3 (E3) transcription region, which affect the functions of a number of cell surface receptors including Fas, intracellular cell signaling events involving NF-kappaB, and the secretion of pro-inflammatory molecules such as chemokines. The successful use of E3 genes in facilitating allogeneic transplantation and in preventing autoimmune diabetes in several transgenic mouse models will also be described.

A non-immunogenic adenoviral vector, coexpressing CTLA4Ig and bilirubin-uridine-diphosphoglucuronateglucuronosyltransferase permits long-term, repeatable transgene expression in the Gunn rat model of Crigler-Najjar syndrome.

Host immune responses limit the duration of expression of transgenes introduced by recombinant adenoviruses, preclude gene transfer upon vector readministration and cause liver injury. CTLA4Ig inhibits immune response by blocking the co-stimulatory interaction between CD28 on T cells and B7 on antigen-presenting cells. We have constructed a recombinant adenovirus, Ad-hUGT1A1-CTLA4Ig that coexpresses human bilirubin-uridinediphosphoglucuronate glucuronosyltransferase (hUGT1A1) and soluble murine CTLA4Ig, both driven by CMV immediate-early promoters. After intravenous injection of this vector (6 x 10(11) p.f.u.) into UGT1A1-deficient jaundiced Gunn rats, serum CTLA4Ig levels peaked at 1.8-2.0 mg/ml on day 7 and declined thereafter to 0.2 mg/ml by day 180. Serum bilirubin declined from mean preinjection levels of 8.0 mg/dl to 0.48-0.6 mg/dl in 3 days, remained normal for 28 weeks, and then gradually increased to 8 mg/dl by day 350. A second injection of Ad-hUGT1A1-CTLA4Ig normalized serum bilirubin. In two rats in this group that were followed longer, serum bilirubin increased to 3.1 and 3.5 mg/dl in 40 weeks, but was normalized again after a third injection. The antibody and cytotoxic lymphocyte (CTL) responses were negligible, and liver biopsy showed no inflammatory cell infiltration. Rats receiving a tertiary challenge with Ad-LacZ (expressing E. coli beta-galactosidase) (5 x 10(11) p.f.u.), 2 months after the second dose of Ad-hUGT1A1-CTLA4Ig, showed beta-galactosidase expression in over 80% of hepatocytes. In contrast, after Ad-hUGT1A1 (which expresses UGT1A1 alone) injection, serum bilirubin remained normal for only 4 weeks, and returned to preinjection levels by day 120. Bilirubin levels did not decline upon reinjection, and beta-galactosidase was not expressed after Ad-LacZ. High levels of adenovirus-specific antibodies and CTL, and hepatic inflammation were found. This is the first demonstration that coexpression of CTLA4Ig permits prolonged expression and repeatable gene transfer by an adenoviral vector.

Sites on FIP-3 (NEMO/IKKgamma) essential for its phosphorylation and NF-kappaB modulating activity.

FIP-3 (NEMO/IKKgamma) is an essential modulator of the activity of NF-kappaB by mechanisms that include alterations in the phosphorylation, ubiquination, and degradation of IkappaBalpha. The multiple protein-protein interactions of FIP-3 (NEMO/IKKgamma) in a high molecular weight IKK complex indicated that this protein may be a link between some of the receptor-proximal upstream signal transduction molecules such as RIP and the downstream effects on IkappaBalpha. Although FIP-3 (NEMO/IKKgamma) has no intrinsic kinase activity, it has been shown to increase the kinase activity of IKKbeta. In this manuscript, the results of serine to alanine mutations at five sites on FIP-3 (NEMO/IKKgamma) are described, and functional assays demonstrated that two of these mutants affect both the phosphorylation and kinase activity of IKKbeta. Protein kinase Calpha appeared to be the kinase that was required for the posttranslational modification of FIP-3 (NEMO/IKKgamma). One of the serine targets of the protein kinase Calpha enzyme at amino acid 141 was within a leucine zipper-like sequence of FIP-3 (NEMO/IKKgamma), which might affect its interactions with other proteins on the signal transduction pathway. The second serine, which augmented the inhibition, was at amino acid 85 within the FIP-3 (NEMO/IKKgamma) interaction site with IKKbeta. When both serines were mutated simultaneously, the effect on IKKbeta and IkappaBalpha phosphorylation was more profoundly affected.

The effect of CD28/B7 blockade on alloreactive T and B cells after liver cell transplantation.

BACKGROUND: Hepatocyte cell lines are beginning to be developed as universal donors for isolated liver cell transplantation, which is a less invasive method than orthotopic liver transplantation for treatment of metabolic liver disease. The immune response to isolated liver cell transplantation and its modification by costimulatory blockade are as yet not well delineated. METHODS: Adenovirus expressing CTLA4Ig was used to study blockade of the costimulatory CD28/B7 pathway in murine models of hepatocyte transplantation, and the effects on alloreactive T and B cells were studied. RESULTS: CTLA4Ig delayed rejection of subcutaneously administered C57L-derived murine hepatoma cells in CBA/J recipients for >50 days. Activation and cytokine secretion by allospecific CD4+ and CD8+ T cells were initially blocked by CTLA4Ig; delayed rejection was associated with tumor infiltration by CD8+ T cells that did not secrete interferon-gamma. CTLA4Ig failed to block transplant rejection in primed mice, indicating that memory effector T cells were resistant to its action. In contrast, CTLA4Ig suppressed both naive and memory alloreactive B cells. High levels of CTLA4Ig mediated acceptance of hepatoma cells delivered directly into the spleen. However, isolated primary C57BL/6 mouse hepatocytes delivered into the spleen were rejected with only moderately delayed kinetics. CONCLUSIONS: Transplant antigenicity, transplant site, and CTLA4Ig dose all affected the survival of transplanted liver cells. CD8+ T cells are significant mediators of hepatocyte transplant rejection and are relatively resistant to costimulatory blockade with CTLA4Ig. Strategies to specifically antagonize CD8+ T cells or to modulate MHC class I expression in association with costimulatory blockade by CTLA4Ig may enhance the clinical feasibility of transplanting allogeneic hepatocytes.

Requirements for viral-mediated autoimmune diabetes: beta-cell damage and immune infiltration.

The induction of autoimmunity by viruses has been attributed to numerous mechanisms. Coxsackievirus B4 (CB4) induces insulin-dependent diabetes mellitus (IDDM) in mice resembling the final step of disease progression in humans. Following viral infection, autoreactive lymphocytes are activated through exposure to damaged islets consequently precipitating IDDM. However, the viral and host requirements leading up to this final step have yet to be elucidated. We provide evidence that disease induction requires a pre-existing accumulation of beta-cell specific autoreactive T cells within the pancreas, as well as the infection of islet beta-cells. Therefore, the primary role of CB4 in the development of IDDM is to infect tissue, resulting in the presentation of sequestered islet antigen, the stimulation of preexisting autoreactive T cells, and the initiation of disease.

A critical role for inducible nitric oxide synthase in host survival following coxsackievirus B4 infection.

Coxsackieviral infections have been linked etiologically to multiple diseases. The serotype CB4 is associated with acute pancreatitis and autoimmune type 1 diabetes. To delineate the mechanisms of host survival after an acute infection with CB4 (strain E2), we have investigated the role of nitric oxide (NO), generated by the inducible form of nitric oxide synthase (NOS2), in viral clearance and pancreatic beta-cell maintenance. Mice deficient in NOS2 (NOS2-/- mice) and their wild-type (wt) counterparts were injected with CB4, after which both groups developed severe pancreatitis, hepatitis, and hypoglycemia within 3 days. Within 4 to 7 days postinfection (p.i.), most of the NOS2-/- mice died and at a strikingly higher mortality rate than wt mice. Histological examination of pancreata from both infected NOS2-/- and infected wt mice revealed early and complete destruction of the pancreatic acinar tissue, but intact, insulin-stained islets. When examined up to 8 weeks p.i., neither surviving NOS2-/-mice nor surviving wt mice developed hyperglycemia. However, the clearance of infectious CB4 was different between the mice. The spleens of NOS2-/- survivors were cleared of infectious virus with kinetics similar to that of wt mice, but the livers, pancreata, kidneys, and hearts of the NOS2-/- groups cleared virus more slowly than those of the wt group. This delayed clearance was particularly prominent in the livers of infected NOS2-/- mice, which also showed prolonged histopathological features of viral hepatitis. Taken together, this outcome suggests that NOS2 (and NO) is not required for the prevention of pancreatic beta-cell depletion after CB4 infection. Instead the critical actions of NOS2 apparently occur early in the host immune response, allowing mice to survive and clear virus. Moreover, the data support the existence of an organ-specific dependency on NO for a rapid clearance of CB4.

DAP kinase activates a p19ARF/p53-mediated apoptotic checkpoint to suppress oncogenic transformation.

DAP kinase is a pro-apoptotic calcium-regulated serine/threonine kinase, whose expression is frequently lost in human tumours. Here we show that DAP kinase counteracts oncogene-induced transformation by activating a p19ARF/p53-dependent apoptotic checkpoint. Ectopic expression of DAP kinase suppressed oncogenic transformation of primary embryonic fibroblasts by activating p53 in a p19ARF-dependent manner. Consequently, the fibroblasts underwent apoptosis, characterized by caspase activation and DNA fragmentation. In response to c-Myc or E2F-1, the endogenous DAP kinase protein was upregulated. Furthermore, functional or genetic inactivation of the endogenous DAP kinase reduced the extent of induction of p19ARF/p53 and weakened the subsequent apoptotic responses to c-Myc or E2F-1. These results establish a role for DAP kinase in an early apoptotic checkpoint designed to eliminate pre-malignant cells during cancer development.

Pancreatic expression of interferon-gamma protects mice from lethal coxsackievirus B3 infection and subsequent myocarditis.

Cardiovascular disease is one of the leading causes of death worldwide, and has been associated with many environmental risk factors. Recent evidence has indicated the involvement of pathogens such as viruses as causative agents, and specifically identified the coxsackievirus B serogroup as the leading culprit. Not only has coxsackievirus B3 (CB3) been identified from patients with cardiovascular disease, but also infection of mice with CB3 strains can reproduce human clinical heart disease in rodents. Several mechanisms have been proposed in an attempt to distinguish between pathology mediated by direct viral destruction of cardiac muscle cells or by the virus-induced immune response directed at infected myocytes or at 'mimicked' epitopes shared between viral and cardiac antigens. To distinguish between these mechanisms, we infected a unique mouse that diminishes the extent of infection and spread of the virus, but allows complete immunity to the virus. Transgenic mice expressing interferon-gamma in their pancreatic beta cells failed to develop CB-3-induced myocarditis. This work challenges the idea of the function of the immune response and 'molecular mimicry' in the CB-3-induced autoimmune myocarditis model, and instead favors the idea of virus-mediated damage. These results emphasize the benefit of reducing the level of viremia early during infection, thereby reducing the incidence of virus-mediated heart damage and autoimmunity.

An adenovirus inhibitor of tumor necrosis factor alpha-induced apoptosis complexes with dynein and a small GTPase.

Adenoviruses (Ad) code for immunoregulatory and cytokine regulatory proteins, one of which is the early region 3, 14.7-kDa protein (Ad E3-14.7K), which has been shown to inhibit tumor necrosis factor alpha-induced apoptosis. In an effort to understand the mechanism of action of Ad E3-14.7K, we previously searched for cell proteins with which it interacted. Three Ad E3-14.7K-interacting proteins (FIP-1, -2, and -3) were isolated. FIP-1 is a small GTPase which was used in this report as bait in the yeast two-hybrid system to find other interacting cell targets. The search resulted in the isolation of a protein, which we called GIP-1 (GTPase-interacting protein) that subsequently was shown to be identical to one of the light-chain components of human dynein (TCTEL1). FIP-1 was able to bind both TCTEL1 and Ad E3-14.7K simultaneously and was necessary to form a complex in which the viral protein was associated with a microtubule-binding motor protein. The functional significance of these interactions is discussed with respect to the steps of the Ad life cycle which are microtubule associated.

Regulation of the NF-kappaB activation pathway by isolated domains of FIP3/IKKgamma, a component of the IkappaB-alpha kinase complex.

FIP3, isolated as a type 2 adenovirus E3-14.7-kDa interacting protein, is an essential component of the multimeric IkappaB-alpha kinase (IKK) complex and has been shown to interact with various components (Fas receptor-interacting protein, NF-kappaB-inducing kinase, IKKbeta) of the NF-kappaB activation pathway. FIP3 has also been shown to repress basal and tumor necrosis factor (TNF) alpha-induced NF-kappaB activity as well as to induce cell death when overexpressed. The adenovirus E3-14.7-kDa protein (E3-14.7K) is an inhibitor of TNFalpha-induced cell death. In the current study, we generated deletion mutants to map the domains of FIP3, which are responsible for its various functions. The NF-kappaB inhibitory activity and the E3-14.7K binding domains were mapped at the carboxyl half of the FIP3 protein. We also found that the carboxyl-terminal half of FIP3 blocked TNFalpha-induced IkappaB-alpha phosphorylation and subsequent degradation, which suggests that the stabilization of the cytoplasmic inhibitor of NF-kappaB underlies the FIP3 inhibition of NF-kappaB activity. The amino-terminal 119 amino acids were responsible for the FIP3-IKKbeta and FIP3-IKKalpha interaction, and the middle of the protein (amino acids 201-300) appeared to be both the FIP3 self-association domain as well as the FIP3-Fas receptor-interacting protein interaction domain. Thus, FIP3 might serve as a scaffold protein to organize the various components of the IkappaB-alpha kinase complex. Whereas the full-length protein is required for efficient cell death, the amino-terminal 200 amino acids are sufficient to cause rounding and detachment of the cells from the monolayer.

Viruses, host responses, and autoimmunity.

Conceptually, the initiation of autoimmune disease can be described as a three-stage process involving both genetic and environmental influences. This process begins with the development of an autoimmune cellular repertoire, followed by activation of these autoreactive cells in response to a localized target and, finally, the immune system's failure to regulate these self-reactive constituents. Viruses have long been associated with inciting autoimmune disorders. Two mechanisms have been proposed to explain how a viral infection can overcome immunological tolerance to self-components and initiate an organ-specific autoreactive process; these mechanisms are molecular mimicry and bystander activation. Both pathways, as discussed here, could play pivotal roles in the development of autoimmunity without necessarily excluding each other. Transgene technology has allowed us and others to examine more closely the roles of these mechanisms in mice and to dissect the requirements for initiating disease. These results demonstrate that bystander activation is the most likely explanation for disease development. Additional evidence suggests a further role for viruses in the reactivation and chronicity of autoimmune diseases. In this scenario, a second invasion by a previously infecting virus may restimulate already existing autoreactive lymphocytes, and thereby contribute to the diversity of the immune response.

Differential chemokine induction by the mouse adenovirus type-1 in the central nervous system of susceptible and resistant strains of mice.

Mouse adenovirus-type 1 (MAV-1) has recently been shown to cause a fatal hemorrhagic encephalopathy in certain strains of mice whereas other strains are resistant. Morbidity is associated with a productive infection of cerebrovascular endothelial cells, resulting in necrosis of the vasculature, infarction, hemorrhage and death within 4 - 6 days. Previous studies were not able to define a role for the innate or acquired immune response. In the current study we have addressed the effect of MAV-1 on chemokine and chemokine receptor expression in the central nervous system (CNS) and spleen of susceptible (C57BL/6) and resistant (BALB/c) strains of mice. Intra-peritoneal infection with MAV-1 in C57BL/6 animals resulted in early and prominent induction of IP-10/crg-2 in the spleen and CNS. Increased expression of MCP-1, MIP-1alpha, MIP-1beta and RANTES was also noted in the CNS of MAV-1-infected C57BL/6 animals commencing around 72 h post-infection. In contrast, chemokine expression in BALB/c animals was more restricted with prominent upregulation only of MIP-2 in the CNS. In situ hybridization identified the vascular endothelium and CNS glia as the principal site of IP-10/crg-2 production in the C57BL/6 animals. The chemokine receptors CCR1-5 were upregulated in the CNS of both strains of mice. These data show that productive infection of the CNS with MAV-1 leads to the upregulation of a characteristic pattern of chemokines and their receptors, which may point to a role for these factors in disease pathogenesis.

Detailed in vivo analysis of interferon-gamma induced major histocompatibility complex expression in the the central nervous system: astrocytes fail to express major histocompatibility complex class I and II molecules.

To recognize and respond immunologically to foreign antigens, T lymphocytes require the presentation of foreign peptides by MHC molecules. To determine which cells of the central nervous system (CNS) are capable of expressing MHC molecules, we used confocal microscopy and dual immunofluorescence with cell-specific and MHC-specific antibodies to study brain sections of adult mice. We took advantage of transgenic mice that initiate CNS-specific expression of IFN-gamma at 8 weeks of age. This inflammatory cytokine is a strong inducer of MHC expression both in culture and in vivo. From this analysis, we clearly found MHC class I and II expression on endothelial, microglial, and oligodendrocyte cell types, but did not find astrocytes or neurons capable of expressing either MHC class I or II molecules under these conditions. This finding suggests that, although microglia and oligodendrocytes may participate in the antigen presentation process in the organism, we found no in vivo evidence to support the concept that astrocytes act as antigen-presenting cells.

Protection from lethal coxsackievirus-induced pancreatitis by expression of gamma interferon.

Coxsackievirus infection causes severe pancreatitis and myocarditis in humans, often leading to death in young or immunocompromised individuals. In susceptible strains of mice, coxsackievirus strain CB4 causes lethal hypoglycemia. To investigate the potential of gamma interferon (IFN-gamma) in protection and clearance of the viral infection, IFN-gamma knockout mice and transgenic (Tg) mice specifically expressing IFN-gamma in their pancreatic beta cells were infected with CB4. Lack of IFN-gamma in mice normally resistant to CB4-mediated disease resulted in hypoglycemia and rapid death. However, expression of IFN-gamma in the beta cells of Tg mice otherwise susceptible to lethal infection allowed for survival and protected them from developing the accompanying hypoglycemia. While all the mice had high levels of viral replication in their pancreata and comparable tissue pathology following viral infection, the Tg mice had significantly lower levels of virus at the peak of infection, significantly higher numbers of activated macrophages before and after infection, and less damage to their acinar tissue. Additionally, despite having increased levels of inducible nitric oxide synthetase (iNOS) expression, treatment of Tg mice with the iNOS inhibitor aminoguanidine did not alter the level of protection afforded by IFN-gamma expression. In conclusion, IFN-gamma protects from lethal coxsackievirus infection by activating macrophages in an iNOS-independent manner.

Identification of a cell protein (FIP-3) as a modulator of NF-kappaB activity and as a target of an adenovirus inhibitor of tumor necrosis factor alpha-induced apoptosis.

FIP-3 (14.7K interacting protein) was discovered during a search for cell proteins that could interact with an adenovirus protein (Ad E3-14.7K) that had been shown to prevent tumor necrosis factor (TNF)-alpha-induced cytolysis. FIP-3, which contains leucine zippers and a zinc finger domain, inhibits both basal and induced transcriptional activity of NF-kappaB and causes a late-appearing apoptosis with unique morphologic manifestations. Ad E3-14.7K can partially reverse apoptotic death induced by FIP-3. FIP-3 also was shown to bind to other cell proteins, RIP and NIK, which previously had been described as essential components of TNF-alpha-induced NF-kappaB activation. In addition, FIP-3 inhibited activation of NF-kappaB induced by TNF-alpha, the TNFR-1 receptor, RIP, NIK, and IKKbeta, as well as basal levels of endogenous NF-kappaB in 293 cells. Because the activation of NF-kappaB has been shown to inhibit apoptosis, FIP-3 appears both to activate a cell-death pathway and to inhibit an NF-kappaB-dependent survival mechanism.