Autologous tumor cells engineered to express bacterial antigens.

Cancer immunotherapies are emerging as promising treatment modalities in the management of the disease. As a result, cancer vaccines are considered to be immensely crucial in preventing recurrence, a well-known nemesis in cancer patients because they have the potential to activate memory antitumor immunity. Due to poor antigenicity and self-tolerance, most tumor antigens require interventional vaccine therapies to provide an adequate "danger" signal to the immune system in order to activate a robust, clinically meaningful antitumor immunity. It has been postulated that this requirement may be achieved by providing bacterial and/or viral immunogens to prime this type of immune response. Briefly, we provide here a method of transfecting whole tumor cells with plasmid DNA encoding an immunogenic bacterial protein such as Emm55, which was derived from Streptococcus pyogenes (S. pyogenes). Subsequent inactivation of the transfected cells by irradiation (100 Gray) prevents replication. This type of whole-cell vaccine, e.g., ImmuneFx™, has demonstrated activity in a murine neuroblastoma model, in canine lymphoma patients with naturally occurring disease, and in many cancer types in companion animals. The protocols described in this chapter provide the necessary materials and methodologies to manufacture such a vaccine.

Localized gene expression following administration of adeno-associated viral vectors via pancreatic ducts.

Gene transfer into pancreatic cells in vivo could be of immense therapeutic benefit in cases of type 1 diabetes (T1D) through the production of molecules capable of interrupting the progression of autoimmunity or promoting regeneration of insulin-secreting beta cells. We adapted a clinically relevant surgical technique (endoscopic retrograde cholangiopancreatography) to deliver rAAV encoding human alpha1-antitrypsin (approved gene symbol SERPINA1) to the pancreas of 3-week-old Fisher 344 rats and C57BL/6 mice. We compared natural as well as bioengineered serotypes of rAAV (rAAV1, rAAV2/Apo, rAAV8) as well as different promoters (chicken beta-actin, human insulin) for their expression in vivo. Rats injected with rAAV1 showed the highest hAAT expression (week 2, rAAV1/CB-AT, 579 +/- 457 ng/ml). In mice, rAAV8 vector delivered the highest serum concentration of hAAT (week 2, rAAV8/CB-AT, 19 +/- 6 microg/ml). The chicken beta-actin promoter provided the highest expression in both rodent experiments. Immunohistochemical staining indicated transduction primarily of pancreatic acinar cells with either the rAAV1/CB-AT vector in the rat or the rAAV8/CB-AT vector in the mouse. This study demonstrates that rAAV vectors can be designed to deliver therapeutic genes efficiently to the pancreas and achieve high levels of gene expression and may be useful in treating pancreatic disorders, including T1D.

Islet replacement vs. regeneration: hope or hype?

Type 1 diabetes is caused by autoimmune destruction of pancreatic islet beta-cells. Management of this disease is burdensome both to the individual and society, costing over 100 billion US dollars annually. Shortage of pancreatic tissue, together with a lifetime requirement of immunosuppressive drugs to prevent rejection and recurrent disease, remain as major hurdles yet to be overcome prior to widespread applicability. Stem cells, with their potential of developing into pancreatic beta-cells, appear to be the best prospect for overcoming the islet shortage. Current investigation, however (both embryonic and adult stem cells), is still in the preliminary stage and several more years remain before they can potentially be used in the clinical setting. Procedures that reduce in vitro manipulation of cells and allow stem cells to develop into islets in vivo are crucial. Furthermore, the regeneration of existing islets is a distinct possibility. Simplistically, it might be hypothesized that down-regulation of autoimmunity may give the pancreas the breathing space to regenerate islets. Supplementation with factors known to induce beta-cell replication and neogenesis might further augment the regenerative processes. Clearly, islet-regeneration research will soon match the level of interest currently focused on in vitro stem cell-based approaches.

Animal models to study adult stem cell-derived, in vitro-generated islet implantation.

Type 1 diabetes (T1D) is an autoimmune disease characterized by hyperglycemia following the destruction of the insulin-producing beta cells of the pancreatic islets of Langerhans by the body's own immune system. Although routine insulin injections can provide diabetic patients with their daily insulin requirements, this treatment is not always effective in maintaining normal glucose levels. A true "cure" is considered possible only through replacement of the beta cell mass, by pancreas transplantation, islet implantation, or implantation of nonendocrine cells modified to secrete insulin. With the recent success of islet implantation to reverse T1D, this procedure has become a welcome therapy for T1D patients. Unfortunately, this procedure is hampered by the limited number of transplantation quality pancreata available for the harvesting of islets. This shortage has sparked great interest in finding a replacement for organ donation, primarily the possible use of stem cell-derived islets starting with stem cells, or alternatively the harvesting of nonhuman islets. This review focuses on progress with growing islets in the laboratory from stem cells and a comparison between this developing technology and the current use of islets harvested from nonhuman sources.

In vitro-generation of surrogate islets from adult stem cells.

Type 1 diabetes is one of the more costly chronic diseases of children and adolescents throughout North America and Europe, exhibiting an average estimated prevalence rate of nearly 0.2%. It occurs in genetically predisposed individuals when the immune system attacks and destroys specifically the insulin-producing beta cells of the pancreatic islets of Langerhans. While routine insulin therapy can provide diabetic patients with their daily insulin requirements, non-compliance and undetected hyperglycemic excursions often lead to subsequent long-term microvascular and macrovascular complications. The only real cure for type 1 diabetes is replacement of the beta cell mass, currently being accomplished through ecto-pancreatic transplantation and islet implantation. Both of these procedures suffer from a chronic shortage of available donor tissue in comparison to the number of potential recipients. To circumvent this need, three alternative approaches are being intensively investigated: (1) the production of surrogate cells by genetically modifying non-endocrine cells to secrete insulin in response to glucose challenge; (2) the trans-differentiation of non-endocrine stem/progenitor cells or mature cells to glucose-responsive adult tissue; and (3) the regulated differentiation of islet stem/progenitor cells to produce large numbers of mature, functional islets. In recent years, each of these approaches has made impressive advances, leading to the most important question, 'how soon will this new science be available to the patient?' In the present review, we discuss some of the recent advances, focusing primarily on the differentiation of islet stem cells to functional endocrine pancreas that may form the basis for future treatment.

Reduced IL-2 and IL-4 mRNA expression in CD4+ T cells from bovine leukemia virus-infected cows with persistent lymphocytosis.

The role of T-helper (Th) responses in the subclinical progression of bovine leukemia virus (BLV) infection was explored by determining the contribution of CD4+ T cells to the expression of mRNAs encoding interferon-gamma (IFN-gamma), interleukin-2 (IL-2), interleukin-4 (IL-4), and interleukin-10 (IL-10) in BLV-infected cattle. Relative levels of mRNA encoding IFN-gamma, IL-2, IL-4, and IL-10 were measured in fresh and concanavalin A (Con A) activated peripheral blood mononuclear cells (PBMCs) and purified CD4+ T cells from cows seronegative to BLV (BLV-), seropositive without persistent lymphocytosis (BLV+PL-), and seropositive with PL (BLV+PL+) using a semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) assay. The expressions of IFN-gamma, IL-2, and IL-4 mRNAs were significantly reduced in the PBMCs from BLV+PL+ cows as compared to BLV- cows. Reduced levels of IL-2 and IL-4 mRNAs were detected in fresh CD4+ T cells from BLV+PL+ cows. In contrast, Con A stimulated PBMCs and CD4+ T cells did not differ significantly in expression of IFN-gamma, IL-2, IL-10, or IL-4 mRNAs among the BLV infection groups. Using flow-sorted CD4+ T cells and semiquantitative RT-PCR the frequencies of CD4+ T cells transcribing IFN-gamma, IL-2, IL-4, and IL-10 mRNAs in the peripheral blood of BLV-, BLV+PL-, and BLV+PL+ cows were determined. There were no significant differences in the frequencies of CD4+ T cells expressing these cytokine mRNAs among animals in the different BLV infection categories. Thus, the observed differences in IL-2 and IL-4 mRNAs in CD4+ T cells were due to changes in steady-state mRNA levels expressed by individual cells and not to changes in the frequency of cells transcribing IL-2 and IL-4 mRNAs. These results demonstrate that the progression of BLV infection to PL is associated with reduced expression of classical Th1 and Th2 cytokines by CD4+ T cells, thus suggesting aberrant Th regulation in subclinically infected animals.

Generation of islets of Langerhans from adult pancreatic stem cells.

Ductal structures of the adult pancreas contain multipotent stem cells that, under controlled in vitro conditions, are able to self-renew and differentiate into functional islets of Langerhans. In vitro-generated islets, whether derived from stem cells of human, porcine, or mouse origin, exhibit temporal changes in mRNA transcripts for islet-associated markers as well as regulated insulin responses following glucose challenge. When in vitro-generated mouse islets were implanted into diabetic mice, neovascularization of the implant material occurred, followed by reversal of insulin-dependent diabetes. The possibility of growing functional islets from adult stem cells provides new opportunities to produce large numbers of islets, even autologous islets, for use as implants.