Pdx-1 or Pdx-1-VP16 protein transduction induces beta-cell gene expression in liver-stem WB cells.

BACKGROUND: Pancreatic duodenal homeobox-1 (Pdx-1) or Pdx-1-VP16 gene transfer has been shown to induce in vitro rat liver-stem WB cell conversion into pancreatic endocrine precursor cells. High glucose conditions were necessary for further differentiation into functional insulin-producing cells. Pdx-1 has the ability to permeate different cell types due to an inherent protein transduction domain (PTD). In this study, we evaluated liver-to-pancreas conversion of WB cells following Pdx-1 or Pdx-1-VP16 protein transduction. FINDINGS: WB cells were grown in high glucose medium containing Pdx-1 or Pdx-1-VP16 recombinant proteins for two weeks. beta-like cell commitment was analysed by RT-PCR of pancreatic endocrine genes. We found that WB cells in high glucose culture spontaneously express pancreatic endocrine genes (Pdx-1, Ngn3, Nkx2.2, Kir6.2). Their further differentiation into beta-like cells expressing genes related to endocrine pancreas development (Ngn3, NeuroD, Pax4, Nkx2.2, Nkx6.1, Pdx-1) and beta-cell function (Glut-2, Kir6.2, insulin) was achieved only in the presence of Pdx-1(-VP16) protein. CONCLUSION: These results demonstrate that Pdx-1(-VP16) protein transduction is instrumental for in vitro liver-to-pancreas conversion and is an alternative to gene therapy for beta-cell engineering for diabetes cell therapy.

Some ethical issues regarding xenotransfusion.

BACKGROUND: The use of porcine red blood cells has recently been proposed as a possible solution to the shortage of blood for human transfusion. OBJECTIVES: The purpose of this paper is to compare some ethical issues regarding xenotransfusion (XTF) with those relating to xenotransplantation (XT) of organs, tissues and cells. MATERIALS AND METHODS: Various ethical concerns and viewpoints relating to XTF are discussed. RESULTS: The main ethical obstacles to XT do not apply to XTF. It is much more ethically acceptable to raise pigs for regular blood collection as it doesn't damage the health of the animal. Porcine endogenous retrovirus infection, the major concern associated with XT, does not apply to XTF, since red blood cells have no DNA and have a very short lifespan. Clinical trials will be possible in humans once XTF has been demonstrated to be effective and harmless in non-human primates. Transgenesis is acceptable for pig blood donors because only a limited number of genes are involved, and these animals will never enter into the livestock gene pool or the food chain. CONCLUSION: Because the need for blood is less pressing than that for organs, tissues or cells, the use of animal blood for human transfusion is not an absolute necessity. However, it represents a real opportunity. The ability to gain access to an unlimited quantity of blood is a reasonable justification for XTF. Because its technical and ethical hurdles are less stringent, XTF could be the first large-scale clinical application of XT.

Xenotransfusions, past and present.

The first blood transfusions in humans were xenotransfusions, carried out by Jean-Baptiste Denis beginning in 1667. Richard Lower, Matthäus Purmann and Georges Mercklin also experimented with the use of animal blood for transfusion until this practice was forbidden in 1670, after the death of one of Denis's patients. In the middle of the 19th century, xenotransfusion was rescued from oblivion by the work of Pierre Cyprien Oré. Franz Gesellius and Oscar Hasse fervently defended xenotransfusion, but Emil Ponfick and Leonard Landois stressed the potentially harmful effects of inter-species transfusion from 1874 onward. Xenotransfusion was abandoned completely following the discovery of blood groups by Karl Landsteiner in 1900. From 2000, because of progress in xenotransplantation and the need of blood supply, xenotransfusion is again being considered. Pigs are the best potential donors. The development of alpha-1,3-galactosyltransferase gene-knockout pigs has overcome the first hurdle to xenotransfusion. The main obstacle to porcine red blood cell transfusion is now the cellular response involving macrophages or natural killer cells.

Gene-gun biolistic immunization encoding glutamic acid decarboxylase: a model for studying Langerhans cell abnormalities and mimicry in the nonobese diabetic mouse.

Plasmid-DNA gene-gun immunization may be an efficient approach for investigating the role of skin dendritic cells (DCs) in type 1 diabetes (T1D) pathogenesis and the significance of the presentation of peptides that mimic autoantigenic epitopes in aggravating or modulating the autoimmune reaction. Gene-gun immunization has been described as producing long-lasting immune responses elicited by skin DCs, especially Langerhans cells (LCs). Therefore, we tested the immune response and diabetes modulation in nonobese diabetic (NOD) mice and in control BALB/c mice, by gene-gun administration of plasmid-DNA encoding (1) human 65 kDa glutamic acid decarboxylase (hGAD65) mimicking the crucial mouse autoantigen GAD65 (similarity of 95.7%) or (2) beta-galactosidase (betaGAL) as a negative control. Expression of GAD and betaGAL in skin of pc-GAD- and pc-LacZ-injected mice, respectively, was confirmed. It was surprising that both pc-LacZ-injected BALB/c and NOD mice exhibited a betaGAL-specific Th1 immune response: spleen cells of pc-LacZ mice proliferated specifically to betaGAL (P < 10(-4)) and secreted significant amounts of IFNgamma (P < 10(-4)). pc-LacZ mice also developed a betaGAL-specific Th1-related (IgG2a/2c) and Th2-related (IgG1) humoral response. Although pc-GAD BALB/c mice showed Th2-related GAD-specific IgG1 production and a significant secretion of IL4 (P < .03), pc-GAD NOD mice did not generate either an antibody response or a T cell response specific to GAD. Moreover, gene-gun immunization encoding hGAD65 did not clearly modulate diabetes onset in NOD mice. This absence of detectable GAD-specific response may implicate skin DC deficiencies in NOD mice. The gene-gun technique could thus provide an interesting model for studying skin DC abnormalities in NOD mice and their potential implication of presenting mimetic peptides that modulate the autoimmune response in T1D.

Reluctance of French patients with type 1 diabetes to undergo pig pancreatic islet xenotransplantation.

INTRODUCTION: Type 1 diabetes could possibly be treated by transplantation of pig pancreatic islets. In addition to medical difficulties and ethical problems, social hurdles may need to be overcome. We have evaluated the attitude of patients with type 1 diabetes to the xenotransplantation of pig pancreatic islets and to the potential risks associated with such treatment. METHODS: A survey of 214 patients with type 1 diabetes was carried out in France based on a multiple-choice questionnaire. RESULTS: At first, 52.0% of these patients indicated that they would agree to receive pig islet xenografts. The main sources of reluctance were the ''risk of disease transmission'' (55.5%) and ''risks not yet identified'' (48.7%). After they were told of the risk of cancer or infection associated with immunosuppression, 74.9% of the respondents chose to refuse the transplant, compared with 48.0% before they heard of such risks. A 68.1% would refuse the xenotransplant if it would not exempt them completely from being treated by insulin injections. Discontinuing insulin injections was the most important priority for diabetic patients (73.5%), rather than limitation of diabetes-related complications (52.5%) or increase in life expectancy (44.0%). After they were informed of all of the risks associated with the procedure, 70.5% of the respondents decided they would rather not take any risks, and said they would refuse pig islet transplantation. CONCLUSION: When diabetic patients learned about potential infectious risks and other risks associated with immunosuppression, reluctance to undergo xenotransplantation gained in significance or even led to refusal of the procedure.

History of xenotransplantation.

The present historical review reports the clinical experiences of transplantations from animal to human. The first transplantation attempts were made without any knowledge of the species barrier. The pioneers of xenotransplantation realized xenotransfusions as early as the 16th century, then cell and tissue xenotransplantations in the 19th century. At the beginning of the 20th century, xenotransplantation of testicles became the latest craze. At the same time, and later in the 1960s, organ xenotransplantations were attempted, with disappointing results. Mathieu Jaboulay, Serge Voronoff, Keith Reemtsma, James Hardy, Denton Cooley, Thomas Starzl, Christiaan Barnard and Leonard Bailey were among the pionneers of xenotransplantation. Recent trials concerned above all tissue and cell xenotransplantations. Nowadays, with encapsulation, transgenesis, and cloning, great advances have been made for controlling xenograft rejection, but ethical questions linked to the risk of infections have become a major pre-occupation within the scientific community and the general population.

AN69 hollow fiber membrane will reduce but not abolish the risk of transmission of porcine endogenous retroviruses.

As the risk of porcine endogenous retrovirus (PERV) infection is a major obstacle to the xenotransplantation of porcine tissue, we investigated whether an AN69 hollow fibre membrane, used for islets of Langerhans transplantation, could prevent the transfer of PERVs and thus reduce the risk of PERV infection. PK15 cells were used as a PERV source. A specific and highly sensitive RCR was used for detection of a PERV provirus DNA (gag region) and a porcine mtDNA. Human U293 cells were incubated in vitro with encapsulated PK15 cells, concentrated encapsulated PK15 supernatant, or concentrated PK15 supernatant as a control. CD1 mice were implanted in vivo with encapsulated PK15 cells or injected with PK15 supernatant. We found no infection in human cells incubated with either encapsulated PK15 supernatant or in 10 out of 11 samples after coincubation with encapsulated PK15 cells. Infection of human cells was, however, detected in 1 out of 11 samples after coincubation with encapsulated PK15 cells. The presence of PERV provirus DNA and porcine mtDNA was detected in all the investigated tissues of the mice injected with PK15 supematant and in various tissues of the mice implanted with encapsulated PK15 cells. Four weeks after the last injection of PK15 supernatant or a fiber explantation, no mouse showed any presence of PERV provirus DNA or porcine mtDNA. Our results demonstrate that AN69 hollow fiber membrane will reduce but not abolish the risk of PERV infection. Because the real risk of PERV infection still remains unknown, it is necessary to investigate further the real protection that could be provided by hollow fibers to ensure the safety of clinical xenotransplantation.

In vitro induction of inhibitory macrophage differentiation by granulocyte-macrophage colony-stimulating factor, stem cell factor and interferon-gamma from lineage phenotypes-negative c-kit-positive murine hematopoietic progenitor cells.

CD11b+Gr-1+ inhibitory macrophages (iMacs) were implicated in profound depression of T cell functions sometimes observed during cyclophosphamide treatments and overwhelming infections, through a secretion of nitric oxide (NO). Myeloid origin and maturation stages of iMacs are still unknown. As tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) contributed crucially to the activation of inducible NO synthase (iNOS) gene transcription and to the differentiation of macrophages, we tested their roles in the induction of iMacs differentiation from bone marrow hematopoietic progenitor cells (HPC) of uncompromised mice. Lineage phenotypes-negative (lin)) c-kit+ cells of Balb/c mice were cultured 6 days with granulocyte-macrophage colony-stimulating factor (GM-CSF), stem cell factor (SCF, c-kit ligand) in presence or not of TNF-alpha or IFN-gamma. CD11b+Gr-1+ cells only derived in presence of [GM-CSF + SCF + TNF-alpha] or [GM-CSF + SCF + IFN-gamma] could express iNOS upon in vitro stimulation with [IFN-gamma + TNF-alpha] or [IFN-gamma + LPS] known to boost iNOS expression in murine macrophages. However, whereas [GM-CSF + SCF + TNF-alpha] induced only weakly iMacs generation and contributed also to the differentiation of CD11b+Gr-1-CD11c+ myeloid dendritic cells, [GM-CSF + SCF + IFN-gamma] induced exclusively and importantly iMacs differentiation. Moreover [GM-CSF + SCF + IFN-gamma]-generated iMacs were more mature than [GM-CSF + SCF + TNF-alpha]-derived iMacs since IFN-gamma increased more strongly CD11b+Gr-1+ cells expressing Ly-6C and generated lesser cells expressing MHC class II and CD86 molecules. Finally [GM-CSF + SCF + IFN-gamma]-generated CD11b+ cells showing a powerful suppressive activity on T cell proliferations, correlated with NO secretion. In conclusion, our study showed, for the first time, that IFN-gamma induced very efficiently the differentiation of functional iMacs from lin- c-kit+ murine HPC in vitro, and indicated clearly that iMacs progenitors may be present in bone marrow of naïve mice.