Stem cell potential for type 1 diabetes therapy

Stem cells have been considered as a useful tool in Regenerative Medicine due to two main properties: high rate of self-renewal, and their potential to differentiate into all cell types present in the adult organism. Depending on their origin, these cells can be grouped into embryonic or adult stem cells. Embryonic stem cells are obtained from the inner cell mass of blastocyst, which appears during embryonic day 6 of human development. Adult stem cells are present within various tissues of the organism and are responsible for their turnover and repair. In this sense, these cells open new therapeutic possibilities to treat degenerative diseases such as type 1 diabetes. This pathology is caused by the autoimmune destruction of pancreatic β-cells, resulting in the lack of insulin production. Insulin injection, however, cannot mimic β-cell function, thus causing the development of important complications. The possibility of obtaining β-cell surrogates from either embryonic or adult stem cells to restore insulin secretion will be discussed in this review.     

A dual role for genetically modified lymphocytes in cancer immunotherapy

T cells as the ultimate effectors of adaptive immune responses are currently used to treat patients affected by infectious diseases and certain tumors. Recently, T cells have been manipulated ex vivo with viral vectors coding for chimeric antigen receptors, exogenous T cell receptors, or 'suicide' genes to potentiate their efficacy and minimize possible side effects. However, the introduction of exogenous genes into T lymphocytes, particularly bacterial or viral transgene products, has occasionally produced immune-mediated elimination of transduced lymphocytes. This immune effect has recently been exploited in a trial of active immunotherapy in melanoma patients. In this opinion article, we discuss the therapeutic possibilities presented by the dual aspects of genetically modified lymphocytes used to treat cancer patients.     

Complementation cell lines for viral vectors to be used in gene therapy

Viral vectors provide a highly efficient method for the transfer of foreign genes into a variety of quiescent or dividing eukaryotic cells from many animal origins. While recombinant vectors derived from an increasing number of mammalian viruses (herpes simplex virus, autonomous and non-autonomous parvoviruses, poxviruses, retroviruses, adenoviruses available today, vectors based on murine retroviruses and human adenoviruses constitute preferential candidates for the delivery of marker or therapeutic genes into human somatic cells. The availability of such vectors has made possible the recent transition of human gene therapy from laboratory benches to clinical settings. Most current recombinant vectors have been generated by deleting essential viral genes in order to make space available for the introduction of passenger genes. Such vectors are therefore unable to replicate in the absence of these critical gene products and their production relies on the development of stable complementation cell lines providingin trans the missing viral functions. Although complementation (or packaging) cell lines are available for both adenovirus and retrovirus vectors, their respective drawbacks still limit their use to research applications and phase I clinical trials. The future success or failure of human gene therapy will therefore rely on the production of improved generations of packaging cell lines that can produce safer and more efficient vectors which are fully adapted to large scale production and clinical applications.     

Isolation and therapeutic potential of human haemopoietic stem cells

The haemopoietic stem cell (HSC) has long been regarded as an archetypal, tissue specific, stem cell, capable of completely regenerating haemopoiesis after myeloablation. It has proved relatively easy to harvest HSC, from bone marrow or peripheral blood. In turn, isolation of these cells has allowed therapeutic stem cell transplantation protocols to be developed, that capitalise on their prodigious self renewal and proliferative capabilities. Ex vivo approaches have been described to isolate, genetically manipulateand expand pluripotent stem cell subsets. These techniques have been crucial to the development of gene therapy, and may allow adults to enjoy the potential advantages of cord blood transplantation. Recently, huge conceptual changes have occurred in stem cell biology. In particular, the dogma that, in adults, stem cells are exclusively tissue restricted has been questioned and there is great excitement surrounding the potential plasticity of these cells, with the profound implications that this has, for developing novel cellular therapies. Mesenchymal stem cells, multipotent adult progenitor cells and embryonic stem cells are potential sources of cells for transplantation purposes. These cells may be directed toproduce HSC, in vitro and in the future may be used for therapeutic, or drug development, purposes. This revised version was published online in July 2006 with corrections to the Cover Date.     

Rat major histocompatibility RT1.C antigens of restricted tissue distribution consist of two polypeptide chains with molecular weights of about 42 000 and 12 500

The RT1.C region has recently been defined genetically as a third region of the rat major histocompatibility system, RT1, and has been shown to code for cell-surface antigens with restricted tissue distribution (present on lymphocytes and absent from red blood cells and liver) and for target antigens of unrestricted cytotoxic T lymphocytes. Immunoprecipitation with C-antigen-specific alioantisera and SDS-PAGE analysis, reveal that C-region products are glycoproteins composed of molecules of 40 500–43 000 and 12 500 molecular weight, respectively. Thus, the RT1.B region, which codes for class II molecules, is flanked by RT1 regions which determine class I molecules (as defined biochemically), which are either expressed ubiquitously (RT1.A antigens) or in a restricted manner (RT1.C products). The homology to H-2Qa antigens is discussed.     

Anti-self antibodies selected from a human IgD heavy chain repertoire: a novel approach to generate therapeutic human antibodies against tumor-associated differentiation antigens

Human antibodies were isolated by phage display from a naturally expressed human antibody repertoire. Antibody selection was carried out against the epithelial cell adhesion molecule (EpCAM) or 17-1A antigen, that in a clinical trial had been successfully used as a target for antibody therapy of minimal residual colorectal cancer. VH chains were selected from the human IgD repertoire expressed on naive B2 and autoreactive B1 lymphocytes. By guiding the selection through a murine template antibody, two EpCAM-specific human antibodies, HD69 and HD70, were obtained that closely resembled the murine therapeutic 17-1A antibody in their binding properties when expressed as complete huIgG1 molecules in CHO cells. However, both human antibodies recruited human cytotoxic effector cells far more efficiently than the murine 17-1A antibody used for clinical trials. Therefore, and in view of the long in vivo half-life of human IgG1 antibodies, HD69 and HD70 are regarded as highly promising third generation versions of the murine therapeutic antibody. Because of their origin from an evolutionary conserved germline VH repertoire, they are expected to exhibit minimal immunogenicity in patients. Received: 16 November 2000 / Accepted: 11 January 2001     

T-cell gene therapy

In the past year, a number of human gene therapy trials involving the adoptive transfer of genetically modified T lymphocytes have been reported. These include trials of adenosine deaminase gene transfer in children with severe combined immunodeficiency syndrome, a gene-marking study of Epstein—Barr virus-specific cytotoxic T cells, and trials of gene-modified T cells expressing suicide or viral resistance genes in patients infected with HIV. Additional strategies for T-cell gene therapy currently being pursued in the clinic involve the engineering of novel T-cell receptors that impart antigen specificity for virally infected or malignant cells.     

Production of therapeutic glycoproteins through the engineering of glycosylation pathway in yeast

The application of recombinant DNA technology to restructure metabolic networks can change metabolite and protein products by altering the biosynthetic pathways in an organism. Although some success has been achieved, a more detailed and thorough investigation of this approach is certainly warranted since it is clear that such methods hold great potential based on the encouraging results obtained so far. In last decade, there have been tremendous advances in the field of glycobiology and the stage has been set for the biotechnological production of glycoproteins for therapeutic use. Today glycoproteins are one of the most important groups of pharmaceutical products. In this study the attempt was made to focus on identifying technologies that may have general application for modifying glycosylation pathway of the yeast cells in order to produce glycoproteins of therapeutic use. The carbohydrates of therapeutic recombinant glycoproteins play very important roles in determining their pharmacokinetic properties. A number of biological interactions and biological functions mediated by glycans are also being targeted for therapeutic manipulationin vivo. For a commercially viable production of therapeutic glycoproteins a metabolic engineering of a host cell is yet to be established.     

Human somatic cell gene therapy

The prelude to successful human somatic gene therapy, i.e. the efficient transfer and expression of a variety of human genes into target cells, has already been accomplished in several systems. Safe methods have been devised to do this using non-viral and viral vectors. Potentially therapeutic genes have been transferred into many accessible cell types, including hematopoietic cells, hepatocytes and cancer cells, in several different approaches to ex vivo gene therapy. Successful in vivo gene therapy requires improvements in tissuetargeting and new vector design, which are already being sought. Gene-transfer protocols have been approved for human use in inherited diseases, cancer and acquired disorders. Althouth the results of these trials to date have been somewhat disappointing, human somatic cell gene therapy promises to be an effective addition to the arsenal of approaches to the therapy of many human diseases in the 21st century if not sooner.     

Human fibroblasts transduced with CD80 or CD86 efficiently trans-costimulate CD4+ and CD8+ T lymphocytes in HLA-restricted reactions: implications for immune augmentation cancer therapy and autoimmunity.

Augmenting immunogenicity by genetically modifying tumor cells to express costimulatory molecules has proven to be a promising therapeutic strategy in murine tumor models and is currently under investigation in human clinical trials for metastatic cancer. However, there are significant technical and logistic problems associated with implementing strategies requiring direct gene modification of primary tumor cells. In an effort to circumvent these problems, we are developing a strategy in which the costimulatory signal required for tumor-specific T lymphocyte activation is provided by a genetically modified human fibroblast (trans-costimulation). We have evaluated the efficiency of CD80- and CD86-mediated trans-costimulation in the activation of human CD8+ and CD4+ T lymphocytes in MHC class I- and class II-restricted lymphoproliferation reactions. Our studies demonstrate that the efficiency of CD80- or CD86-mediated trans-costimulation of purified human CD8+ and CD4+ T lymphocytes is comparable to cis-costimulation under defined conditions. Moreover, a dose-response relationship consistent with the predicted two-hit kinetics of the reaction was evident in trans-costimulation reactions in which the ratio of target cells expressing either signal 1 or signal 2 was varied incrementally from 1:10 to 10:1. Importantly, the level of cell-surface CD86 required for trans-costimulation is equivalent to that constitutively expressed by human peripheral blood monocytes. These results may have significant implications for the clinical implementation of this type of cancer immunotherapy and also raise questions about the possibility of trans-costimulating autoreactive T lymphocytes in vivo.     

Murine interferon-gamma inducible protein-10 (IP-10) secreted by Lactococcus lactis chemo-attracts human CD3+ lymphocytes

Chemokines are members of the super family of cytokines necessary for leukocyte recruitment in tissues and lymphoid organs. The interferon-gamma inducible protein-10 (IP-10) chemo-attracts CXCR3-expressing cells, such as activated T lymphocytes and monocytes. We have genetically engineered a strain of Lactococcus lactis to secrete a biologically active murine IP-10 that interacts with human CXCR3, its homolog receptor, and chemo-attracts human CD3+ T lymphocytes.     

Targeting the tumor and its associated stroma: One and one can make three in adoptive T cell therapy of solid tumors

Adoptive T cell therapy (ACT) has become a promising immunotherapeutic option for cancer patients. The proof for ACT therapeutic efficacy was first obtained with allogenic T cells and then reproduced with T cells isolated from patients’ tumor samples (i.e. tumor-infiltrating lymphocytes). It is now clear that specificity of ACT products can be educated by genetically engineering T cells with classical T Cell Receptors (TCR) or chimeric antigen receptors (CAR). To date a poor accessibility of the tumor mass and a hostile microenvironment, influenced by genetic and epigenetic instability, mainly limit ACT therapeutic efficacy in the case of solid tumors. Available data indicate that these hurdles might be overcome by combinatorial therapeutic strategies targeting the tumor and its associated stroma. Here we review some of the available dual targeting strategies focusing on given combination of TCR/CAR-redirected T cell products and their association with drugs targeting the tumor-vessel and/or epigenetic modifiers, with the ability to sensitize tumors to T cell recognition. Existing data have proven synergistic effects in combined settings (one and one can indeed make three) and suggest that further benefit might be achieved by additional combinatorial therapeutic approaches (could one + one + one make ten?) in ACT of solid tumor.     

Improvement of a method to reproducibly immortalize human T cells by oncogene transfection

The method to immortalize human T cells efficiently and reproduciblyby oncogene transfection was improved. T cells were first grown selectively from peripheralblood lymphocytes population of healthy donors andatopic asthma patients, and from lymph nodelymphocytes population of lung cancer patients byactivating with mitogens (phytohemagglutinin andconcanavalin A) and recombinant human interleukin-2(rhIL-2) for five days. Plasmids expressingoncogenes, such as c-Ha-ras, c-myc,c-fos, v-myb and v-jun under the controlof human cytomegalovirus promoter, were then introducedinto these stimulated lymphocytes either separately orin various combinations by electropolation. Afterculturing these transfected lymphocytes for recoveryfor 1 day, they were fed every 3–4 days. Although all the control cells died within one month,oncogene-transfected lymphocytes continued toproliferate actively even for more than severalmonths, indicating that oncogene-transfectedlymphocytes were successfully immortalized. Flowcytometric analyses revealed that most of theimmortalized lymphocytes were T cells expressingCD3⁺ surface antigen. The ratios of CD4⁺and CD8⁺ subpopulations in immortalized T cellsderived from healthy donors varied, depending onthe kinds of oncogenes used. However, CD8⁺subpopulation in immortalized T cells derived fromcancer patients and atopic asthma patients weredominant, independent of the kinds of oncogenes. These immortalized T cells showed differentproliferative responses in the presence or absence ofexogenous human rhIL-2, depending on their origin ofdonors. Furthermore, immortalized T cells derivedfrom healthy donors showed stronger cytotoxicityagainst K562 cells, suggesting that MHC-nonrestrictedkiller T cells in T cell population were alsoimmortalized. Immortalized T cell lines, whichproliferate continuously without stimulation of amitogen or antigen in medium containing a lowconcentration of rhIL-2, have been maintained for morethan 2 years without any growth rate decrease.     

Redirecting therapeutic T cells against myelin-specific T lymphocytes using a humanized myelin basic protein-HLA-DR2-zeta chimeric receptor

Therapies that Ag-specifically target pathologic T lymphocytes responsible for multiple sclerosis (MS) and other autoimmune diseases would be expected to have improved therapeutic indices compared with Ag-nonspecific therapies. We have developed a cellular immunotherapy that uses chimeric receptors to selectively redirect therapeutic T cells against myelin basic protein (MBP)-specific T lymphocytes implicated in MS. We generated two heterodimeric receptors that genetically link the human MBP84-102 epitope to HLA-DR2 and either incorporate or lack a TCRzeta signaling domain. The Ag-MHC domain serves as a bait, binding the TCR of MBP-specific target cells. The zeta signaling region stimulates the therapeutic cell after cognate T cell engagement. Both receptors were well expressed on primary T cells or T hybridomas using a tricistronic (alpha, beta, green fluorescent protein) retroviral expression system. MBP-DR2-zeta-, but not MBP-DR2, modified CTL were specifically stimulated by cognate MBP-specific T cells, proliferating, producing cytokine, and killing the MBP-specific target cells. The receptor-modified therapeutic cells were active in vivo as well, eliminating Ag-specific T cells in a humanized mouse model system. Finally, the chimeric receptor-modified CTL ameliorated or blocked experimental allergic encephalomyelitis (EAE) disease mediated by MBP84-102/DR2-specific T lymphocytes. These results provide support for the further development of redirected therapeutic T cells able to counteract pathologic, self-specific T lymphocytes, and specifically validate humanized MBP-DR2-zeta chimeric receptors as a potential therapeutic in MS.     

Anticarcinogenic impact of extracellular vesicles (exosomes) from cord blood stem cells in malignant melanoma: A potential biological treatment

Incidence of Malignant Melanoma has become the 5th in the UK. To date, the major anticancer therapeutics include cell therapy, immunotherapy, gene therapy and nanotechnology-based strategies. Recently, extracellular vesicles, especially exosomes, have been highlighted for their therapeutic benefits in numerous chronic diseases. Exosomes display multifunctional properties, including inhibition of cancer cell proliferation and initiation of apoptosis. In the present in vitro study, the antitumour effect of cord blood stem cell (CBSC)-derived exosomes was confirmed by the CCK-8 assay (p < 0.05) on CHL-1 melanoma cells and improve the repair mechanism on lymphocytes from melanoma patients. Importantly, no significant effect was observed in healthy lymphocytes when treated with the exosome concentrations at 24, 48 and 72 h. Comet assay results (OTM and %Tail DNA) demonstrated that the optimal exosome concentration showed a significant impact (p < 0.05) in lymphocytes from melanoma patients whilst causing no significant DNA damage in lymphocytes of healthy volunteers was 300 μg/ml. Similarly, the Comet assay results depicted significant DNA damage in a melanoma cell line (CHL-1 cells) treated with CBSC-derived exosomes, both the cytotoxicity of CHL-1 cells treated with CBSC-derived exosomes exhibited a significant time-dependent decrease in cell survival. Sequencing analysis of CBSC exosomes showed the presence of the let-7 family of miRNAs, including let-7a-5p, let-7b-5p, let-7c-5p, let-7d-3p, let-7d-5p and two novel miRNAs. The potency of CBSC exosomes in inhibiting cancer progression in lymphocytes from melanoma patients and CHL-1 cells whilst causing no harm to the healthy lymphocytes makes it a potential candidate as an anticancer therapy.     

Bacteria in cancer therapy: a novel experimental strategy

Resistance to conventional anticancer therapies in patients with advanced solid tumors has prompted the need of alternative cancer therapies. Moreover, the success of novel cancer therapies depends on their selectivity for cancer cells with limited toxicity to normal tissues. Several decades after Coley's work a variety of natural and genetically modified non-pathogenic bacterial species are being explored as potential antitumor agents, either to provide direct tumoricidal effects or to deliver tumoricidal molecules. Live, attenuated or genetically modified non-pathogenic bacterial species are capable of multiplying selectively in tumors and inhibiting their growth. Due to their selectivity for tumor tissues, these bacteria and their spores also serve as ideal vectors for delivering therapeutic proteins to tumors. Bacterial toxins too have emerged as promising cancer treatment strategy. The most potential and promising strategy is bacteria based gene-directed enzyme prodrug therapy. Although it has shown successful results in vivo yet further investigation about the targeting mechanisms of the bacteria are required to make it a complete therapeutic approach in cancer treatment.     

Mutagenic and Genotoxic Effects of <Emphasis Type="Italic">cis</Emphasis>-(Dichloro)tetraammineruthenium(III) Chloride on Human Peripheral Blood Lymphocytes

Chemotherapeutic agents play an important role in cancer treatment mostly due their systemic action on human organism allowing access to liquid tumors and even metastases. Among these drugs, ruthenium compounds have been showing promising results to treat tumors and represent an important development of new antitumor therapy. This study presents the evaluation of cis-(dichloro)tetraammineruthenium(III) chloride, cis-[RuCl₂(NH₃)₄]Cl, genotoxic effects using human peripheral blood lymphocytes cultured in vitro. Mitotic index (MI), chromosome aberrations (CA), and DNA damage using the comet assay were analyzed. MI in human peripheral blood lymphocyte cultures treated with 1, 10, 100, and 1,000 μg mL⁻¹ cis-[RuCl₂(NH₃)₄]Cl were 5.9%, 4.6%, 3.9%, and 0%, respectively. Doxorubicin chloridate was used as the positive control. CA derived from 1, 10, and 100 μg mL⁻¹ concentrations were defined as spontaneous when compared with the negative control, and at the concentration of 1,000 μg mL⁻¹, the cell cycle was inhibited (IM = 0%). Results obtained for the comet assay using cis-[RuCl₂(NH₃)₄]Cl suggest that this compound has no genotoxic activity against cultured human peripheral blood lymphocytes.     

Development of pluripotent stem cell‐based human tenocytes

Human pluripotent stem cells (PSCs) are used as a platform for therapeutic purposes such as cell transplantation therapy and drug discovery. Another motivation for studying PSCs is to understand human embryogenesis and development. All cell types that make up the body tissues develop through defined trajectories during embryogenesis. For example, paraxial mesoderm is considered to differentiate into several cell types including skeletal muscle cells, chondrocytes, osteocytes, dermal fibroblasts, and tenocytes. Tenocytes are fibroblast cells that constitute the tendon. The step‐wise narrowing fate decisions of paraxial mesoderm in the embryo have been modeled in vitro using PSCs; however, deriving tenocytes from human‐induced PSCs and their application in cell therapy have long been challenging. PSC‐derived tenocytes can be used for a source of cell transplantation to treat a damaged or ruptured tendon due to injury, disorder, or aging. In this review, we discuss the latest research findings on the use of PSCs for studying the biology of tenocyte development and their application in therapeutic settings.     

Identification of multiple isoforms of the low-affinity human IgG Fc receptor

Two varieties of similar, but structurally distinct, cDNA clones for the human low-affinity receptors for the Fc portion of immunoglobulin G (FcγRII) have been isolated. One type of clone was obtained from human B lymphocytes, and the other from PHA-activated peripheral T cells and monocytes. Transfection of both prototype clones into Cos-7 cells and subsequent specific staining with monoclonal antibodies of the CDw32 group confirmed the identification of the gene products. The nucleotide sequence of the cDNA clone from B lymphocytes contains an open reading frame that encodes a protein of relative mass (M _r) 27000 with an extracellular domain of 179 amino acids containing three potential N-glycosylation sites, a 26 amino acid transmembrane domain, and a 44 amino acid cytoplasmic domain. The clones from peripheral T cells and monocytes both encoded a protein ofM _r 31000 with a 179 amino acid extracellular domain containing two potential N-glycosylation sites and a 26 amino acid transmembrane domain. The two types of clones had similar sequences in their immunoglobulin-like extracellular and transmembrane domains, but differed in their leader sequences and 3′-untranslated regions. The most notable difference between the clones was the presence of a distinctive 76 amino acid cytoplasmic domain in those isolated from T cells and monocytes.     

Rapid engineering of polyketide overproduction by gene transfer to industrially optimized strains

Development of natural products for therapeutic use is often hindered by limited availability of material from producing organisms. The speed at which current technologies enable the cloning, sequencing, and manipulation of secondary metabolite genes for production of novel compounds has made it impractical to optimize each new organism by conventional strain improvement procedures. We have exploited the overproduction properties of two industrial organisms—Saccharopolyspora erythraea and Streptomyces fradiae, previously improved for erythromycin and tylosin production, respectively—to enhance titers of polyketides produced by genetically modified polyketide synthases (PKSs). An efficient method for delivering large PKS expression vectors into S. erythraea was achieved by insertion of a chromosomal attachment site (attB) for φC31-based integrating vectors. For both strains, it was discovered that only the native PKS-associated promoter was capable of sustaining high polyketide titers in that strain. Expression of PKS genes cloned from wild-type organisms in the overproduction strains resulted in high polyketide titers whereas expression of the PKS gene from the S. erythraea overproducer in heterologous hosts resulted in only normal titers. This demonstrated that the overproduction characteristics are primarily due to mutations in non-PKS genes and should therefore operate on other PKSs. Expression of genetically engineered erythromycin PKS genes resulted in production of erythromycin analogs in greatly superior quantity than obtained from previously used hosts. Further development of these hosts could bypass tedious mutagenesis and screening approaches to strain improvement and expedite development of compounds from this valuable class of natural products.