Biomedical subjects

Pat Metharom

Publications and source records attributed to Pat Metharom.

9 recordsLinked to original sources

PubMed2007

Clinical trials in stem cell therapy: pitfalls and lessons for the future.

The first human trial of stem cell therapy for cardiovascular disease was performed 4 years ago. Since that time, almost a dozen studies have reported the early and late clinical effects of cell therapy in acute myocardial infarction and chronic ischemic cardiomyopathy. Initial nonrandomized trials universally showed slight improvement in the left ventricular ejection fraction. Later randomized, controlled trials, however, suggested a less significant effect. They showed either no difference between cell therapy and control treatment or a slight treatment benefit with cell therapy that is lost by 12 months' follow-up. These results have dampened the enthusiasm of some members of the scientific community for the continuation of clinical trials. Because early phase I trials should not be judged on issues other than safety, however, research is unlikely to be hindered. Indeed, the clinical studies reported so far have already taught us a lot about the biology of myocardial repair. Achieving clinical success will, however, probably require much more investment in basic and experimental research. Here, we address some of the current pitfalls in clinical cell therapy trials and lessons that should be learned as we face the challenges of the future.

Cardiomyopathies↗

PubMed2005

Immunosuppressive agents mediate reduced allostimulatory properties of myeloid-derived dendritic cells despite induction of divergent molecular phenotypes.

Immunosuppressive drugs such as glucocorticoids (dexamethasone (Dexa)), cyclosporin A (CsA) and tacrolimus (Tacro) have been shown to impair differentiation and/or function of immunostimulatory dendritic cells (DC(ims)). Phenotypes and functions of the resultant myeloid dendritic cells, however, have not yet been thoroughly elucidated. We show here that all DC subsets generated by treatment with immunosuppressive agents exhibited considerably reduced allostimulatory properties as measured in the primary mixed lymphocyte reaction (tacrolimus>cyclosporin A>dexamethasone, used at equimolar concentrations). In the MLR, all these DC subsets furthermore inhibited secretion of the T-helper type 1 cytokine IFN-gamma; in addition, DC-Tacro and, less so, DC-CsA induced the T-helper type 2 cytokine IL-4. Upon FACS analysis, DC-Tacro and DC-CsA exhibited phenotypic features similar to DC(ims). In addition, DC-CsA and, to a smaller extent, DC-Tacro were characterized by increased mRNA expression of the novel costimulatory molecule B7-H2 (ICOS-ligand). In contrast, dexamethasone induced the generation of DC characterized by decreased expression of CD83 and CD86, by de novo expression of plasmacytoid and myeloid cell markers CD123 and CD14, respectively, and by sustained expression of Toll-like receptor 2. Interestingly, activation of DC-Dexa with a specific TLR2 ligand induced a strong up-regulation of IL-10 along with TNF-alpha and IL-6, a combination of cytokines that allow amplification of regulatory DC populations. In conclusion, myeloid DC induced by dexamethasone as well as by CsA or tacrolimus show reduced allostimulatory properties; however, they are equipped with different molecular repertoires to exert these functions.

CD4-Positive T-Lymphocytes↗

PubMed2005

Gene transfer to dendritic cells induced a protective immunity against melanoma.

Lentiviral vectors have shown promises for efficient gene transfer to dividing as well as nondividing cells. In this study, we explored lentiviral vector-mediated, the entire mTRP-2 gene transfer and expression in dendritic cells (DCs). Adoptive transfer of DCs-expressing mTRP-2 (DC-HR'CmT2) into C57BL/6 mouse was also assessed. Dendritic cells were harvested from bone marrow and functional DCs were proved by allogeneic mixed lymphocyte reaction. Lentiviral vectors were produced by transient transfection of 293T cells. Transduction of DCs was proved by marker gene expression and PCR and RT-PCR amplification. Implantation of the transduced DCs, depletion of immune cells as well as the survival of the mice after tumour challenge were investigated. High efficiency of gene transfer into mature DCs was achieved. The high level expression of the functional antigen (TRP-2) and induction of protective immunity by adoptive transfer of TRP-2 gene modified DCs were demonstrated. In vivo study showed a complete protection of mice from further melanoma cell challenge. In comparison, only 83% of mice survived when mTRP-2 peptide-pulsed DCs were administered, suggesting the generation of specific protection. Together, these results demonstrated the usefulness of this gene transfer to DC approach for immunotherapy of cancer and indicated that using tumour associated antigens (TAAs) for gene transfer may be potentially beneficial for the therapy of melanoma.

Adoptive Transfer↗

PubMed2005

Search for "weapons of mass destruction" for cancer -- immuno/ gene therapy comes of age.

The complexity of a cancer, such as cell heterogeneity, and the existence of hypoxia, stromal cells and stem cells has so far prevented successful development and treatment of patients suffering from the later stages of cancers. At present, the use of conventional therapies, such as chemo/radio therapy is limited, and only therapies that are focused on utilizing the patient's immune response to combat against the disease appear to be the most reliable and promising. Two decades ago, cytokines were discovered to be able to activate the immune systems and mount an anti-tumour response. Then, dendritic cells were hailed as the most significant regulators of immunity and are employed in a variety of cancer management schemes. This review introduces current development in the field, focusing on combination of the components of the rapidly growing fields of immunotherapy and gene transfer/therapy, providing useful and significant detailed information for readers of cellular and molecular immunology.

Animals↗

PubMed2004

High molecular weight plasma proteins induce apoptosis and Fas/FasL expression in human proximal tubular cells.

BACKGROUND: In proteinuria, proximal tubular epithelial cells (PTECs) are exposed to abnormally high protein concentrations, eventually leading to tubular atrophy and end-stage renal disease. The mode of cell death leading to tubular atrophy in proteinuria has not been fully established. This study examines the role of protein overload on apoptosis, necrosis and cell proliferation in primary cultures of human PTECs using plasma protein fractions representative of selective and non-selective proteinuria. The involvement of the Fas/Fas ligand (FasL) system was also investigated. METHODS: Plasma was collected from healthy volunteers and fractionated into albumin-rich (30-100 kDa), high molecular weight (100-440 kDa) and combined (30-440 kDa) fractions. PTECs were exposed to 10 mg/ml of the protein fractions for 24, 48 and 72 h. Apoptosis was measured using fluorescein isothiocyanate (FITC)-annexinV and TUNEL. Necrosis was measured using propidium iodide, metabolic activity by MTT and cell proliferation by bromodeoxyuridine incorporation. Fas and FasL expression was analysed by western blotting. RESULTS: Exposure to the 100-440 and 30-440 kDa fractions produced significant increases in apoptosis at all time points, whereas PTECs exposed to the 30-100 kDa fraction were not significantly different from control cells. There were no changes in the rates of necrosis as a result of protein loading. A significant reduction in metabolic activity was observed in PTECs exposed to the 100-440 and 30-440 kDa fractions, but not to the 30-100 kDa fraction. Cell proliferation was significantly reduced by 24 h in cells exposed to the 100-440 and 30-440 kDa fractions. By 48 and 72 h, all the three fractions had inhibited cell proliferation. PTECs exposed to the 100-440 and the 30-440 kDa fractions showed a significant upregulation in the expression of Fas and FasL. Overall, the high molecular weight fraction was more 'toxic' than the albumin-rich or combined fraction. CONCLUSION: Increased apoptosis and decreased cell proliferation are the major mechanisms of cell death in human PTECs in response to protein overload. These effects may be mediated at least in part by overexpression of the Fas/FasL system. The severity of such changes is largely determined by the high molecular weight fraction (100-440 kDa) rather than the albumin-rich fraction.

Apoptosis↗

PubMed2004

The significance of controlled conditions in lentiviral vector titration and in the use of multiplicity of infection (MOI) for predicting gene transfer events.

BACKGROUND: Although lentiviral vectors have been widely used for in vitro and in vivo gene therapy researches, there have been few studies systematically examining various conditions that may affect the determination of the number of viable vector particles in a vector preparation and the use of Multiplicity of Infection (MOI) as a parameter for the prediction of gene transfer events. METHODS: Lentiviral vectors encoding a marker gene were packaged and supernatants concentrated. The number of viable vector particles was determined by in vitro transduction and fluorescent microscopy and FACs analyses. Various factors that may affect the transduction process, such as vector inoculum volume, target cell number and type, vector decay, variable vector - target cell contact and adsorption periods were studied. MOI between 0-32 was assessed on commonly used cell lines as well as a new cell line. RESULTS: We demonstrated that the resulting values of lentiviral vector titre varied with changes of conditions in the transduction process, including inoculum volume of the vector, the type and number of target cells, vector stability and the length of period of the vector adsorption to target cells. Vector inoculum and the number of target cells determine the frequencies of gene transfer event, although not proportionally. Vector exposure time to target cells also influenced transduction results. Varying these parameters resulted in a greater than 50-fold differences in the vector titre from the same vector stock. Commonly used cell lines in vector titration were less sensitive to lentiviral vector-mediated gene transfer than a new cell line, FRL 19. Within 0-32 of MOI used transducing four different cell lines, the higher the MOI applied, the higher the efficiency of gene transfer obtained. CONCLUSION: Several variables in the transduction process affected in in vitro vector titration and resulted in vastly different values from the same vector stock, thus complicating the use of MOI for predicting gene transfer events. Commonly used target cell lines underestimated vector titre. However, within a certain range of MOI, it is possible that, if strictly controlled conditions are observed in the vector titration process, including the use of a sensitive cell line, such as FRL 19 for vector titration, lentivector-mediated gene transfer events could be predicted.

Journal Article↗

PubMed2004

Breaking immune tolerance to the prion protein using prion protein peptides plus oligodeoxynucleotide-CpG in mice.

The absence of a detectable immune response during transmissible spongiform encephalopathies is likely due to the fact that the essential component of infectious agents, the prion protein (PrP), is a self Ag expressed on the surface of many cells of the host. To overcome self-tolerance to PrP, we used 30-mer PrP peptides previously shown to be immunogenic in Prnp(-/-) mice, together with CFA or CpG-oligodeoxynucleotides (CpG) in IFA. Generation of anti-PrP T and B cell responses was analyzed in the spleen, lymph nodes, and serum of immunized C57BL/6 wild-type mice. Immunization with PrP peptides emulsified in CFA did not trigger an immune response to PrP. When CpG were used, vaccination with peptides P143-172 and P158-187 generated IFN-gamma-secreting splenic T cells, and only P158-187 significantly stimulated IL-4-secreting T cells. Both peptides induced few Ab-producing B cells, and low and variable serum Ab titers. In contrast, immunization with peptide P98-127 did not induce significant levels of T cell responses but elicited specific peptide Abs. T cell epitope mapping, performed using 15-mer peptides covering PrP segment 142-182, revealed that an immunogenic motif lies between positions 156 and 172. These results demonstrate that T and B cell repertoires against PrP can be stimulated in C57BL/6 when adjuvant of the innate immunity such as CpG, but not CFA, is added to PrP peptides, and that the pattern of immune responses varies according to the epitope.

Adjuvants, Immunologic↗

PubMed2004

Identification of two immunogenic domains of the prion protein--PrP--which activate class II-restricted T cells and elicit antibody responses against the native molecule.

Recent reports suggest that immunity against the prion protein (PrP) retards transmissible spongiform encephalopathies progression in infected mice. A major obstacle to the development of vaccines comes from the fact that PrP is poorly immunogenic, as it is seen as self by the host immune system. Additional questions concern the immune mechanisms involved in protection and the risk of eliciting adverse reactions in the central nervous system of treated patients. Peptide-based vaccines offer an attractive strategy to overcome these difficulties. We have undertaken the identification of the immunogenic regions of PrP, which trigger helper T cells (Th) associated with antibody production. Our results identify two main regions, one between the structured and flexible portion of PrP (98-127) and a second between alpha 1 and alpha 2 helix (143-187). Peptides (30-mer) corresponding to these regions elicit class II-restricted Th cells and antibody production against native PrP and could therefore be of potential interest for a peptide-based vaccination.

Amino Acid Sequence↗

PubMedDate not supplied

Endothelial progenitor cells.

The identification of circulating endothelial progenitor cells (EPCs) has prompted an explosion of interest in postnatal vasculogenesis and the role of this mechanism in human health and disease. Previously considered restricted to the embryonic phase, the differentiation in situ of progenitor cells to vascular endothelium is now known to occur in the adult. A role for EPCs in the modulation of angiogenesis has also been recognized. These cells are enriched in the mononuclear cell fraction of peripheral blood but have also been isolated from bone marrow, the vessel wall, and a number of other organs and tissues. Accumulating data suggest an important vasculoprotective function for EPCs, although a maladaptive role underpinning a variety of angiogenesis-dependent diseases is also being investigated. Encouraging results observed with experimental and early human trials of EPC-based regenerative therapies have further underscored the significance of this recently discovered cell type. Notwithstanding the scope and pace of these developments, a number of challenges remain: the precise ontogeny and lineage of these cells is unknown, the true extent to which EPCs participate in neovascularization and vascular repair is still uncertain, and the efficacy of EPC-based regenerative therapies has yet to be proven in randomized controlled trials.

Cell Lineage↗