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P Robbins

Publications and source records attributed to P Robbins.

84 records · Page 5Linked to original sources
PubMedDate not supplied

Dendritic cell-based genetic immunization in mice with a recombinant adenovirus encoding murine TRP2 induces effective anti-melanoma immunity.

BACKGROUND: The induction of cellular immune responses to melanocyte-specific enzymes such as the tyrosinase family of proteins is the goal of various clinical studies for the immunotherapy of melanoma. Tyrosinase-related protein-2 (TRP2) is an attractive model antigen for preclinical studies in C57BL/6 mice because it is naturally expressed by the murine B16 melanoma and can be recognized by self-reactive cytolytic T lymphocytes (CTL). Here we describe efforts to develop genetic immunization with dendritic cells (DC) for the immunotherapy of melanoma in this clinically relevant system. METHODS: Recombinant adenoviruses encoding green fluorescent protein (Ad-EGFP) and murine TRP2 (Ad-mTRP2) were constructed using Cre-loxP-mediated recombination. DC were generated in vitro from precursors in bone marrow and transduced with Ad-EGFP or Ad-mTRP2. Mice were immunized by direct injection of adenovirus or by injection of Ad-transduced DC. Induction of tumor immunity was assessed by intravenous challenge with B16 melanoma cells and enumeration of experimentally induced lung metastases. RESULTS: Flowcytometric analysis of DC transduced with Ad-EGFP demonstrated endogenous fluorescence due to cytoplasmatic expression of EGFP in 30-60% of cells. Ad-EGFP-transduced DC simultaneously displayed the DC-specific marker NLDC145 and high levels of MHC and costimulatory molecules on their cell surface. Transduction of DC with Ad-mTRP2 resulted in strong intracellular expression of TRP2 which could be readily detected by immunostaining. Importantly, immunization of mice with cultured Ad-mTRP2-transduced DC completely prevented the development of lung metastases following an intravenous challenge with B16 melanoma cells. This striking protective effect was observed with both the intravenous and the subcutaneous route of DC immunization. In vivo depletion of T-cell subsets suggested that the protective effect of an immunization with Ad-mTRP2-transduced DC involved both CD8+ and CD4+ T-cells. CONCLUSIONS: Our results demonstrate that DC-based genetic immunization of mice with TRP2, a clinically relevant melanocyte-specific self-antigen, induces effective cellular immunity and prevents metastatic growth of B16 melanoma cells in vivo.

Adenoviridae↗
PubMedDate not supplied

Transfer of proalpha2(I) cDNA into cells of a murine model of human Osteogenesis Imperfecta restores synthesis of type I collagen comprised of alpha1(I) and alpha2(I) heterotrimers in vitro and in vivo.

The oim mouse is a model of human Osteogenesis Imperfecta (OI) that has deficient synthesis of proalpha2(I) chains. Cells isolated from oim mice synthesize alpha1(I) collagen homotrimers that accumulate in tissues. To explore the feasibility of gene therapy for OI, a murine proalpha2(I) cDNA was inserted into an adenovirus vector and transferred into bone marrow stromal cells isolated from oim mice femurs. The murine cDNA under the control of the cytomegalovirus early promoter was expressed by the transduced cells. Analysis of the collagens synthesized by the transduced cells demonstrated that the cells synthesized stable type I collagen comprised of alpha1(I) and alpha2(I) heterotrimers in the correct ratio of 2:1. The collagen was efficiently secreted and also the cells retained the osteogenic potential as indicated by the expression of alkaline phosphatase activity when the transduced cells were treated with recombinant human bone morphogenetic protein 2. Injection of the virus carrying the murine proalpha2(I) cDNA into oim skin demonstrated synthesis of type I collagen comprised of alpha1 and alpha2 chains at the injection site. These preliminary data demonstrate that collagen genes can be transferred into bone marrow stromal cells as well as fibroblasts in vivo and that the genes are efficiently expressed. These data encourage further studies in gene replacement for some forms of OI and use of bone marrow stromal cells as vehicles to deliver therapeutic genes to bone.

Adenoviridae↗
PubMedDate not supplied

[Refined adenoviral transduction for controlled gene transfer into human adult mesenchymal stem cells].

AIM: Adenoviral gene transfer remains a powerful tool for basic research purposes. We hypothesize that adenoviral transduction of human mesenchymal stem cells (hMSC) in vitro can be improved by refined use of experimental parameters. METHODS: hMSCs were transduced by adenoviral vectors encoding Luciferase or BMP-2 at a selection of multiplicities of infection (MOI) and exposure times. Transgene production and total protein content were measured. To determine practical relevance, expression of the bone marker genes Runx2 and Type I collagen was analyzed by quantitative PCR. As a phenotypic marker alkaline phosphatase was assessed. ANOVA and post hoc statistical analyses were used to determine differences among data (p < 0.05). RESULTS: Prolonged exposure led to a decrease in transgene production and total protein content. Increasing MOI at exposure of up to 4 hours resulted in a higher production of the transgene. Transfer of the hBMP-2 gene promoted an enhanced lineage progression to the osteoblast phenotype indicating biological activity. CONCLUSION: Time of exposure is of major importance for toxicity in vitro and should not exceed 4 hours for hMSC. While increase in exposure time leads to cell death, surviving cells, up to a certain limit, seem to compensate by increasing production of the transgene indicating that transduction efficiency cannot be positively measured in a binary yes-or-no scheme.

Adenoviridae↗
PubMedDate not supplied

[Osteoblastic differentiation of human adult mesenchymal stem cells after through gene transfer of BMP-2 in the absence of dexamethasone].

AIM: Mesenchymal stem cells (MSC) of various species appear to require different cues to differentiate towards the osteoblastic lineage. For MSC of human origin, recombinant hBMP-2 is reported to be not sufficient but dexamethasone seems to be essential. The aim of this study was to analyse changes in genotype and phenotype of hMSC after adenoviral transfer of the BMP-2 gene in the absence of dexamethasone. METHODS: We employed hMSC and analysed changes in expression of the Runx2, Osterix and type I collagen gene by quantitative PCR after adenoviral transfer of the human BMP-2 gene in the absence of dexamethasone. As a phenotypic marker alkaline phosphatase activity was assessed. ANOVA and post hoc statistical analyses were used to determine differences among data (p < 0.05). RESULTS: Transfer of the hBMP-2 gene and consecutive production of transgenic BMP-2 up-regulated bone marker gene expression and increased alkaline phosphatase activity and thus promoted an enhanced lineage progression to the osteoblast phenotype without the addition of dexamethasone. CONCLUSION: These findings are noteworthy in the light of a possible superiority of endogenous transgenic proteins compared to exogenous recombinant proteins.

Adult↗
PubMedDate not supplied

[In vitro transfer of genes in spinal tissue].

INTRODUCTION: It is well known that cytokines are involved in the induction of intervertebral disc and articular cartilage destruction. Therapeutic proteins are of great potential as locally produced drugs after transfer of their cognate genes to the sites of interest. METHODS: Chondrocytic cells from bovine os coccygis and discal chondrocytes from 6 wistar rats were isolated and cultured in vitro. The bacterial beta-galactosidase (LacZ) gene and the cDNA of the human interleukin-1 receptor antagonist (IL-1ra) were introduced into the cells by retrovirus mediated gene transfer. LacZ activity was determined by Xgal staining, IL-1ra protein was determined by ELISA. RESULTS: Our study confirms that isolation and cultivation of bovine chondrocytic end plate cells and of rat discal cells in possible. Transfer of both LacZ and of the IL-1ra cDNA to cultured cells was successfull. DISCUSSION: The introduction of exogenous therapeutical genes into cells from the intervertebral disc and the end plate opens the possibility for a local gene therapy of IVD degeneration. This therapy has the potential to be specific, effective and appropriate to the chronicity of the disease.

Animals↗
PubMedDate not supplied

Pituitary carcinoma: an ultrastructural study of eleven cases.

Pituitary carcinomas are very rare. Defined as adenohypophysial tumors that undergo craniospinal and/or systemic metastasis, most are PRL- or ACTH-producing. Their ultrastructural features, particularly relative to benign adenomas of similar functional type, have not been sufficiently explored. Eleven cases of immunohistochemically characterized pituitary carcinoma with documented cerebrospinal and/or systemic metastases were collected from various institutions and studied by transmission electron microscopy. The tumors were surgically removed from 7 women and 4 men ranging in age between 28 and 74 years (mean, 50 years). All were endocrinologically functioning. Six tumors secreted PRL; three were ACTH-producing; one each was GH/PRL- and TSH-producing. The patients with the ACTH-producing tumors had all presented with Cushing's disease and two of them had undergone adrenalectomy (Nelson syndrome). In most cases significant cellular atypia and mitotic activity were observed. In terms of morphologic features of functional differentiation, electron microscopy revealed that in 9 cases the tumor cells maintained at least some ultrastructural markers of their basic phenotype. A unique feature in 2 ACTH carcinomas was the variable admixture of smooth endoplasmic reticulum with intermediate (cytokeratin) filaments. In 2 cases, both PRL-producing carcinomas, the cell type comprising the tumor could not be identified on an ultrastructural basis alone. Ultrastructural investigation of pituitary carcinomas confirms their endocrine nature and, in most but not all cases, reveals their functional differentiation. Despite the diagnostic utility of electron microscopy in the assessment of these rare tumors, the distinction of pituitary carcinoma from pituitary adenoma cannot be firmly made on ultrastructural grounds alone.

Adenoma↗
PubMedDate not supplied

Papillary tumor of the pineal region--a new pathological entity.

Papillary tumor of the pineal region has recently been proposed as a new, distinct clinicopathological entity. On the basis of the immunophenotypic and ultrastructural properties of these lesions, origin from specialized ependymocytes of the subcommissural organ was postulated. We present the third publication on a papillary pineal tumor and describe the morphological, immunohistochemical and ultrastructural features of this neoplasm. The patient was a young woman who presented with signs of raised intracranial pressure and Parinaud syndrome. Magnetic resonance imaging revealed a neoplastic lesion in the pineal region. She underwent surgical resection of the tumor through a midline infratentorial-supracerebellar approach. Papillary tumor of the pineal region represents a new, distinct clinicopathological entity. The differential diagnosis, possible histogenesis and management of these lesions are discussed.

Adult↗
PubMedDate not supplied

Cathepsin-D in human breast cancer: correlation with vascular invasion and other clinical and histopathological characteristics.

The relationship between the Cathepsin-D concentration in breast cancer cytosols and clinical and histopathological characteristics of the tumours was investigated, including vascular invasion, histological type, histological grade, lymph node involvement and tumour size. The median cathepsin-D concentration of a series of 738 primary breast carcinomas was used to define "low" and "high" cathepsin-D. High cathepsin-D concentration was associated with peritumoral vascular invasion, with high grade infiltrating duct carcinomas, with tumours of > or = 2 diameter, and with metastases in the axillary lymph nodes. Low cathepsin-D concentration was associated with in-situ carcinomas.

Breast Neoplasms↗