Biomedical subjects

Antonio Bernad

Publications and source records attributed to Antonio Bernad.

25 records · Page 2Linked to original sources

PubMed2003

Novel interfering bifunctional molecules against the CCR5 coreceptor are efficient inhibitors of HIV-1 infection.

CCR5 is the major coreceptor for the HIV-1 strains responsible for primary infection. Individuals homozygous for a 32-bp deletion in the CCR5 coding region are resistant to HIV-1 infection. Strategies to delete CCR5 functionally could thus be of substantial benefit in preventing HIV-1 infection or delaying disease. We evaluated new molecules for their ability to inhibit cell membrane CCR5 expression and to prevent HIV-1 infection. These inhibitors include several truncated forms of CCR5 that may act as negative transdominants, as well as bifunctional molecules resulting from the combination of a previously described anti-CCR5 ribozyme or a truncated CCR5 variant with an intracellular chemokine (RANTES-KDEL). These constructs efficiently blocked membrane CCR5 expression when cotransfected into HEK 293 cells. When expressed by retroviral transduction, some of these molecules significantly inhibited CCR5-dependent chemotaxis in the MCF-7 cell line and reduced CCR5 expression and HIV-1 infection in human T cells. Analysis of inhibitors with different efficiencies showed a strong linear correlation between CCR5 expression inhibition and prevention of HIV-1 infection. This study indicates the potential clinical application of several new CCR5 inhibitory molecules for HIV-1 gene therapy.

Anti-HIV Agents↗

PubMed2002

A preclinical model for the analysis of genetically modified human skin in vivo.

Although skin is perhaps the most accessible of all somatic tissues for therapeutic gene transfer, it is a challenging site when attempting gene delivery. In addition to the transience of gene expression, important obstacles to cutaneous gene therapy have included the inability to sustain gene expression in a large proportion of keratinocytes within a given skin compartment. In this study, we have developed a novel experimental strategy that allows long-term regeneration of entirely genetically engineered human skin on the backs of NOD/SCID mice. Primary human keratinocytes were infected with a retroviral vector encoding the enhanced green fluorescent protein (EGFP) produced by transient transfection of 293T cells. EGFP expression allowed cell-sorting selection of a polyclonal population of productively transduced keratinocytes that were assembled in a live fibroblast-containing fibrin dermal matrix and orthotopically grafted onto mice. Epifluorescent illumination of the transplanted zone allowed in vivo monitoring of the genetically modified graft. EGFP-positive human skin was present on mice for 22 weeks after grafting. In addition, frozen sections prepared from the grafts displayed consistently strong EGFP-based fluorescence in all epidermal strata at every time point examined. Persistence of transgene expression was further confirmed through EGFP protein immunodetection. Purified EGFP-positive keratinocytes grafted as part of the fibrin-based artificial skin were capable of generating multilayer human epidermis on mice, with well-developed granulosum and corneum strata, and clearly defined rete ridges. Finally, the large proportion of transduced keratinocytes in our grafts allowed us to study, for the first time, the long-term in vivo clonal reconstitution pattern of the regenerated skin. Analysis of the provirus insertion sites indicates that a discrete number of epidermal stem cell clones was responsible for the maintenance of human skin regenerated in NOD/SCID recipients.

Animals↗

PubMed2002

Long-term repopulating ability of telomerase-deficient murine hematopoietic stem cells.

Telomere length must be tightly regulated in highly proliferative tissues, such as the lymphohematopoietic system. Under steady-state conditions, the levels and functionality of hematopoietic-committed or multipotent progenitors were not affected in late-generation telomerase-deficient mice (mTerc(-/-)) with critically short telomeres. Evaluation of self-renewal potential of mTerc(-/-) day-12 spleen colony-forming units demonstrated no alteration as compared with wildtype progenitors. However, the replating ability of mTerc(-/-) granulocyte-macrophage CFUs (CFU-GMs) was greatly reduced as compared with wildtype CFU-GMs, indicating a diminished capacity of late-generation mTerc(-/-) committed progenitors when forced to proliferate. Long-term bone marrow cultures of mTerc(-/-) bone marrow (BM) cells show a reduction in proliferative capacity; this defect can be mainly attributed to the hematopoietic, not to the stromal, mTerc(-/-) cells. In serial and competitive transplantations, mTerc(-/-) BM stem cells show reduced long-term repopulating capacity, concomitant with an increase in genetic instability compared with wildtype cells. Nevertheless, in competitive transplantations late-generation mTerc(-/-) precursors can occasionally overcome this proliferative impairment and reconstitute irradiated recipients. In summary, our results demonstrate that late-generation mTerc(-/-) BM cells with short telomeres, although exhibiting reduced proliferation ability and reduced long-term repopulating capacity, can still reconstitute myeloablated animals maintaining stem cell function.

Animals↗

PubMed2002

Inhibition of programmed cell death impairs in vitro vascular-like structure formation and reduces in vivo angiogenesis.

Tissue remodeling during embryonic development and in the adult organism relies on a subtle balance between cell growth and apoptosis. As angiogenesis involves restructuring of preexisting endothelium, we examined the role of apoptosis in new vessel formation. We show that apoptosis occurs before capillary formation but not after vessels have assembled. Using the human umbilical vein endothelial cell (HUVEC) in vitro Matrigel angiogenesis model, we show that vascular-like structure formation requires apoptotic cell death through activation of a caspase-dependent mechanism and mitochondrial cytochrome c release. Vascular-like structure formation was further blocked by caspase inhibitors such as z-VAD or Ac-DEVD-CHO, using HUVEC and human lung microvascular endothelial cells. Overexpression of anti-apoptotic human Bcl-2 or baculovirus p35 genes in HUVEC altered endothelial cell rearrangement during in vitro angiogenesis, causing impaired vessel-like structure formation. Caspase inhibitors blocked VEGF- or bFGF-induced HUVEC angiogenesis on 2- or 3-D collagen gels, respectively, confirming that apoptosis was not the result of nonspecific cell death after seeding on the matrix. In an in vivo angiogenesis assay, caspase inhibitors blocked VEGF-dependent vascular formation at the alignment step, as demonstrated histologically. This evidence indicates that endothelial cell apoptosis may be relevant for precise vascular tissue rearrangement in in vitro and in vivo angiogenesis.

Amino Acid Chloromethyl Ketones↗

PubMed2001

Functional interaction between the Ser/Thr kinase PKL12 and N-acetylglucosamine kinase, a prominent enzyme implicated in the salvage pathway for GlcNAc recycling.

PKL12 (STK16) is a ubiquitously expressed Ser/Thr kinase, not structurally related to the well known subfamilies, with a putative role in cell adhesion control. Yeast two-hybrid protein interaction screening was used to search for proteins that associate with PKL12 and to delineate signaling pathways and/or regulatory circuits in which this kinase participates. One positive clone contained an open reading frame highly similar to N-acetylglucosamine kinase (GlcNAcK) of several species. The PKL12/GlcNAcK interaction was further confirmed both in vitro and in vivo. Protein expression analysis of GlcNAcK using a specific rabbit antiserum displayed a ubiquitous pattern in cell lines and animal tissues. Subcellular localization studies showed that GlcNAcK is a cytoplasmic protein with a dual subcellular localization, distributed between the perinuclear and peripheral cell reservoirs. After overexpression, GlcNAcK localizes in vesicular structures associated mainly with the cell membrane and colocalizes with the PKL12 protein. GlcNAcK is not otherwise a substrate for PKL12 activity and PKL12 does not appear to influence GlcNAcK activity either in vitro or in vivo. In vitro kinase assays have nonetheless revealed that functional GlcNAcK, although not able to modulate autophosphorylation of PKL12, greatly influences PKL12 kinase activity on a defined substrate protein. These results are interpreted to indicate a potential in vivo role for GlcNAcK in PKL12 translocation and a tentative regulatory role for PKL12-mediated phosphorylation on substrate proteins.

3T3 Cells↗

PubMedDate not supplied

Glycodendritic structures based on Boltorn hyperbranched polymers and their interactions with Lens culinaris lectin.

Multivalent scaffolds bearing carbohydrates have been prepared to mediate biological processes where carbohydrates are involved. These systems consist of dendritic structures based on Boltorn H20 and H30 hyperbranched polymers to which carbohydrates are linked through a convenient spacer. Mannose has been chosen as a sugar unit to test the viability of this strategy. These glycodendritic compounds have been prepared in a few steps with good yields, showing a high solubility in physiological media and low toxicity. The binding of these dendritic polymers to the mannose-binding lectin Lens culinaris (LCA) was studied using STD-NMR experiments and quantitative precipitation assays. The results demonstrate the existence of a clear interaction between the mannose derivative systems and the Lens lectin where the dendritic scaffold does not have an important role in mannose binding but supplies the necessary multivalence for lectin cluster formation. These glycodendritic structures are able to interact with a receptor, and therefore they can be considered as promising tools for biological studies.

Animals↗

PubMedDate not supplied

Single-step, multiple retroviral transduction of human T cells.

BACKGROUND: Retroviral transduction of human peripheral blood T cells has considerable potential in the development of gene therapy strategies for immunological disorders. New vectors and experimental procedures have been developed for efficient transduction of several genes into human T cells. METHODS: Bicistronic retroviral vectors encoding distinct cell markers were used for the simultaneous multiple transduction of a human T-cell line (MT-2), as well as of human peripheral blood T cells from normal donors. Transduction efficiencies were evaluated by flow cytometry and double- and triple-transduced cells were isolated by fluorescence cell sorting. RESULTS: Four new bicistronic retroviral vectors were developed that express different gene markers under the control of the internal ribosome entry site (IRES) of the encephalomyocarditis virus. These markers are, respectively, enhanced green fluorescent protein (EGFP), beta-galactosidase, and truncated versions of human nerve growth factor receptor (Delta NGFR) and human growth hormone receptor (Delta GHR). A single 1 h spinoculation infection, performed in the presence of polybrene and using transiently produced amphotropic retroviral particles, was sufficient to obtain transduction efficiencies consistently greater than 50% on human peripheral blood T lymphocytes which had been previously stimulated for 3 days with immobilized anti-CD3. The transient production of viral particles encoding EGFP, Delta NGFR, and Delta GHR markers in the same viral supernatant has allowed up to three different genes to be introduced simultaneously into human T cells. CONCLUSIONS: This study describes new experimental conditions for efficient single-step multiple transduction of human primary T lymphocytes. The procedure could be of interest for the development of gene therapy approaches.

Chemokine CCL5↗