Selection, enrichment, and culture expansion of murine mesenchymal progenitor cells by retroviral transduction of cycling adherent bone marrow cells.

It has been difficult to characterize murine bone marrow (BM)-derived mesenchymal progenitor cells (MPCs) because of contamination with hematopoietic cells. We took advantage of the rapid proliferation of MPCs after replating to enrich murine MPCs by transfection with a retroviral vector carrying both LacZ and the selective neomycin resistance (neoR) gene. Freshly harvested BM cells from mice were incubated with BAG retroviral vector produced by amphotropic psi-CRIP or ecotropic psi-CRE producer cells for 48 hours and grown in the presence of G418.

Human mesenchymal stem cells engraft and demonstrate site-specific differentiation after in utero transplantation in sheep.

Mesenchymal stem cells are multipotent cells that can be isolated from adult bone marrow and can be induced in vitro and in vivo to differentiate into a variety of mesenchymal tissues, including bone, cartilage, tendon, fat, bone marrow stroma, and muscle. Despite their potential clinical utility for cellular and gene therapy, the fate of mesenchymal stem cells after systemic administration is mostly unknown. To address this, we transplanted a well-characterized human mesenchymal stem cell population into fetal sheep early in gestation, before and after the expected development of immunologic competence.

Photodynamic therapy of early squamous cell cancers of the esophagus.

The conventional treatment for the cure of esophageal cancer is surgical resection. Esophageal cancer, when detected at an early stage, has a very good probability of being eradicated by less aggressive procedures, and photodynamic therapy has proven to be a safe and effective treatment modality in some carefully selected patients. The indications, outcomes, and future considerations regarding the use of photodynamic therapy for the treatment of superficial squamous cell carcinomas of the esophagus are discussed in this article.

Fluorescence pharmacokinetics of Lutetium Texaphyrin (PCI-0123, Lu-Tex) in the skin and in healthy and tumoral hamster cheek-pouch mucosa.

We have investigated the pharmacokinetics (PK) of Lutetium Texaphyrin (Lu-Tex), a second-generation photosensitizer, in the Syrian hamster cheek pouch early cancer model. Ten male hamsters, five with chemically induced early squamous cell cancer of the left cheek pouch, received an intracardiac injection of a 10 mg/ml Lu-Tex solution, resulting in a dose of 12 mg Lu-Tex per kg of bodyweight. The PK of the dye have been measured during the 24 h following the injection with an optical-fiber-based spectrofluorometer on the ventral skin, the healthy and the tumoral cheek-pouch mucosa.

Adenoviral-mediated gene transfer in wound healing: acute inflammatory response in human skin in the SCID mouse model.

The use of an adenoviral vector as a means of therapeutic protein delivery for the treatment of impaired wound healing is a potentially effective application of current gene transfer techniques. This study was designed to investigate the ability of adenovirus to mediate gene transfer in healing wounds in human skin in vivo. The human skin/severe combined immunodeficient mouse chimera model was used to study both the response of human tissue to adenoviral infection and the nature of the acute inflammatory response.

Pulse oximeter as a cause of skin burn during photodynamic therapy.

Photodynamic therapy is increasingly being used for the treatment of various cancers. However, this technique has a major adverse effect, namely skin photosensitization. An unusual case of skin burn associated with pulse oximetry during photodynamic therapy in a patient treated for an early esophageal tumor is described.

Adenoviral-mediated overexpression of platelet-derived growth factor-B corrects ischemic impaired wound healing.

Chronic wounds represent a major clinical problem with significant morbidity and healthcare expenditures, but no effective therapies. Topical platelet-derived growth factor-BB trials have required large and repeated doses to achieve only a modest effect. We examined the ability of an adenovirus containing the platelet-derived growth factor-B transgene to improve the rate of wound healing through induction of platelet-derived growth factor-B overexpression in cells participating in the wound healing response.

Update on lumbar spinal stenosis. Retrospective study of 62 patients and review of the literature.

Purpose And Methods: Although lumbar spinal stenosis syndrome is extremely common, considerable controversy continues to surround its classification, diagnosis, and treatment. We retrospectively reviewed the medical charts of 62 patients admitted for lumbar spinal stenosis syndrome, and we compared our findings to those in the literature. There were 31 women and 31 men.

RanGTP-mediated nuclear export of karyopherin alpha involves its interaction with the nucleoporin Nup153.

Using binding assays, we discovered an interaction between karyopherin alpha2 and the nucleoporin Nup153 and mapped their interacting domains. We also isolated a 15-kDa tryptic fragment of karyopherin beta1, termed beta1*, that contains a determinant for binding to the peptide repeat containing nucleoporin Nup98. In an in vitro assay in which export of endogenous nuclear karyopherin alpha from nuclei of digitonin-permeabilized cells was quantitatively monitored by indirect immunofluorescence with anti-karyopherin alpha antibodies, we found that karyopherin alpha export was stimulated by added GTPase Ran, required GTP hydrolysis, and was inhibited by wheat germ agglutinin.

Karyopherin beta2 mediates nuclear import of a mRNA binding protein.

We have cloned and sequenced cDNA for human karyopherin beta2, also known as transportin. In a solution binding assay, recombinant beta2 bound directly to recombinant nuclear mRNA-binding protein A1. Binding was inhibited by a peptide representing A1's previously characterized M9 nuclear localization sequence (NLS), but not by a peptide representing a classical NLS.

Nuclear protein import: Ran-GTP dissociates the karyopherin alphabeta heterodimer by displacing alpha from an overlapping binding site on beta.

The alpha subunit of the karyopherin heterodimer functions in recognition of the protein import substrate and the beta subunit serves to dock the trimeric complex to one of many sites on nuclear pore complex fibers. The small GTPase Ran and the Ran interactive protein, p10, function in the release of the docked complex. Repeated cycles of docking and release are thought to concentrate the transport substrate for subsequent diffusion into the nucleus.

The binding site of karyopherin alpha for karyopherin beta overlaps with a nuclear localization sequence.

By using proteolysis, recombinant mutant proteins, or synthetic peptides and by testing these reagents in liquid phase binding or nuclear import assays, we have mapped binding regions of karyopherin alpha. We found that the C-terminal region of karyopherin alpha recognizes the nuclear localization sequence (NLS), whereas its N-terminal region binds karyopherin beta. Surprisingly, karyopherin alpha also contains an NLS.

Gene transfer to the thymus. A means of abrogating the immune response to recombinant adenovirus.

Objective: The authors investigated whether adenoviral gene transfer to the thymus could be accomplished in vivo and whether immunologic unresponsiveness to recombinant adenovirus could be induced by intrathymic inoculation.

Summary Background Data: A major barrier to the clinical application of adenovirus-mediated gene therapy for diseases requiring long-lasting gene expression is the immunogenicity of adenoviral vectors, which limits the duration of its effects. In other experimental models, intrathymic inoculation of foreign proteins or cells has proven to be an effective means to induce immunologic tolerance.

Mammalian karyopherin alpha 1 beta and alpha 2 beta heterodimers: alpha 1 or alpha 2 subunit binds nuclear localization signal and beta subunit interacts with peptide repeat-containing nucleoporins.

Although only 44% identical to human karyopherin alpha 1, human karyopherin alpha 2 (Rch1 protein) substituted for human karyopherin alpha 1 (hSRP-1/NPI-1) in recognizing a standard nuclear localization sequence and karyopherin beta-dependent targeting to the nuclear envelope of digitonin-permeabilized cells. By immunofluorescence microscopy of methanol-fixed cells, karyopherin beta was localized to the cytoplasm and the nuclear envelope and was absent from the nuclear interior. Digitonin permeabilization of buffalo rat liver cells depleted their endogenous karyopherin beta.

The peptide repeat domain of nucleoporin Nup98 functions as a docking site in transport across the nuclear pore complex.

We report the cDNA deduced primary structure of a wheat germ agglutinin-reactive nuclear pore complex (NPC) protein of rat. The protein, termed Nup98 (for nucleoporin of 98 kDa), contains numerous GLFG and FG repeats and some FXFG repeats and is thus a vertebrate member of a family of GLFG nucleoporins that were previously discovered in yeast. Immunoelectron microscopy showed Nup98 to be asymmetrically located at the nucleoplasmic side of the NPC.

Previously identified protein of uncertain function is karyopherin alpha and together with karyopherin beta docks import substrate at nuclear pore complexes.

Previously, we had purified a cytosolic protein complex, termed karyopherin, that functions in docking import substrate at the nuclear envelope in digitonin-permeabilized cells and also had molecularly cloned and sequenced its 97-kDa beta subunit. We now report that the karyopherin alpha subunit is the previously identified protein NPI-1/SRP-1 of hitherto uncertain function. Using purified recombinant karyopherin alpha or beta subunit, we showed that neither karyopherin alpha nor karyopherin beta alone was sufficient for docking of import substrate at the nuclear envelope.

[Comparison between immunofluorescence techniques and ELISA, using whole neutrophil extract and primary granules, for the detection of antineutrophil cytoplasma antibodies (ANCA)].

The detection of antineutrophil cytoplasmic antibodies is a very important tool for the diagnosis of systemic vasculitis. The specificity and sensitivity of these antibodies depends on the assay utilized for their detection. Therefore we have compared the immunofluorescence test (IF) with the ELISA using two different antigens: total neutrophil extract and isolated primary granules.

Identification of a protein complex that is required for nuclear protein import and mediates docking of import substrate to distinct nucleoporins.

We have identified and characterized a 9S protein complex from a Xenopus ovary cytosolic subfraction (fraction A) that constitutes this fraction's activity in recognizing a model nuclear import substrate and docking it at the nuclear pore complex. Because of its function, the complex is termed karyopherin. The 54- and 56-kDa subunits of the complex are termed alpha 1 and alpha 2, respectively, and the 97-kDa subunit is termed beta.

Nup107 is a novel nuclear pore complex protein that contains a leucine zipper.

We have previously described procedures for the isolation of potential nuclear pore complex proteins (nucleoporins or Nups) from rat liver nuclear envelopes and their subsequent subfractionation into those binding to wheat germ agglutinin (WGA) and those that do not. One of these non-WGA-reactive proteins, termed Nup155, was previously molecularly cloned and sequenced and by immunoelectron microscopy shown to be a bona fide nucleoporin. Here we have characterized a second protein of the non-WGA-reactive type and show that it is a Nup as well.

The human CAN protein, a putative oncogene product associated with myeloid leukemogenesis, is a nuclear pore complex protein that faces the cytoplasm.

We have carried out partial amino acid sequence analysis of a putative nuclear pore complex protein (nucleoporin) of rat that reacts with wheat germ agglutinin and with the polyspecific monoclonal antibody 414. Surprisingly, these partial amino acid sequence data revealed a high degree of similarity with the human CAN protein, the complete cDNA-derived primary structure of which was reported by Von Lindern et al. [Von Lindern, M.

[Antineutrophil cytoplasmic antibodies in systemic vasculitis].

The term systemic vasculitis concerns a group of diseases characterized by inflammation of vessels. The diagnosis and follow-up of these conditions is a serious challenge since their classification is difficult and the therapy is usually empiric. Perhaps the greatest breakthrough in the management of these diseases is the recent discovery of serological markers for some vasculitic syndromes.

Nup155 is a novel nuclear pore complex protein that contains neither repetitive sequence motifs nor reacts with WGA.

We have molecularly cloned and sequenced a rat liver nuclear pore complex (NPC) protein of calculated molecular mass of 155 kD. Consistent with recently proposed nomenclature this protein is termed nucleoporin 155, or nup155. Unlike other nups that have so far been molecularly cloned and sequenced, nup155 does not contain repetitive sequence domains.

Autologous melanoma vaccine induces inflammatory responses in melanoma metastases: relevance to immunologic regression and immunotherapy.

Human primary malignant melanoma is often accompanied by a host response of infiltrating lymphocytes suggestive of tumor antigen-induced immunity and correlated in some tumors with prognosis. Whereas metastatic melanoma deposits typically are not inflamed and contain relatively few lymphocytes and dendritic immune cells, immunization with autologous melanoma-cell vaccine may induce a clinical inflammatory response associated with mononuclear-cell infiltration. In this study, we characterize immune responses to dermal and subcutaneous melanoma metastases in dinitrophenyl (DNP)-pre-sensitized patients immunized with DNP-conjugated melanoma cells.

A simple polymerase chain reaction apparatus based on a computer printer.

We describe a very simple laboratory-made polymerase chain reaction (PCR) apparatus. The reaction tubes are placed in a holder fixed through a mechanical arm to the tape cartridge of a computer printer. A computer controls the horizontal movement of the tube carrier by sending the proper printing commands.