Human adult bone marrow contains both hematopoietic stem cells that generate cells of all hematopoietic lineages and human mesenchymal stem cells (hMSCs), which support hematopoiesis and contribute to the regeneration of multiple connective tissues. The goal of the current study was to demonstrate that transduced hMSCs maintain transgene expression after stem cell differentiation in vitro and in vivo. We have introduced genes into cultured hMSCs by retroviral vector transfer and demonstrated long-term in vitro and in vivo expression of human interleukin 3 (hIL-3) and green fluorescent protein (GFP). Protocols were developed to achieve transduction efficiencies of 80-90% in these stem cells. In vitro expression of hIL-3 averaged 350 ng/10(6)cells/24 h over 17 passages (> 6 months) and GFP expression was stable over the same time period. Transduced hMSCs were able to differentiate into osteogenic, adipogenic, and chondrogenic lineages and maintained transgene expression after differentiation. Parallel studies were performed in vivo using NOD/SCID mice. Human MSCs expressing hIL-3 were cultured on several matrices and then delivered by subcutaneous, intravenous, and intraperitoneal routes. Sampling of peripheral blood demonstrated that systemic hIL-3 expression was maintained in the range of 100-800 pg/ml over a period of 3 months. These results illustrate the ability of hMSCs to express genes of therapeutic potential and demonstrate their potential clinical utility as cellular vehicles for systemic gene delivery.
Helicobacter pylori urease requires nickel ions in the enzyme active site for catalytic activity. Nickel ions must, therefore, be actively acquired by the bacterium. NixA (high-affinity nickel transport protein)-deficient mutants of H. pylori retain significant urease activity, suggesting the presence of alternate nickel transporters. Analysis of the nucleotide sequence of the H. pylori genome revealed a homolog of NikD, a component of an ATPdependent nickel transport system in Escherichia coli. Based on this sequence, a 378-bp DNA fragment was PCR amplified from H. pylori genomic DNA and used as a probe to identify an H. pylori ZAPII genomic library clone that carried these sequences. Four open reading frames of 621, 273, 984, and 642 bp (abcABCD) were revealed by sequencing and predicted polypeptides of 22.7, 9.9, 36.6, and 22.8 kDa, respectively. The 36.6-kDa polypeptide (AbcC) has significant homology (56% amino acid sequence identity) to an E. coli ATP-binding protein component of an ABC transport system, while none of the other putative proteins are significantly homologous to polypeptides in the available databases. To determine the possible contribution of these genes to urease activity, abcC and abcD were each insertionally inactivated with a kanamycin resistance (aphA) cassette and allelic exchange mutants of each gene were constructed in H. pylori UMAB41. Mutation of abcD resulted in an 88% decrease in urease activity to 27 ؎ 31 mol of NH 3 /min/mg of protein (P < 0.0001), and a double mutant of nixA and abcC resulted in the near abolishment of urease activity (1.1 ؎ 1.4 mol of NH 3 /min/mg of protein in the double mutant versus 228 ؎ 92 mol of NH 3 /min/mg of protein in the parent [P < 0.0001]). Synthesis of urease apoenzyme, however, was unaffected by mutations in any of the abc genes. We conclude that the abc gene cluster, in addition to nixA, is involved in production of a catalytically active urease.Peptic ulcer disease is a widespread ailment that may affect as much as 4% of women and 10% of men over their lifetimes (48). Helicobacter pylori, a gram-negative, microaerophilic, spiral-shaped bacterium, has been established as an important cause of gastritis (7,58). Infection with this organism is strongly associated with the development of gastric and duodenal ulcers (10,33,36); chronic infection has also been correlated with the progression to gastric carcinoma (44). The mechanisms by which H. pylori colonizes the acidic gastric mucosa and causes these diseases are not yet fully understood; however, one cellular protein, urease, is required for colonization (14) and most likely contributes directly to peptic ulcer disease (for a review, see reference 42). Urease catalyzes the hydrolysis of urea to generate carbon dioxide and ammonia. The ammonia produced by this reaction is hypothesized to allow H. pylori to survive in the gastric mucosa by neutralizing gastric acid around the bacterium and raising the local pH (14,37,40,54). The contribution of urease to virulence, however, appears to go well be...
The marginal zone (MZ) is largely composed of a unique subpopulation of B cells, the so-called MZ-B cells. At a molecular level, memory B cells are characterized by the presence of somatically mutated IGV genes. The earliest studies in the rat have documented the presence of hapten-specific MZ-B cells after immunization in the MZ. This work later received experimental support demonstrating that the IGHV-Cµ transcripts expressed by phenotypically defined splenic MZ-B cells (defined as CD90negIgMhighIgDlow B cells) can carry somatic hypermutation. However, only a minor fraction (< 10%–20%) of these MZ-B cells is mutated and is considered to represent memory B cells. Memory B cells can either be class-switched (IgG, IgA, IgE), or non–class-switched (IgM) B cells. B cells in the MZ are a heterogeneous population of cells and both naïve MZ-B cells; class switched and unswitched memory MZ-B cells are present at this unique site in the spleen. Naïve MZ-B cells carry unmutated Ig genes, produce low-affinity IgM molecules and constitute a first line of defense against invading pathogens. Memory MZ-B cells express high-affinity Ig molecules, directed to (microbial) antigens that have been encountered. In this review, we report on the memory compartment of splenic MZ-B cells in the rat to provide insights into the origin and function of these memory MZ-B cells.
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