Neointimal formation by circulating bone marrow cells

The origin of smooth muscle cells involved in vascular healing was examined. Eighteen C57BL/6 (Ly 5.2) female mice underwent whole body irradiation followed by transfusion with 10(6) bone nucleated marrow cells from congenic (Ly 5.1) male donors. Successful repopulation by donor marrow was demonstrated after 4 weeks by flow cytometry with FITC-conjugated A20.1/Ly 5.1 monoclonal antibody. The iliac artery of six of the chimeric mice was scratch-injured by five passes of a probe, causing severe medial damage. After 4 weeks the arterial lumen was obliterated by a cell-rich neointima, with alpha-smooth muscle actin-containing cells present around the residual lumen. Approximately half of these cells were of male donor origin, as evidenced by in situ hybridization with a Y chromosome-specific probe. An organized arterial thrombus was formed in the remaining 12 chimeric mice by inserting an 8.0 silk suture into the left common carotid artery. Donor cells staining with alpha-smooth muscle actin were found in those arteries sustaining serious damage but not in arteries with minimal damage. Our results suggest that bone marrow-derived cells are recruited in vascular healing as a complementary source of smooth muscle-like cells when the media is severely damaged and few resident smooth muscle cells are available to effect repair.

Constitutively Active Mutant D816VKit Induces Megakayocyte and Mast Cell Differentiation of Early Haemopoietic Cells from Murine Foetal Liver

Mutations of Kit at position D816 have been implicated in mastocytosis, acute myeloid leukaemia and germ cell tumours. Expression of this mutant Kit in cell lines results in factor-independent growth, differentiation and increased survival in vitro and tumourigenicity in vivo. Mutant D816VKit and wild-type Kit were expressed in murine primary haemopoietic cells and grown in stem cell factor (SCF) or the absence of factors. Expression of D816VKit did not lead to transformation as assessed by a colony assay, but resulted in enhanced differentiation of cells when compared to control cells. D816VKit induced an increase in the number of cells differentiating along the megakaryocyte lineage in the absence of factors. SCF had an added effect with an increase in differentiation of mast cells. Expression of wild-type Kit in the presence of SCF also failed to cause transformation and induced differentiation of mast cells and megakaryocytes. We conclude that constitutive expression of D816VKit in primary haemopoietic cells is not a sufficient transforming stimulus but leads to the survival and maturation of cells whose phenotype is influenced by the presence of SCF. (C) 2003 Elsevier Science Ltd. All rights reserved.

The mechanism of inflammation in RelB deficient mice

RelB, NIK and TRAF6-deficient mice die prematurely with multi-organ inflammatory disease and apparent excessive myelopoiesis. While thymic development of CD4+CD25+ regulatory T cells (Treg) is reduced in TRAF6 deficient mice, the impact of this on inflammation is not known. Here we show that while RelB deficient thymic stroma is unable to sustain the development of Treg, surprisingly, FoxP3hi Treg are increased in the periphery. Peripheral expansion of Treg is driven by GITRligand, expressed by immature monocytes maintained by RelBdeficient stroma. RelB-deficient DC fail to activate Treg suppressor function. The data reveal the dual roles of RelB in both hemopoietic and stromal cells to maintain tolerance and contain inflammation through Treg and DC.

Identification and characterization of a G protein-coupled receptor homolog encoded by murine cytomegalovirus

This report describes the identification of a murine cytomegalovirus (MCMV) G protein-coupled receptor (GCR) homolog. This open reading frame (M33) is most closely related to, and collinear with, human cytomegalovirus UL33, and homologs are also present in human herpesvirus 6 and 7 (U12 for both viruses). Conserved counterparts in the sequenced alpha- or gammaherpesviruses have not been identified to date, suggesting that these genes encode proteins which are important for the biological characteristics of betaherpesviruses. We have detected transcripts for both UL33 and M33 as early as 3 or 4 h postinfection, and these reappear at late times. In addition, we have identified N-terminal splicing for both the UL33 and M33 RNA transcripts. For both open reading frames, splicing results in the introduction of amino acids which are highly conserved among known GCRs. To characterise the function of the M33 in the natural host, two independent MCMV recombinant viruses were prepared, each of which possesses an M33 open reading frame which has been disrupted with the beta-galactosidase gene. While the recombinant M33 null viruses showed no phenotypic differences in replication from wild-type MCMV in primary mouse embryo fibroblasts in vitro, they showed severely restricted growth in the salivary glands of infected mice. These data suggest that M33 plays an important role in vivo, in particular in the dissemination to or replication in the salivary gland, and provide the first evidence for the function of a viral GCR homolog in vivo.

Chinese hamster ovary cells produce sufficient recombinant insulin-like growth factor I to support growth in serum-free medium - Serum-free growth of IGF-I producing CHO cells

Insulin-like growth factor I has similar mitogenic effects to insulin, a growth factor required by most cells in culture, and it can replace insulin in serum-free formulations for some cells. Chinese Hamster Ovary cells grow well in serum-free medium with insulin and transferrin as the only exogenous growth factors. An alternative approach to addition of exogenous growth factors to serum-free medium is transfection of host cells with growth factor-encoding genes, permitting autocrine growth. Taking this approach, we constructed an IGF-I heterologous gene driven by the cytomegalovirus promoter, introduced it into Chinese Hamster Ovary cells and examined the growth characteristics of Insulin-like growth factor I-expressing clonal cells in the absence of the exogenous factor. The transfected cells secreted up to 500 ng/10(6) cells/day of mature Insulin-like growth factor I into the conditioned medium and as a result they grew autonomously in serum-free medium containing transferrin as the only added growth factor. This growth-stimulating effect, observed under both small and large scale culture conditions, was maximal since no further improvement was observed in the presence of exogenous insulin.

Muscle-specific regulation of tropomyosin gene expression and myofibrillogenesis differs among muscle systems examined at metamorphosis of the gastropod Haliotis rufescens

The spatial and temporal association of muscle-specific tropomyosin gene expression, and myofibril assembly and degradation during metamorphosis is analyzed in the gastropod mollusc. Haliotis rufescens. Metamorphosis of tile planktonic larva to the benthic juvenile includes rearrangement and atrophy of specific larval muscles, and biogenesis of the new juvenile muscle system. The major muscle of the larva - the larval retractor muscle - reorganizes at metamorphosis, with two suites of cells having different fates. The ventral cells degenerate, while the dorsal cells become part of the developing juvenile mantle musculature. Prior to these changes in myofibrillar structure, tropomyosin mRNA prevalence declines until undetectable in the ventral cells, while increasing markedly in the dorsal cells. In the foot muscle and right shell muscle, tropomyosin mRNA levels remain relatively stable, even trough myofibril content increases. In a population of median mesoderm cells destined to form de novo the major muscle of the juvenile and adult (the columellar muscle), tropomyosin expression is initiated at 45 h after induction of metamorphosis. Myofibrillar filamentous actin is not detected in these cells until about 7 days later. Given that patterns of tropomyosin mRNA accumulation in relation to myofibril assembly and disassembly differ significantly among the four major muscle systems examined, we suggest that different regulatory mechanisms, probably operating at both transcriptional and post-transcriptional levels, control the biogenesis and atrophy of different larval and postlarval muscles at metamorphosis.

Myelin proteolipid protein expressed in COS-1 cells is targeted to actin-associated surfaces

The compact myelin sheath represents one of the largest expanses of membrane-membrane contact in the body and, in the central nervous system, requires the myelin proteolipid protein (PLP) for assembly, To determine whether the molecular properties of PLP promote membrane adhesion and direct its subcellular localization in the absence of oligodendrocyte-specific targeting mechanisms, PLP was expressed in COS-I fibroblasts, Immunofluorescence staining indicated that PUP was translated effectively, transited the rough endoplasmic reticulum and Golgi apparatus, was delivered to the cell surface, and was endocytosed, In the plasma membrane, the PLP distribution was patchy and only sporadically coincided with sites of membrane-membrane contact between PLP-expressing cells, PLP was not randomly distributed, however, but correlated closely with microfilament locations in leading edge membranes and microvilli, as demonstrated by phalloidin double labeling, Our results indicate that even in non-myelinating cells, PLP can be concentrated in membranes associated with movement and growth, and suggest possible roles for the actin cytoskeleton in PLP localization, As PLP, DM20, and the DM20-like M6 protein all associate with actin-enriched membranes, this may be a common feature of PLP/DM20 gene family members. (C) 1997 Wiley-Liss, Inc.

In vivo effects of chicken interferon-gamma during infection with Eimeria

Newly hatched chickens are highly susceptible to infection by opportunistic pathogens during the first 1 or 2 weeks of life, The use of cytokines as therapeutic agents has been studied in animal models as well as in immunosuppressed patients, This approach has become more feasible in livestock animals, in particular poultry, with the recent cloning of cytokine genes and the development of new technologies, such as live delivery vectors, We have recently cloned the gene for chicken interferon-gamma (Ch-IFN-gamma), Poly-HIS-tagged recombinant Ch-IFN-gamma was expressed in Escherichia coil, was purified by Ni chromatography, and was found to be stable at 4 degrees C and an ambient temperature for at least several months and Several weeks, respectively, Ch-IFN-gamma was capable of protecting chick fibroblasts from undergoing virus-mediated lysis, induced nitrite secretion from chicken macrophages in vitro, and enhanced MHC class II expression on macrophages, Administration of recombinant Ch-IFN-gamma to chickens resulted in enhanced weight gain over a 12-day period, Furthermore, the therapeutic potential of Ch-IFN-gamma was assessed using a coccidial challenge model, Birds were treated with Ch-IFN-gamma or a diluent control and then infected with Eimeria acervulina. Infected birds treated with Ch-IFN-gamma showed improved weight gain relative to noninfected birds, The ability of Ch-IFN-gamma to enhance weight gain in the face of coccidial infection makes it an excellent candidate as a therapeutic agent.

Enamel matrix derivative induces matrix synthesis by cultured human periodontal fibroblast cells

Background: Periodontal wound healing and regeneration require that new matrix be synthesized, creating an environment into which cells can migrate. One agent which has been described as promoting periodontal regeneration is an enamel matrix protein derivative (EMD). Since no specific growth factors have been identified in EMD preparations, it is postulated that EMD acts as a matrix enhancement factor. This study was designed to investigate the effect of EMD in vitro on matrix synthesis by cultured periodontal fibroblasts. Methods: The matrix response of the cells was evaluated by determination of the total proteoglycan synthesis, glycosaminoglycan profile, and hyaluronan synthesis by the uptake of radiolabeled precursors. The response of the individual proteoglycans, versican, decorin, and biglycan were examined at the mRNA level by Northern blot analysis. Hyaluronan synthesis was probed by identifying the isotypes of hyaluronan synthase (HAS) expressed in periodontal fibroblasts as HAS-2 and HAS-3 and the effect of EMD on the levels of mRNA for each enzyme was monitored by reverse transcription polymerase chain reaction (RTPCR). Comparisons were made between gingival fibroblast (GF) cells and periodontal ligament (PDLF) cells. Results: EMD was found to significantly affect the synthesis of the mRNAs for the matrix proteoglycans versican, biglycan, and decorin, producing a response similar to, but potentially greater than, mitogenic cytokines. EMD also stimulated hyaluronan synthesis in both GF and PDLF cells. Although mRNA for HAS-2 was elevated in GF after exposure to EMD, the PDLF did not show a similar response. Therefore, the point at which the stimulation of hyaluronan becomes effective may not be at the level of stimulation of the mRNA for hyaluronan synthase, but, rather, at a later point in the pathway of regulation of hyaluronan synthesis. In all cases, GF cells appeared to be more responsive to EMD than PDLF cells in vitro. Conclusions: EMD has the potential to significantly modulate matrix synthesis in a manner consistent with early regenerative events.

Growth factors and cytokines modulate gene expression of cell-surface proteoglycans in human periodontal ligament cells

Cell-surface proteoglycans have been known to be involved in many functions including interactions with components of the extracellular microenvironment, and act as co-receptors which bind and modify the action of various growth factors and cytokines. The purpose of this study was to determine the regulation by growth factors and cytokines on cell-surface proteoglycan gene expression in cultured human periodontal ligament (PDL) cells. Subconfluent, quiescent PDL cells were treated with various concentrations of serum, bFGF, PDGF-BB, TGF-beta1, IL-1 beta, and IFN-gamma. RT-PCR technique was used, complemented with Northern blot for syndecan-1, to examine the effects of these agents on the mRNA expression of five cell-surface proteoglycans (syndecan-1, syndecan-2, syndecan-4, glypican and betaglycan). Syndecan-1 mRNA levels increased in response to serum, bFGF and PDGF-BB, but decreased in response to TGF-beta1, IL-1 beta and IFN-gamma. In contrast, syndecan-2 mRNA levels were upregulated by TCF-beta1 and IL-1 beta stimulation, but remained unchanged with the other agents. Betaglycan gene expression decreased in response to serum, but was upregulated by TCF-beta1 and unchanged by the other stimulants. Additionally, syndecan-4 and glypican were not significantly altered in response to the regulator molecules studied, with the exception that glypican is decreased in response to IFN-gamma. These data demonstrate that the gene expression of the five cell-surface proteoglycans studied is differentially regulated in PDL cells lending support to the nation of distinct functions for these cell-surface proteoglycans. (C) 2001 Wiley-Liss, inc.

Matrix metalloproteinases and their inhibitors in oral lichen planus

Background: Oral lichen planus (OLP) is characterized by a subepithelial lymphocytic infiltrate, basement membrane (BM) disruption, intra-epithelial T-cell migration and apoptosis of basal keratinocytes. BM damage and T-cell migration in OLP may be mediated by matrix metalloproteinases (MMPs). Methods: We examined the distribution, activation and cellular sources of MMPs and their inhibitors (TIMPs) in OLP using immunohistochemistry, ELISA, RT-PCR and zymography. Results: MMP-2 and -3 were present in the epithelium while MMP-9 was associated with the inflammatory infiltrate. MMP-9 and TIMP-1 secretion by OLP lesional T cells was greater than OLP patient (p

HPP1: A transmembrane protein-encoding gene commonly methylated in colorectal polyps and cancers

Adenomas are the precursors of most colorectal cancers. Hyperplastic polyps have been linked to the subset of colorectal cancers showing DNA microsatellite instability, but little is known of their underlying genetic etiology. Using a strategy that isolates differentially methylated sequences from hyperplastic polyps and normal mucosa, we identified a 370-bp sequence containing the 5' untranslated region and the first exon of a gene that we have called HPP1. Rapid amplification of cDNA ends was used to isolate HPP1 from normal mucose. Using reverse transcription-PCR, HPP1 was expressed in 28 of 30 (93%) normal colonic samples but in only seven of 30 (23%) colorectal cancers (P < 0.001). The 5' region of HPP1 included a CpG island containing 49 CpG sites, of which 96% were found to be methylated by bisulfite sequencing of DNA from colonic tumor samples. By COBRA analysis, methylation was detected in six of nine (66%) adenomas, 17 of 27 (63%) hyperplastic polyps, and 46 of 55 (84%) colorectal cancers. There was an inverse relationship between methylation level and mRNA expression in cancers (r = -0.67; P < 0.001), and 5-aza-2-deoxycytidine treatment restored HPP1 expression in two colorectal cancer cell lines. In situ hybridization of HPP1 indicated that expression occurs in epithelial and stromal elements in normal mucosa but is silenced in both cell types in early colonic neoplasia. HPP1 is predicted to encode a transmembrane protein containing follistatin and epidermal growth factor-like domains. Silencing of HPP1 by methylation may increase the probability of neoplastic transformation.

Visualising the activity of the cystine-glutamate antiporter in glial cells using antibodies to aminoadipic acid, a selectively transported substrate

The cystine-glutamate antiporter is a transport system that facilitates the uptake of cystine, concomitant with the release of glutamate. The cystine accumulated by this transporter is generally considered for use in the formation of the cysteine-containing antioxidant glutathione, which is abundant in many glial cells. This study used the simple strategy of generating an antibody to aminoadipic acid, a selective substrate for the cystine-glutamate antiporter. Stereospecific accumulation of aminoadipic acid into specific cell types in rat brain slice preparations was detected immunocytochemically. Strong accumulation was detected in astroglial cells in all brain regions studied including those in white matter tracts. Strong accumulation into radial glial cells, including the retinal Muller cells and the Bergmann glial cells was also observed. Glial accumulation was observed not only in cells within the blood brain barrier, but also outside such; anterior pituitary folliculostellate cell and intermediate lobe pituitary glial cells exhibited strong accumulation of aminoadipic acid. Interestingly, some glial cells such as the posterior pituitary glial cells (pituicytes) exhibited very little if any accumulation of aminoadipic acid. Within the brain labelling was not uniform. Particularly strong labelling was noted in some regions, such as the glial cells surrounding the CA1 pyramidal cells. By contrast, neurons never exhibited uptake of aminoadipic acid. Because cystine uptake is associated with glutamate release, it is suggested that this antiporter might contribute to release of glutamate from glial cells under some pathophysiological conditions. (C) 2001 Wiley-Liss, Inc.

Physiological roles and regulation of mammalian sulfate transporters

All cells require inorganic sulfate for normal function. Sulfate is among the most important macronutrients; in cells and is the fourth most abundant anion in human plasma (300 muM). Sulfate is the major sulfur source in many organisms, and because it is a hydrophilic anion that cannot passively cross the lipid bilayer of cell membranes, all cells require a mechanism for sulfate influx and efflux to ensure an optimal supply of sulfate in the body. The class of proteins involved in moving sulfate into or out of cells is called sulfate transporters. To date, numerous sulfate transporters have been identified in tissues and cells from many origins. These include the renal sulfate transporters NaSi-1 and sat-1, the ubiquitously expressed diastrophic dysplasia sulfate transporter DTDST, the intestinal sulfate transporter DRA that is linked to congenital chloride diarrhea, and the erythrocyte anion exchanger AE1. These transporters have only been isolated in the last 10-15 years, and their physiological roles and contributions to body sulfate homeostasis are just now beginning to be determined. This review focuses on the structural and functional properties of mammalian sulfate transporters and highlights some of regulatory mechanisms that control their expression in vivo, under normal physiological and pathophysiological states.

The expression of plasminogen activator system in a rat model of periodontal wound healing

Background: The plasminogen activator system has been proposed to play a role in proteolytic degradation of extracellular matrices in tissue remodeling, including wound healing. The aim of this study was to elucidate the presence of components of the plasminogen activator system during different stages of periodontal wound healing. Methods: Periodontal wounds were created around the molars of adult rats and healing was followed for 28 days. Immunohistochemical analyses of the healing tissues and an analysis of the periodontal wound healing fluid by ELISA were carried out for the detection of tissue-type plasminogen activator (t-PA), urokinase-type plasminogen activator (u-PA), and 2 plasminogen activator inhibitors (PAI-1 and PAI-2). Results: During the early stages (days 1 to 3) of periodontal wound healing, PAI-1 and PAI-2 were found to be closely associated with the deposition of a fibrin clot in the gingival sulcus. These components were strongly associated with the infiltrating inflammatory cells around the fibrin clot. During days 3 to 7, u-PA, PAI-1, and PAI-2 were associated with cells (particularly monocytes/macrophages, fibroblasts, and endothelial cells) in the newly formed granulation tissue. During days 7 to 14, a new attachment apparatus was formed during which PAI-1, PAI-2, and u-PA were localized in both periodontal ligament fibroblasts (PDL) and epithelial cells at sites where these cells were attaching to the root surface. In the periodontal wound healing fluid, the concentration for t-PA increased and peaked during the first week. PAI-2 had a similar expression to t-PA, but at a lower level over the entire wound-healing period. Conclusions: These findings indicate that the plasminogen activator system is involved in the entire process of periodontal wound healing, in particular with the formation of fibrin matrix on the root surface and its replacement by granulation tissue, as well as the subsequent formation of the attachment of soft tissue to the root surface during the later stages of wound repair.