Beta-positive Cells Research Articles

Airway pathology in severe asthma is related to airflow obstruction but not symptom control.

Patients with asthma present structural and inflammatory alterations that are believed to play a role in disease severity. However, airway remodeling and inflammation have not been extensively investigated in relation to both symptom control and airflow obstruction in severe asthmatics. We aimed to investigate several inflammatory and structural pathological features in bronchial biopsies of severe asthmatics that could be related to symptom control and airflow obstruction after standardized treatment. Fifty severe asthmatics received prednisone 40 mg/d for 2 weeks and maintenance therapy with budesonide/formoterol 400/12 μg twice daily + budesonide/formoterol 200/6 μg as needed for 12 weeks. Endobronchial biopsies were performed at the end of 12 weeks. We performed extensive immunopathological analyses of airway tissue inflammation and remodeling features in patients stratified by asthma symptom control and by airflow obstruction. Airway tissue inflammation and remodeling were not associated with symptom control. Asthmatics with persistent airflow obstruction had greater airway smooth muscle (Asm) area with decreased periostin and transforming growth factor beta-positive cells within Asm bundles, in addition to lower numbers of chymase-positive mast cells in the submucosa compared to patients with nonpersistent obstruction. Symptom control in severe asthmatics was not associated with airway tissue inflammation and remodeling, although persistent airflow obstruction in these patients was associated with bronchial inflammation and airway structural changes.

Platelet-Derived Growth Factor Receptor-Positive Pericytic Cells of White Adipose Tissue from Critical Limb Ischemia Patients Display Mesenchymal Stem Cell-Like Properties.

BackgroundThe pericytes in the blood vessel wall have recently been identified to be important in regulating vascular formation, stabilization, remodeling, and function. We isolated and identified pericyte-like platelet-derived growth factor receptor beta-positive (PDGFRβ+) cells from the stromal vascular fraction (SVF) of adipose tissue from critical limb ischemia (CLI) patients and investigated their potential as a reliable source of stem cells for cell-based therapy.MethodsDe-identified subcutaneous fat tissues were harvested after amputation in CLI patients. Freshly isolated SVF cells and culture-expanded adipose-derived stem cells (ADSCs) were quantified using flow cytometry. A matrigel tube formation assay and multi-lineage differentiation were performed to assess pericytic and mesenchymal stem cell (MSC)-like characteristics of PDGFRβ+ ADSCs.ResultsPDGFRβ+ cells were located in the pericytic area of various sizes of blood vessels and coexpressed mesenchymal stem cell markers. PDGFRβ+ cells in freshly isolated SVF cells expressed a higher level of stem cell markers (CD34 and CXCR4) and mesenchymal markers (CD13, CD44, CD54, and CD90) than PDGFRβ– cells. In vitro expansion of PDGFRβ+ cells resulted in enrichment of the perivascular mesenchymal stem-like (PDGFRβ+/CD90+/CD45–/CD31–) cell fractions. The Matrigel tube formation assay revealed that PDGFRβ+ cells were located in the peritubular area.ConclusionsPDGFRβ+ ADSCs cells demonstrated a good multilineage differentiation potential. Pericyte-like PDGFRβ+ cells from the SVF of adipose tissue from CLI patients had MSC-like characteristics and could be amplified by in vitro culture with preservation of their cell characteristics. We believe PDGFRβ+ cells in the SVF of adipose tissue can be used as a reliable source of stem cells even in CLI patients.

Normal and cancer stem cells of the human female reproductive system

The female reproductive system (FRS) has a great capacity for regeneration. The existence of somatic stem cells (SSC) that are likely to reside in distinct tissue compartments of the FRS is anticipated. Normal SSC are capable of regenerating themselves, produce a progeny of cells that differentiate and maintain tissue architecture and functional characteristics, and respond to homeostatic controls. Among those SSC of the FRS that have been identified are: a) undifferentiated cells capable of differentiating into thecal cells and synthesizing hormones upon transplantation, b) ovarian surface epithelium stem cells, mitotically responsive to ovulation, c) uterine endometrial and myometrial cells, as clonogenic epithelial and stromal cells, and d) epithelial and mesenchymal cells with self-renewal capacity and multipotential from cervical tissues. Importantly, these cells are believed to significantly contribute to the development of different pathologies and tumors of the FRS.It is now widely accepted that cancer stem cells (CSC) are at the origin of many tumors. They are capable of regenerating themselves, produce a progeny that will differentiate aberrantly and do not respond adequately to homeostatic controls. Several cell surface antigens such as CD44, CD117, CD133 and MYD88 have been used to isolate ovarian cancer stem cells. Clonogenic epithelial and stromal endometrial and myometrial cells have been found in normal and cancer tissues, as side population, label-retaining cells, and CD146/PDGF-R beta-positive cells with stem-like features. In summary, here we describe a number of studies supporting the existence of somatic stem cells in the normal tissues and cancer stem cells in tumors of the human female reproductive system.

Protective effect of quercetin on liver damage induced by chronic toluene exposure in rats

The aim of this study was to evaluate the possible protective effects of quercetin (QE) on liver injury caused by chronic toluene exposure in rats. The rats were randomly allotted into one of three experimental groups: control, toluene treated and toluene treated with QE; each group contained 10 animals. The control group received 1 ml physiologic serum, and toluene treatment was performed by inhalation of 3000 ppm toluene, for 8 h/day and 6 days/week for 12 weeks. The rats in the QE-treated groups were given QE (15 mg/kg, intraperitoneal) once a day for 12 weeks starting just after toluene exposure. Tissue samples were obtained for histopathological investigation. To date, no histopathological changes in the liver of rats after chronic toluene exposure by QE treatment have been reported. Light microscopic evaluation of liver tissue samples of toluene-exposed rats revealed enlarged sinusoids filled with blood. In addition, some of the hepatocytes showed loss of cytoplasm, and some had a hyperchromatic nucleus. QE treatment attenuated alterations in liver histology. The alpha smooth muscle actin, transforming growth factor beta-positive cells and the activity of terminal deoxynucleotidyl transferase dUTP nick end labeling in the toluene-treated group were observed to be reduced with QE treatment. The data indicate that QE attenuates toluene-induced liver injury, but further investigation is still absolutely necessary.

14-3-3 beta in the healthy and diseased male reproductive system

Dysfunction of cellular processes in the testes can lead to infertility, tumourigenesis or other testicular disorders. 14-3-3 proteins are known to play pivotal roles in cellular communication, signal transduction, intracellular trafficking, cell-cycle control, transcription and cytoskeletal structure and have been implicated in several diseases including tumourigenesis. Here we investigated the expression of the 14-3-3 beta isoform in healthy testicular tissues of humans, rats and mice as well as in tissues of Sertoli-cell-only (SCO) syndrome, intratubular germ cell neoplasia (IGCN) and classical seminoma. Samples of healthy and diseased testes from humans, rats and mice were analysed by immunohistochemistry. For PCR, human testis cell lysates were used. Immunoblot analyses of rats and humans healthy testes were performed. Duolink proximity ligation assay (PLA) and co-immunoprecipitation (Co-IP) were carried out to investigate interactions between 14-3-3 beta and vimentin in human, rat and mouse testes. In healthy testes and SCO syndrome, strong 14-3-3 beta-positive cells could be identified as Sertoli cells. Furthermore, 14-3-3 beta proteins were detected in cells of the peritubular stroma. In samples of IGCN and classical seminoma, the malignant transformed cells stained positive for 14-3-3 beta antigen. Immunoblot analyses revealed the presence of 14-3-3 beta in healthy testicular tissues. 14-3-3 beta mRNA transcripts were detected in cell lysates of healthy human testes. Interaction of 14-3-3 beta with the intermediate filament vimentin was revealed by Duolink PLA and Co-IP. Co-IP experiments identified tubulin as another 14-3-3 beta binding partner. Our data suggest that 14-3-3 beta expression is essential for normal spermatogenesis by interacting with vimentin in Sertoli cells. Additionally, 14-3-3 beta expression in malignant transformed cells in IGCN and classical seminoma may lead to tumourigenesis and cell survival.

FoxL2 and Smad3 Coordinately Regulate Follistatin Gene Transcription

Follistatin is a transcriptional target and a modulator of activin action. Through an autocrine/paracrine loop, activin controls follistatin levels and thus regulates its own bioavailability. In gonadotropic alphaT3-1 cells, activin induces follistatin transcription primarily through the action of Smad3 at an intronic Smad-binding element (SBE1). Using a proteomics approach, we searched for endogenous alphaT3-1 proteins that participate in SBE1-mediated transcription. We identified FoxL2, a member of the forkhead family, as a candidate modulator of SBE1 function. Mutations of FoxL2 are associated with the blepharophimosis/ptosis/epicanthus inversus syndrome characterized with craniofacial defects and premature ovarian failure. FoxL2 localizes to alpha-glycoprotein subunit- and follicle-stimulating hormone beta-positive cells of the adult mouse pituitary and is present in alphaT3-1 and LbetaT2 cells, but its pituitary actions remain largely unknown. We have determined that FoxL2 binds to a forkhead-binding element (FKHB) located just downstream of the SBE1 site of the follistatin gene and functions as a Smad3 partner to drive SBE1-mediated transcription in alphaT3-1 cells treated with activin. Chromatin immunoprecipitation assays confirm that endogenous FoxL2 and Smad3 are recruited to the intronic enhancer of the follistatin gene where the SBE1 and FKHB sites are located. Exogenous FoxL2 enhances SBE1-mediated transcription, and short hairpin RNA-mediated knockdown of endogenous FoxL2 protein compromises this effect in alphaT3-1 cells. FoxL2 directly associates with Smad3 but not Smad2 or Smad4. This association between Smad3 and FoxL2 is mediated by the MH2 domain of Smad3 and is dependent on an intact forkhead domain in FoxL2. Altogether, these observations highlight a novel role for FoxL2 and suggest that it may function as a transcriptional regulator and a coordinator of Smad3 targets.

Molecular Cloning of Paired Related Homeobox 2 (Prx2) as a Novel Pituitary Transcription Factor

This study aimed to identify protein(s) that bind(s) to the highly AT-rich sequence of porcine Fshb promoter region -852/-746 (named Fd2) by the Yeast One-Hybrid Cloning System and finally a paired related homeodomain transcription factor, Prx2, known as a key factor for skeletogenesis was cloned. RT-PCR analysis of fetal and postnatal porcine pituitaries demonstrated that Prx2 starts to be expressed at around fetal days 40-50 just before the beginning of Lhb-expression and that the level of Prx2 increases after birth. Immunohistochemical analysis of the prepubertal porcine pituitary revealed that some Prx2-positive cells overlap some Lh beta-positive cells. Transient transfection assay using non-pituitary CHO cells and pituitary tumor-derived LbetaT2 cells revealed that Prx2 plays a cell-type dependent role in modulation of the Fshb promoter, showing stimulation in CHO cells and repression in LbetaT2 cells via the regions of Fd2 and -596/-239. The binding ability of Prx2 to the regions of Fd2 and -596/-239 was confirmed by electrophoretic mobility shift assay. DNase I footprinting revealed that broad regions of Fd2 were bound by Prx2 and that -596/-239 contained seven Prx2-binding sites. The SELEX method using a random N15-mer oligonucleotide pool demonstrated that Prx2 monomer binds to a TAATT motif, which is present in Fd2 and -596/-239. However, the binding of Prx2 to TAATT with a single molecule and its inverted repeat with two molecules could not induce transcriptional activation, indicating that the Prx2-dependent transcriptional modulation demonstrated in cultured cells is not introduced by Prx2 alone. Thus, this study demonstrated for the first time that Prx2 is expressed in the pituitary gland and at least in a part of gonadotropes in which Prx2 may play a role in repression of the Fshb gene.

Binding and uptake of Aβ1‐42 by primary human astrocytes in vitro

Clearance of the amyloid-beta peptide (A beta) as a remedy for Alzheimer's disease (AD) is a major target in on-going clinical trials. In vitro studies confirmed that A beta is taken up by rodent astrocytes, but knowledge on human astrocyte-mediated A beta clearance is sparse. Therefore, by means of flow cytometry and confocal laser scanning microscopy (CLSM), we evaluated the binding and internalization of A beta1-42 by primary human fetal astrocytes and adult astrocytes, isolated from nondemented subjects (n = 8) and AD subjects (n = 6). Furthermore, we analyzed whether alpha1-antichymotrypsin (ACT), which is found in amyloid plaques and can influence A beta fibrillogenesis, affects the A beta uptake by human astrocytes. Upon over night exposure of astrocytes to FAM-labeled A beta1-42 (10 microM) preparations, (80.7 +/- 17.7)% fetal and (52.9 +/- 20.9)% adult A beta-positive astrocytes (P = 0.018) were observed. No significant difference was found in A beta1-42 uptake between AD and non-AD astrocytes, and no influence of ApoE genotype on A beta1-42 uptake was observed in any group. There was no difference in the percentage of A beta-positive cells upon exposure to A beta1-42 (10 microM) combined with ACT (1,000:1, 100:1, and 10:1 molar ratio), versus A beta1-42 alone. CLSM revealed binding of A beta1-42 to the cellular surfaces and cellular internalization of smaller A beta1-42 fragments. Under these conditions, there was no increase in cellular release of the proinflammatory chemokine monocyte-chemoattractant protein 1, as compared with nontreated control astrocytes. Thus, primary human astrocytes derived from different sources can bind and internalize A beta1-42, and fetal astrocytes were more efficient in A beta1-42 uptake than adult astrocytes.

The effect of tamoxifen on bladder functions and histology, and the role of estrogen receptor β in a rat chemical cystitis model

The purpose of this study was to investigate the effect of tamoxifen citrate on bladder functions and histology, and also to investigate the role of estrogen receptor beta (ER beta) in a rat chemical cystitis model. The study included 37 female Sprague-Dawley rats. Chemical cystitis was induced by intravesical instillation of hydrochloric acid in 32 rats, and the treatment group (n = 15) received daily 0.4 mg/kg of tamoxifen citrate with orogastric tube, and the control group (n = 17) received no treatment. The sham group consisted of five rats having no acid instillation and no treatment. Cystometric studies were performed in all rats at the beginning and end of the experiment. The rats were euthanized at 2 months. The bladders were removed and examined histologically for mast cells, inflammatory cells, and ER beta. The mean maximal bladder volume increased by 73.6% +/- 25.2 in the treatment group and decreased by 7.2% +/- 10.8 in the control group, revealing a significant difference (P = 0.007). The mean bladder compliance increased by 81.2% +/- 25.2 in the treatment group and decreased by 4.8% +/- 12.7 in the control group, revealing a significant difference between the two groups (P = 0.005). ER beta positive cells were significantly lower in the bladders with chronic cystitis than in the sham group (P = 0.038). Tamoxifen citrate may be an alternative choice, as easy, to other treatment options in the treatment of chronic inflammatory condition to improve deteriorated bladder function. In addition, ER beta may have a role on chronic bladder inflammation in a rat chemical cystitis model.

In vivo allograft rejection in a bony fish Dicentrarchus labrax (L.): characterisation of effector lymphocytes

Lymphoid cell subpopulations involved in allograft rejection in the teleost Dicentrarchus labrax were characterised at the ultrastructural level and quantified by using monoclonal antibodies against T- and B-lymphocytes. T-cells positive for T-cell receptor beta-chain (TcR beta) were detected by reverse transcription/polymerase chain reaction (RT-PCR) and in situ hybridisation by using RNA probes for TcR beta. Flow cytometry detected a similar percentage of T- and B-lymphocytes (around 17%) in the leucocyte-enriched fraction from allografts. Two different types of T-lymphocytes (DLT 15-immunoreactive) infiltrating the allografts were identified by cytomorphology: small cells with high nuclear/cytoplasmic ratio and cells with a higher cytoplasmic content. RT-PCR revealed a single band (513 bp) corresponding to the TcR beta. In situ hybridisation showed that TcR beta-positive cells in the grafted muscle fibres were less numerous compared with DLT 15-positive cells, as evidenced in parallel sections, suggesting that cytotoxic cells might express different TcR phenotypes. DLIg 3-immunoreactive Ig-producing lymphocytes had: 1) a high nuclear/cytoplasmic ratio or 2) a larger size similar to that of pre-plasma cells (plasma cells lacked any membrane labelling).

Age-related changes in estrogen receptor beta in rat hypothalamus: a quantitative analysis.

Although the estrogen receptor beta (ER beta) is a major target for actions of estrogen on the brain, little is known about its neural expression during aging, when levels and the mode of estrogen release undergo substantial changes. Therefore, in the present study we examined effects of aging and estrogen treatment on the number of cells expressing the ER beta in female rats. Two regions relevant to reproductive function were analyzed: the anteroventral periventricular nucleus (AVPV) and the principal nucleus of the bed nucleus of the stria terminalis (pBST). The numbers of ER beta-expressing cells were quantified using an unbiased stereological approach. Female rats were used at three ages [young (3-4 months), middle-aged (10-12 months), and old (24-26 months)], with or without estrogen replacement. Because the estrogen milieu impacts the function of neurotransmitter receptors such as the N-methyl-D-aspartate receptor in the brain, we also investigated the colocalization of ER beta and the obligatory N-methyl-D-aspartate receptor subunit, NR1. We observed a significant age-related decrease in ER beta cell number in the AVPV, but not the pBST. No significant effect of estrogen on ER beta cell number was detected in either brain region at any age. Approximately 10% and 3% of cells expressing ER beta also coexpressed NR1 in AVPV and pBST, respectively, and this did not differ with age or treatment. Taken together, our results demonstrate 1) there are age-related changes in ER beta cell number that are region specific; 2) this expression is not altered by estrogen replacement; and 3) a subset of ER beta-positive cells coexpresses NR1.

Ovulation-induced DNA damage in ovarian surface epithelial cells of ewes: prospective regulatory mechanisms of repair/survival and apoptosis.

Oxidative base (8-oxoguanine) damage, DNA fragmentation, and apoptosis occurred among ovarian surface epithelial cells within the formative site of ovulation in sheep. The incidence of 8-oxoguanine adducts in surviving antiapoptotic Bcl-2/base excision repair polymerase beta-positive cells at the margins of ruptured follicles (which avoid the focal point of the ovulatory assault) was intermediate between apoptotic and outlying healthy epithelium. Cells containing perturbations to DNA expressed the tumor suppressor p53. Localized reactions of DNA injury and programmed cellular death were averted by ovulation blockade with indomethacin. Progesterone enhanced the biosynthesis of polymerase beta in ovarian surface epithelial cells exposed in vitro to a sublethal concentration of H(2)O(2). Ovulation is a putative etiological factor in common epithelial ovarian cancer. A genetically altered progenitor cell, with unrepaired DNA, but not committed to death, could give rise to a transformed phenotype that is hence propagated upon healing of the ovulatory wound; it appears that this incongruity is normally reconciled by up-regulation of the base excision repair pathway during the ensuing luteal phase.

Expression and localization of leptin receptor in the normal rat pituitary gland.

Leptin receptor (leptin-R) is a polypeptide consisting of a single transmembrane-spanning component. Recent studies performed by reverse transcriptase polymerase chain reaction (RT-PCR) have shown the production of leptin-R in various tissues including the pituitary, hypothalamus and reproductive organs. The localization of leptin-R protein in the pituitary gland, however, has not been extensively studied. This study deals with the expression of leptin-R in the normal rat pituitary gland, which was disclosed primarily in the plasma membrane fraction by immunoblotting and immunohistochemical staining methods. Double immunohistochemical staining revealed that the colocalization of leptin-R and anterior pituitary hormone expression was seen mainly in growth hormone (GH)-secreting cells (97.4 +/- 1.3%; GH-positive cells/leptin-R-positive cells), but in less than 1% of prolactin (PRL)-, adrenocorticotropic hormone (ACTH)-, thyroid-stimulating hormone-beta (TSH beta)- and follicle-stimulating hormone-beta (FSH beta)/luteinizing hormone-beta (LH beta)-positive cells. In contrast, leptin was localized most frequently in FSH beta/LH beta- and less frequently in TSH beta-positive cells. The above findings suggest that, in the rat anterior pituitary gland, there are paracrine relationships between leptin-producing cells and cells with leptin-R, which may regulate the function of GH cells.

Cytokines and cell cycle regulation in the fibrous progression of crescent formation in antiglomerular basement membrane nephritis of WKY rats.

Cytokines may regulate cell proliferation by cell-cycle-regulatory proteins, in which cyclin-dependent kinase inhibitors (CDKI) inhibit cell proliferation. We investigated whether CDKI p21 or p27, both of which are potentially regulated by transforming growth factor (TGF)-beta, a key cytokine in fibrogenesis, are involved together with TGF-beta and/or platelet-derived growth factor (PDGF) in the fibrous progression of glomerular crescent formation and examined the sequential change in the cell type and the cellular background of myofibroblasts in crescent formation. Crescentic glomerulonephritis (GN) was induced by i.v. injection of rabbit antirat glomerular basement membrane antiserum in WKY rats. Animals were killed 1, 2, 3 and 4 weeks after the induction of GN, and their kidneys were processed for immunohistochemical examination. After 1 week more than 85% of glomeruli showed cellular crescents, which became fibrocellular with decreased cellularity by 4 weeks. ED 1-positive macrophages were components of crescent cells in about 44% at 1-2 weeks, and this proportion declined markedly afterwards. Alpha smooth muscle actin (alpha SMA, a marker for myofibroblasts)-positive cells were found in Bowman's epithelial cells (BEP) and in some crescent cells at 1 week, becoming major components of crescent cells by 4 weeks (about 40%). It was 2 weeks before invasion of alpha SMA-positive interstitial cells into glomeruli was evident. PDGF-B and PDGF receptor beta-positive cells, indicating possible targets for PDGF, were found in BEP adjoining crescent formation almost exclusively from 1 to 2 weeks. By contrast, both TGF-beta receptor types I- and II-positive cells, indicating possible effectors for TGF-beta, were found in BEP and crescent formation, and the percentage of these in the crescent formation did not change until 4 weeks (about 32%). Cells with positive immunostaining for proliferating cell nuclear antigen and cyclin A, markers for cell proliferation, in the crescent formation peaked in number and proportion at 1-2 weeks, then decreased. In contrast, cells with positive immunostaining for p21 and p27, CDKI, were sparse at 1 week, and then increased markedly in number and in proportion, peaking at 3 (39.6%) or 2-3 weeks (about 25-30%), respectively. The present study demonstrates that restrained expression or a transient increase in p21 and p27 may be associated with proliferation or with inhibited proliferation of crescent cells, most of which are macrophages and myofibroblasts. The action, of PDGF and TGF-beta may contribute to the recruitment of myofibroblasts into the crescent. The action of TGF-beta on crescent cells might be linked to the expression of p21 and/or p27.

Differential regulation and action of estrogen receptors alpha and beta in GH3 cells.

The pituitary lactotroph, a well established target for estrogens, expresses estrogen receptor-alpha (ER alpha) and -beta (ER beta). A truncated isoform of ER alpha, named TERP, is expressed in the pituitary, but not in the uterus. In this study we used the somatolactotroph cell line, GH3 cells, to examine 1) the expression of ER alpha, TERP, or ER beta and their regulation by estradiol; 2) the presence of receptor proteins; and 3) the effects of overexpressing ER beta or TERP on estrogen induction of the PRL gene and activation of the estrogen response element (ERE). Incubation of GH3 cells with estradiol (0.1-10 nM) produced dose-dependent increases in messenger RNA levels of ER beta and TERP, but not ER alpha, as determined by quantitative RT-PCR. Cell incubation with 1 nM estradiol resulted in a time-dependent biphasic increase in TERP and a delayed rise in ER beta, suggesting activation by both direct and indirect mechanisms. A polyclonal ER beta antibody directed against an N-terminal synthetic peptide was generated. This antibody detected ER beta-positive cells in ovarian granulosa cells and in many cells throughout the pituitary; its specificity was demonstrated by preabsorption with the synthetic peptide. The antibody detected a 58- to 60-kDa protein by Western blotting of ovarian, pituitary, and GH3 cell extracts. Cotransfection of ER beta and reporter genes (PRL promoter/luciferase or ERE/luciferase) into GH3 cells resulted in a dose-dependent increase in estrogen-induced PRL gene expression, with a lesser activation of the ERE. A 20-kDa TERP protein was undetectable in untreated GH3 cells and was weakly induced by estradiol. Overexpression of TERP had no effect on estrogen induction of either PRL or ERE. We conclude that 1) both ER beta and TERP messenger RNAs in GH3 cells are increased by estradiol in a dose- and time-dependent manner, whereas ER alpha is not altered; 2) a 58-kDa ER beta protein is expressed in both the pituitary and GH3 cells; and 3) overexpression of ER beta increases estrogen-induced PRL gene expression.

Accumulation of Alzheimer amyloid-beta peptide in cultured myocytes is enhanced by serum and reduced by cerebrospinal fluid.

Smooth muscle cells cultured from leptomeningeal vessels from old dogs with amyloid-angiopathy accumulate intracellular deposits that are immunoreactive for amyloid-beta peptide (A beta). We used this cellular model in the present study to examine the influence of sera and cerebrospinal fluid on intracellular accumulation of A beta-immunoreactive deposits and on secretion of soluble A beta into culture media. We found that sera from old dogs significantly increased the percentage of A beta-positive smooth muscle cells in culture. The enhanced accumulation of A beta was associated with (a) lower secretion of A beta into media, (b) altered maturation of amyloid-beta-precursor protein (A betaPP) into A betaPP751-770 with faster electrophoretic mobility, (c) increased accumulation of C-terminal fragments of A betaPP (12-15 kD, 10kD and less), and (d) increased secretion of A betaPP into culture media. These findings suggest that age- or disease-related serum factors increase accumulation of A beta by affecting production and processing of A betaPP In contrast, cerebrospinal fluids reduced accumulation of A beta. Involvement of A beta-carrier proteins-apolipoprotein E and transthyretin-in accumulation of A beta is demonstrated. Accumulation of A beta in cultured smooth muscle cells-a model of beta-amyloidosis-may be regulated by factors that alter production and processing of A betaPP as well as the fate of soluble A beta in extracellular space.

Thymic nurse cells forming a dynamic microenvironment in spontaneous thymoma BUF/Mna rats.

Thymic nurse cells (TNCs) were studied using an animal model, BUF/Mna rats, which spontaneously develop benign thymomas of epithelial origin with age. The unusual increment and high availability of TNCs in this thymus enabled us to analyze TNCs directly either in tissue sections or on smears after enzymatic isolation. No structural or phenotypical abnormality in these TNCs was detected as assessed by electron microscopy and immunohistochemistry. Typical TNCs were widely distributed in the cortical areas but not in the medullary areas. They showed characteristic euchromatic bright nuclei and enclosed intra-TNC cells with an investment of relatively light cytoplasm with abundant small vesicles and rough-endoplasmic reticulum. The intra-TNC cells were mostly double positive (CD4+CD8+) cortical thymocytes, though macrophage populations could also be distinguished by their content of membrane-bounded phagosomes, multivesicular bodies and other inclusion bodies, and by their lack of cytoskeletal keratin filaments. High voltage electron microscopy revealed that intra-TNC cells were separated into several compartments by extremely thin internal veils of the TNC processes. The outer veils of the TNCs were continuous with occasional small gaps through which intra-TNC cells could migrate in and out of the compartments. Immunohistochemical analyses revealed that the TNCs per se were positive for MHC class I and class II, keratin and thymulin, but lacked both lymphocyte and macrophage markers. Among all adhesion molecules tested, ICAM-1 was strongly expressed on almost all TNCs. A minority of TNCs also contained either LFA-1 alpha or LFA-1 beta positive cells. These results suggest that TNCs may form a rather dynamic microenvironment for T cell development where either nursing or clearance of thymocytes take place, depending on the cellular components of intra-TNC cells. Macrophage populations may also play crucial roles as the third component within TNCs.

Abrogation of the allelic exclusion in a T cell receptor beta chain gene transgenic mouse strain.

The expression of endogenous T cell receptor (TcR) beta chains in a TcR beta chain gene transgenic mouse (TGM) strain was examined. Unlike many other TGM strains reported, a considerable proportion of T cells from the thymus and spleen as well as organ cultured fetal thymus from our TGM express endogenous TCR beta chains on their surface. Compatible with this was the elucidation of VDJ rearrangement of endogenous beta chain genes by PCR. Three color flow cytometric analysis of thymus cell subpopulations revealed that the expression levels of both endogenous and transgenic TcR beta genes are regulated in a maturational stage specific manner. Splenic T cells contained a several fold higher percentage of endogenous TcR beta positive cells than thymus cells, suggesting a role of TcR on T cell peripherization. V beta 6 positive cells were deleted in the TGM carrying minor lymphocyte stimulating (Mls)-la antigen, indicating that the endogenous TcR beta is functional in terms of transmitting a signal for clonal deletion.

In situ hybridization analysis of anterior pituitary hormone gene expression during fetal mouse development.

We used 35S-labeled oligonucleotides and cRNAs (riboprobes) to detect the temporal order and spatial pattern of anterior pituitary hormone gene expression in (B6CBF1 x B6CBF1)F2 fetal mice from embryonic Day 9.5 (E9.5) to postnatal Day 1 (P1). Pro-opiomelanocortin (POMC) mRNA was expressed in the basal diencephalon on Day E10.5, in the ventromedial zone of the pars distalis on Day E12.5, and in the pars intermedia on Day E14.5. The common alpha-glycoprotein subunit (alpha-GSU) mRNA first appeared in the anterior wall of Rathke's pouch on Day E11.5 and extended to the pars tuberalis and ventromedial zone of the pars distalis on Day E12.5. Thyroid-stimulating hormone-beta (TSH beta) subunit mRNA was expressed initially in both the pas tuberalis and ventromedial pars distalis on Day E14.5, with an identical spatial distribution to alpha-GSU at the time. In contrast, luteinizing hormone-beta (LH beta) subunit and follicle-stimulating hormone beta (FSH beta) subunit mRNAs were detected initially only in the ventromedial pars distalis on Days E16.5 and E17.5, respectively, in an identical distribution to each other. POMC-, alpha-GSU-, TSH beta, LH beta-, and FSH beta-positive cells within the pars distalis all increased in number and autoradiographic signal with differing degrees of spatial expansion posteriorly, laterally, and dorsally up to Day P1. POMC expression was typically the most intense and extended circumferentially to include the entire lateral and dorsal surfaces of the pars distalis. The expression of both growth hormone (GH) and prolactin (PRL) started coincidentally on Day E15.5. However PRL cells localized in the ventromedial area similarly to POMC and the glycoprotein hormone subunits, whereas GH cells were found initially in a more lateral and central distribution within the lobes of the pars distalis. Somatotrophs increased dramatically in number and autoradiographic signal, extending throughout the pars distalis except for the most peripheral layer of cells on Day E17.5. Mammotrophs also increased in number but less abundantly than somatotrophs, and PRL expression remained more confined to central-medial and ventrolateral areas of the pars distalis up to Day P1. These data demonstrate distinctive patterns of expression for each of the major anterior pituitary hormone genes during development of the mouse pituitary gland and suggest that different groups of committed cells are the immediate precursors to the terminally differentiated hormone-secreting cell types.

Histological evaluation of interleukin-1 beta and collagen in gingival tissue from untreated adult periodontitis.

Interleukin-1 beta (IL-1 beta) may be related to the pathological processes associated with periodontitis, primarily due to its ability to induce collagenase, increase neutrophil chemotaxis, and stimulate bone resorption. This study was designed to histologically quantitate IL-1 beta positive cells from various histologic fields in untreated gingivitis/early periodontitis (G/EP) versus moderate/severe periodontitis (M/SP) gingival tissues, and associate these with collagen loss. Two gingival biopsies from 8 patients were collected, one from a G/EP site and one from a M/SP site. Mouse monoclonal antibodies in combination with an avidin-biotin-peroxidase system were used to stain for IL-1 beta, while the van Gieson method was used to stain for collagen in serial sections. Collagen loss in G/EP (35%) and M/SP (52%) fields was consistent with gingivitis and periodontitis, respectively. IL-1 beta positive cells in combined coronal/sulcular (Co/Su) and apical/sulcular (Ap/Su) fields (nearest the bacterial insult) were significantly more numerous compared to combined coronal/middle (Co/Mi) and apical/middle (Ap/Mi) fields (p < 0.05). While numbers and percentages of IL-1 beta positive cells were generally higher in M/SP biopsies, differences were not significant. Further, there was no correlation between the number of IL-1 beta positive cells and percent collagen loss. However, a significant correlation between IL-1 beta positive cells and corresponding gingival crevicular fluid IL-1 beta concentrations was noted (r = 0.65, p = 0.01). Through the use of immunohistochemistry, this study demonstrated that the presence of IL-1 beta + cells does not appear to have a direct association with collagen loss.