Growth factor assays for normal human hepatocytes and hepatoma cells

Summary An improved assay procedure of growth factors for normal human hepatocytes and hepatoma cells is reviewed. The combined use of improved cell isolation and dispersion procedures and a selective culture medium supports the response of human parenchymal cells homogenous to growth factors under serum-free culture conditions. Differentiated human hepatoma cells (HepG2), which synthesize and secrete the liver-specific plasma proteins and enzymes, provide a useful model for comparative study of growth factor requirements of hepatoma cells to normal hepatocytes. HepG2 cells grow in protein-free, defined medium at high cell density. This indicates that the hepatoma cells may secrete autocrine growth factors into the medium. HepG2 cells at low cell density permit detection of exogeneous growth factors that may be secreted by HepG2 cells at high cell density. HepG2 cells at high cell density are a useful assay for growth inhibitory activities for hepatoma cells.     

Preparation of quiescent human monocytes for studies of colony-stimulating factor responsiveness

Summary A method is described for isolating by adherence human peripheral blood monocytes and rendering them quiescent. Quiescence is achieved by depriving the cells of human serum and hematopoietic growth factors. These quiescent monocytes express very low levels of immediate early response gene mRNAs and are thus useful in studies of signal transduction with colony stimulating factors.     

Age-dependent changes of the normal human spine during adulthood.

The impact of aging on the morphology of the osseous spine is still debated. Clinical studies usually record combined aging effects, as well as age-related degenerative changes. The aim of this study was to determine the impact of (degeneration-independent) aging on the morphology of the osseous human spine during adulthood. Various osseous dimensions of human spinal landmarks at all major vertebral levels have been assessed in macroscopically normal Swiss skeletons (N = 71), with historically known sex and age at death, as well as in larger Central European skeletal samples (N = 277) with anthropologically determined individual age and sex. All measurements were correlated with individual age (or age group) by linear regression and analyzed separately for each sex. Only few osseous spinal dimensions, and only in men, correlate significantly with individual age. Generally, the significant dimensions show an increase in size during adulthood. Similar tendencies, but with significant alterations of spinal measurements in women as well, can be found in the larger samples with anthropologically determined sex and age group. Increase of certain spinal dimensions found in this study may be a reflection of an increase in the robustness of individuals with age. Because of the absence of a significant secular alteration of stature within the well-recorded sample, we exclude secular change in body dimensions as a major bias.     

The effects of endothelin-1 on degranulation, cytokine, and growth factor production by skin-derived mast cells

Endothelin-1 (ET-1), originally described as a vasoconstrictor, is now known to be involved in pathogenesis of various disorders including vascular, inflammatory, and fibrotic diseases. Recent studies suggest that mast cells are also involved in the same pathological conditions. In this study, we tested a hypothesis that ET-1 would affect mast cell functions and contribute to such disease conditions, using fetal skin-derived cultured mast cells (FSMC) and bone marrow-derived cultured mast cells (BMMC). FSMC expressed ET receptors (ET(A) and ET(B)) at mRNA and protein levels, whereas BMMC expressed lower levels of ET(A), and little, if any, ET(B). ET-1 induced degranulation by FSMC, but not by BMMC through ET(A)-mediated pathways. ET-1 at different concentrations exerted the reciprocal effects on degranulation by IgE-bound FSMC. Furthermore, ET-1 induced TNF-alpha and IL-6 production by FSMC, but not by BMMC, and significantly enhanced VEGF production and TGF-beta1 mRNA expression by FSMC. Finally, ET-1 was produced by FSMC, but not by BMMC in response to Toll-like receptor ligands. These results indicate contrasting impacts of ET-1 on distinct mast cell populations. We propose that ET-1 may participate in pathological conditions of various disorders via its multi-functional effects on mast cells under certain conditions.     

Maturation decreases responsiveness of human bone marrow B lineage cells to stromal-derived factor 1 (SDF-1).

We compared the chemotactic responsiveness of different subsets of human B lineage cells to stromal derived factor-1 (SDF-1). High percentages (30-40% of input) of purified bone marrow progenitors including non-B lineage progenitors, pro-B cells, and pre-B cells migrated to SDF-1alpha, demonstrating that SDF-1 is an efficacious chemoattractant of these cells. Pro-B cells responded optimally to a lower concentration of SDF-1 than other subsets, demonstrating that SDF-1 is a more potent chemoattractant of this subset. A lower percentage (10-15% of input) of mature B lymphocytes migrated to SDF-1alpha than pro-B cells, demonstrating that responsiveness of B lineage cells to SDF-1 decreases during differentiation. Inhibition by anti-CXCR4 mAb demonstrated that migration of B lineage cells to SDF-1 was completely dependent on CXC chemokine receptor-4 (CXCR4). Mature B cells expressed higher levels of CXCR4 receptors than uncommitted progenitors and pro-B cells, despite differences in responsiveness to SDF-1. CXCR4 receptors expressed by unresponsive and SDF-1-responsive B cells bound SDF-1alpha with similar affinities (K(D) = 1.7-3.3 x 10(-9) M). Therefore, elements downstream from CXCR4 appear to regulate responsiveness of B cells to SDF-1. We speculate that SDF-1 and CXCR4 direct migration of progenitor cells in microenvironments that promote B lymphopoiesis.     

Growth factor priming of synovium-derived stem cells for cartilage tissue engineering

This study investigated the potential use of synovium-derived stem cells (SDSCs) as a cell source for cartilage tissue engineering. Harvested SDSCs from juvenile bovine synovium were expanded in culture in the presence (primed) or absence (unprimed) of growth factors (1?ng/mL transforming growth factor-β(1), 10?ng/mL platelet-derived growth factor-ββ, and 5?ng/mL basic fibroblast growth factor-2) and subsequently seeded into clinically relevant agarose hydrogel scaffolds. Constructs seeded with growth factor-primed SDSCs that received an additional transient application of transforming growth factor-β(3) for the first 21 days (release) exhibited significantly better mechanical and biochemical properties compared to constructs that received sustained growth factor stimulation over the entire culture period (continuous). In particular, the release group exhibited a Young's modulus (267±96?kPa) approaching native immature bovine cartilage levels, with corresponding glycosaminoglycan content (5.19±1.45%ww) similar to native values, within 7 weeks of culture. These findings suggest that SDSCs may serve as a cell source for cartilage tissue engineering applications.     

Psoriatic scales: a promising source for the isolation of human skin-derived antimicrobial proteins

Patients with psoriasis, a chronic, hyperproliferative and noninfectious skin disease, suffer surprisingly fewer cutaneous infections than would be expected. This observation led us to the hypothesis that a local "chemical shield" in the form of antimicrobial proteins provides psoriatic skin with resistance against infection. We subsequently began a systematic analysis of in vitro antimicrobially active proteins in psoriatic-scale extracts. A biochemical approach with rigorous purification and characterization combined with antimicrobial testing identified a number of mostly new human antibiotic peptides and proteins. In this review, we will focus on the most prominent antimicrobial proteins in psoriatic-scale extracts, which we identified as the S100-protein psoriasin, human beta-defensin 2 (hBD-2), RNase 7, lysozyme, and human neutrophil defensin 1-3. Apart from these cutaneous, antimicrobial proteins, only a few others, including hBD-3, have been characterized. A great number of minor antimicrobial proteins await further structural characterization.     

Modulation of chemokine receptor expression and chemotactic responsiveness during differentiation of human naive T cells into Th1 or Th2 cells

Chemokines and their receptors direct movements and encounters of lymphocytes and professional APC into specific microenvironments of lymphoid tissues. Chemokine receptors such as CCR7, CXCR5 and CCR4 that are differentially expressed and modulated in distinct subsets of T cells contribute to establish functionally and spatially segregated microenvironments within secondary lymphoid tissues where T cell activation and differentiation occur. Here, we have explored the modulation of CCR7, CCR4, CCR8 and CXCR5 expression and chemotactic responsiveness to their ligands during commitment of human naive T cells along the Th1 or Th2 differentiation pathway in vitro. Our results document that activation of human naive T cells and differentiation in Th1 or Th2 cells result in progressive down-modulation of CCR7 expression and CCL19 responsiveness. By contrast, expression of CCR4 and responsiveness to CCL22 is rapidly induced at the early stages of both Th1/Th2 cell development. However, while CCR4 expression is further up-regulated upon differentiation into Th2 cells, it is lost on fully differentiated Th1 cells. CCR8 is detected at later time points than CCR4 and exclusively on differentiated Th2 cells as revealed by analysis of mRNA expression and responsiveness to CCL1. Expression of CXCR5 is transiently induced at the early stages of Th cell differentiation, but with distinct kinetics in developing Th1 and Th2 cells. Analysis of human tonsillar CD4(+) T cells reveals a consistent pattern of chemotactic responsiveness and chemokine receptor expression in distinct transitional stages of human T cell activation and differentiation in vivo.     

Growth characteristics of and virulence factor production by group A Streptococcus during cultivation in human saliva

Group A Streptococcus (GAS) commonly infects the human oropharynx, but the initial molecular events governing this process are poorly understood. Saliva is a major component of the innate and acquired immune defense in this anatomic site. Although landmark studies were done more than 60 years ago, investigation of GAS-saliva interaction has not been addressed extensively in recent years. Serotype M1 GAS strain MGAS5005 cultured in human saliva grew to approximately 10(7) CFU/ml and, remarkably, maintained this density for up to 28 days. Strains of several other M-protein serotypes had similar initial growth patterns but did not maintain as high a CFU count during prolonged culture. As revealed by analysis of the growth of isogenic mutant strains, the ability of GAS to maintain high numbers of CFU/ml during the prolonged stationary phase in saliva was dependent on production of streptococcal inhibitor of complement (Sic) and streptococcal pyrogenic exotoxin B (SpeB). During cultivation in human saliva, GAS had growth-phase-dependent production of multiple proven and putative extracellular virulence factors, including Sic, SpeB, streptococcal pyrogenic exotoxin A, Mac protein, and streptococcal phospholipase A(2). Our results clearly show that GAS responds in a complex fashion to growth in human saliva, suggesting that the molecular processes that enhance colonization and survival in the upper respiratory tract of humans are well under way before the organism reaches the epithelial cell surface.     

Alexithymia in young adulthood: a risk factor for pathological gambling

BACKGROUND: Pathological gambling is more prevalent among postsecondary students than among the general adult population. While the prevalence of pathological gambling in this group has risen over the past decade, factors underlying the development of problem gambling among university students remain largely unexplored. One early study found alexithymia to be associated with pathological gambling. The aim of the present study was to further examine the relationship between alexithymia and gambling among postsecondary students. METHODS: The relationship between alexithymia and pathological gambling was examined in 562 postsecondary students who completed the South Oaks Gambling Screen (SOGS) and the 20-item Toronto Alexithymia Scale (TAS-20). RESULTS: Approximately 12% of the sample was classified as alexithymic according to the TAS-20. These individuals were found to have significantly more gambling problems, as measured by the SOGS, than nonalexithymic individuals. Approximately 9% of the sample was classified as pathological gamblers according to the SOGS. These individuals were found to have significantly higher levels of alexithymia, as measured by the TAS-20, than nonproblem gamblers. CONCLUSIONS: Alexithymia is associated with pathological gambling and may be a risk factor among postsecondary students for developing severe gambling problems.     

Interleukin 7 is a growth factor for mature human T cells

We have studied the capacity of interleukin (IL) 7 to support the growth and expansion of human T cell clones of different phenotype and function. All the clones studied (CD4+CD8+, CD4-CD8- Tcell receptor alpha/beta or gamma/delta) responded to IL 7. The proliferative response of all the T cell clones induced by IL 7 was routinely less than to IL2, but comparable to the IL4 response. IL 7 also induced the proliferation of resting, freshly prepared peripheral blood mononuclear cells (PBMC) or Tcell-enriched (E+) cells. The pattern of proliferation observed in the presence of IL 7 was similar, but lower in magnitude, to that induced by IL 2. In both these cells populations the response to lymphokines alone was always less than the response to lymphokines plus insolubilized anti-CD3 monoclonal antibody. In contrast IL4 produced a different pattern of responsiveness, as significant proliferation was observed only on PBMC costimulated with anti-CD3. The possibility that IL 7 mediates its growth stimulation by the IL2 pathway was excluded by the incapacity of anti-IL2 or anti-Tac monoclonal antibody, in concentrations which blocked IL2-dependent proliferation, to inhibit IL 7-dependent growth. We conclude that IL 7 is a major growth factor for human mature T cells, and its activity is not limited to lymphocyte progenitors.     

The effect of adherent and phagocytic cells on human lymphocyte PHA responsiveness.

The effect of small numbers of adherent and phagocytic cells on the human peripheral blood lymphocyte response to PHA was examined by depleting these cells from lymphocyte preparations. Lymphocyte preparations obtained by centrifugation on Ficoll--Triosil, which contained on average 85% lymphocytes, responded well to PHA. Depletion of cells adhering to nylon fibre, giving a population containing on average 95% lymphocytes, resulted in a considerably reduced response. Depletion of cells that adhered to plastic or ingested iron powder to give populations containing on average 90% lymphocytes, also reduced the PHA response, but to a lesser extent. Reduction in PHA responsiveness correlated with increasing lymphocyte purity. The responsiveness of nylon-column-filtered cells could be restored by adding a small number of cells from a monocyte-rich population.     

Antigen-independent responsiveness to interleukin-4 demonstrates differential regulation of newborn human T cells

The low incidence of graft-versus-host disease following clinical use of umbilical cord blood compared to adult bone marrow as a source of stem cells for bone marrow reconstitution, leads to questions concerning the level of immunocom-petence of newborn T cells. The maturation and functional status of newborn CD4⁺ T cells, which are almost exclusively CD45RA⁺ naive T cells, compared with their adult phenotypic counterparts, is poorly understood. We examined the proliferative response to mitogens and cytokines of CD4/CD45RA⁺ T cells from adults and newborns, with and without accessory cells. Newborn CD4/CD45RA⁺ T cells demonstrated a distinct proliferative response profile which was determined by the number of accessory cells present in co-cultures with various stimuli. Newborn CD4/CD45RA⁺ T cells were particularly responsive to interleukin (IL)-4, IL-4 plus anti-CD2 monoclonal antibodies (mAb) and IL-4 plus phytohemagglutinin (PHA), whereas adult CD4/CD45RA⁺ T cells were unresponsive under similar conditions. The mitogenic responses of newborn and adult CD4/CD45RA⁺ T cells to PHA and anti-CD2 mAb, which were equivalent, were directly proportional to the number of accessory cells present, whereas the responsiveness to cytokines was inversely proportional to the number of co-cultured accessory cells. Anti-CD2 responses were much more sensitive to low numbers of accessory cells than PHA. The particular sensitivity of newborn CD4/CD45RA⁺ T cells to IL-4 represents an antigen-independent T cell activation response which could help promote a Th2 immune response resulting in the newborn.     

Growth factor and receptor malfunctions associated with human genetic deafness

A variety of different signaling pathways are necessary for development and maintenance of the human auditory system. Normal hearing allows for the detection of soft sounds within the frequency range of 20 to 20 000 Hz, but more importantly to perceive the human voice frequency band of 250 to 6000 Hz. Loss of hearing is common, and is a clinically heterogeneous disorder that can be caused by environmental factors such as exposure to loud noise, infections and ototoxic drugs. In addition, variants of hundreds of genes have been reported to disrupt processes required for hearing. Noncoding regulatory variants and variants of additional genes necessary for hearing remain to be discovered as many individuals with inherited deafness are without a genetic diagnosis, despite the advent of whole exome sequencing. Here, we discuss in detail some of these deafness-causing variants of genes encoding a ligand or its receptor. Spotlighted in this review are three growth factor-receptor-pairs EDN3/EDNRB, HGF/MET and JAG/NOTCH, which individually are necessary for normal hearing. We also offer our perspective on unanswered questions, future challenges and potential opportunities for treatments emerging from molecular genetic and mechanistic studies of deafness due to these causes.     

Growth characteristics of rhinoviruses in human gingival cells

Summary Type M and H rhinoviruses have been shown to replicate in human gingival tissue culture and under selective conditions to produce CPE. Both of these viruses replicate best at approximately 33° C with TCID₅₀/ml of 10⁵ and 10⁴ for rhinoviruses 2060 and 11,757, respectively.In gingival cells the type H rhinovirus (strain 11,757) has an eclipse phase from 1/2 to 6 hours and a maximum intracellular viral titer at 16 hours, whereas the type M rhinovirus (strain 2060) has an eclipse phase from 2 to 8 hours and a maximum intracellular viral titer at 24 hours. A more rapid viral replication of the type H virus was indicated by its rapid attachment to the host cell, short latent period and early virus release.This report was presented in part at the 46th General Meeting of the International Association for Dental Research, San Francisco, California, March 21–24, 1968.This investigation was supported by a General Research Support Grant awarded to the School of Dental Medicine, University of Pittsburgh by the National Institutes of Health, U.S. Public Health Service.     

Cellular and growth factor requirements for the direct and indirect oxidative induction of interleukin 2 responsiveness in human thymocytes

Human thymocytes were separated by peanut agglutinin (PNA) into PNA+ and PNA- cell fractions, and directly oxidized by the combined action of the enzymes neuraminidase and galactose oxidase (NAGO). Such treatment resulted in the induction of interleukin 2 (IL-2) responsiveness in PNA- cells but not in PNA+ cells. The molecular basis for the poor IL-2 responsiveness of PNA+ cells resides in their inability to express sufficient amounts of IL-2 receptors in response to NAGO treatment. Irradiated oxidized PNA+ or PNA- cells are able to transmit an oxidative mitogenic signal to autologous native PNA- cells but not to PNA+ cells. Depletion of plastic adherent cells from the PNA+ subpopulation totally abolished its high potency for the indirect signal transduction, whereas accessory cell depleted PNA- cells were affected to a lesser extent. Nonspecific esterase staining indicated that human thymic macrophages/monocytes are PNA+ cells. In spite of their small number (less than 0.5% of the total thymus cells) they appear to be very active in the indirect oxidative signal transmission. Unlike the indirect system, direct oxidative mitogenesis is independent of accessory cells. Attempts to detect NAGO-dependent IL-2 receptor inducing soluble factors were fruitless and there is a strict need for cell-cell interaction for the indirect transmission of the oxidative mitogenic signal.