Altered expression of CD1d molecules and lipid accumulation in the human hepatoma cell line HepG2 after iron loading.

Iron overload in the liver may occur in clinical conditions such as hemochromatosis and nonalcoholic steatohepatitis, and may lead to the deterioration of the normal liver architecture by mechanisms not well understood. Although a relationship between the expression of ICAM-1, and classical major histocompatibility complex (MHC) class I molecules, and iron overload has been reported, no relationship has been identified between iron overload and the expression of unconventional MHC class I molecules. Herein, we report that parameters of iron metabolism were regulated in a coordinated-fashion in a human hepatoma cell line (HepG2 cells) after iron loading, leading to increased cellular oxidative stress and growth retardation. Iron loading of HepG2 cells resulted in increased expression of Nor3.2-reactive CD1d molecules at the plasma membrane. Expression of classical MHC class I and II molecules, ICAM-1 and the epithelial CD8 ligand, gp180 was not significantly affected by iron. Considering that intracellular lipids regulate expression of CD1d at the cell surface, we examined parameters of lipid metabolism in iron-loaded HepG2 cells. Interestingly, increased expression of CD1d molecules by iron-loaded HepG2 cells was associated with increased phosphatidylserine expression in the outer leaflet of the plasma membrane and the presence of many intracellular lipid droplets. These data describe a new relationship between iron loading, lipid accumulation and altered expression of CD1d, an unconventional MHC class I molecule reported to monitor intracellular and plasma membrane lipid metabolism, in the human hepatoma cell line HepG2.This work was funded by the Inova Foundation for Medical Research/The American Portuguese Biomedical Research Fund (APBRF, USA). The authors would like to thank L. Mayer and K. Uchida for providing antibodies, A. do Vale, F. Pisarra and M.T. Silva for help and comments on TEM, and M. Santos and R. Hultcrantz for critical reading of the manuscript. We also thank M. de Sousa for mentoring this work. M.C. and C.F.P. were supported by a fellowship from Inova/APBRF. E.M.C. was partially supported by EU grant (QLG1-CT-1999–00665)

Hepatocytes and IL-15: A Favorable Microenvironment for T Cell Survival and CD8+ T Cell Differentiation

Human intrahepatic lymphocytes are enriched in CD1d-unrestricted T cells coexpressing NKR. Although the origin of this population remains controversial, it is possible to speculate that the hepatic microenvironment, namely epithelial cells or the cytokine milieu, may play a role in its shaping. IL-15 is constitutively expressed in the liver and has a key role in activation and survival of innate and tissue-associated immune cells. In this in vitro study, we examined whether hepatocyte cell lines and/or IL-15 could play a role in the generation of NK-like T cells. The results show that both HepG2 cells and a human immortalized hepatocyte cell line increase survival and drive basal proliferation of T cells. In addition, IL-15 was capable of inducing Ag-independent up-regulation of NKR, including NKG2A, Ig-like receptors, and de novo expression of CD56 and NKp46 in CD8(+)CD56(-) T cells. In conclusion, our study suggests that hepatocytes and IL-15 create a favorable microenvironment for T cells to growth and survive. It can be proposed that the increased percentage of intrahepatic nonclassical NKT cells could be in part due to a local CD8(+) T cell differentiation.This work was supported by grants from the American Portuguese Biomedical Research Fund (Inova Grant) and from ISCSN-CESPU (Grants CESPU 1F/05/2005 and CESPU 2F/03/2006). M.P.C. was supported by a fellowship from Fundação para a Ciência e a Tecnologia (SFRH/BD/24396/2005)

Spatial Division Multiplexing for Multiplex Coherent Anti-Stokes Raman Scattering

We demonstrate how a narrowband pump and a broadband spectrum can be spatially multiplexed by selective coupling them in two distinct modes of a few-mode microstructure fiber. The first mode carries most of the input pump energy, and experiences spectral broadening. Whereas the second mode preserves the narrow bandwidth of the remaining part of the pump. Bimodal propagation, with a power unbalance strongly in favor of the fundamental mode, is naturally obtained by maximizing coupling into the fundamental mode of the fiber. At the fiber output, the nearly monochromatic beam and the supercontinuum carried by the two different modes are combined by a microscope objective, and used as a pump and a Stokes wave for self-referenced multiplex coherent anti-Stokes Raman scattering micro-spectroscopy. The spectral resolution, the signal-to-noise-ratio, and the possible amplification of the remaining pump beam are discussed.Comment: 10 pages, 9 figure

HLA-C and HIV-1: friends or foes?

The major histocompatibility complex class I protein HLA-C plays a crucial role as a molecule capable of sending inhibitory signals to both natural killer (NK) cells and cytotoxic T lymphocytes (CTL) via binding to killer cell Ig-like receptors (KIR). Recently HLA-C has been recognized as a key molecule in the immune control of HIV-1. Expression of HLA-C is modulated by a microRNA binding site. HLA-C alleles that bear substitutions in the microRNA binding site are more expressed at the cell surface and associated with the control of HIV-1 viral load, suggesting a role of HLA-C in the presentation of antigenic peptides to CTLs. This review highlights the role of HLA-C in association with HIV-1 viral load, but also addresses the contradiction of the association between high cell surface expression of an inhibitory molecule and strong cell-mediated immunity. To explore additional mechanisms of control of HIV-1 replication by HLA-C, we address specific features of the molecule, like its tendency to be expressed as open conformer upon cell activation, which endows it with a unique capacity to associate with other cell surface molecules as well as with HIV-1 proteins

Total blood lymphocyte counts in hemochromatosis probands with HFE C282Y homozygosity: relationship to severity of iron overload and HLA-A and -B alleles and haplotypes

BACKGROUND: It has been reported that some persons with hemochromatosis have low total blood lymphocyte counts, but the reason for this is unknown. METHODS: We measured total blood lymphocyte counts using an automated blood cell counter in 146 hemochromatosis probands (88 men, 58 women) with HFE C282Y homozygosity who were diagnosed in medical care. Univariate and multivariate analyses of total blood lymphocyte counts were evaluated using these variables: sex; age, transferrin saturation, and serum ferritin concentration at diagnosis; units of blood removed by phlebotomy to achieve iron depletion; and human leukocyte antigen (HLA)-A and -B alleles and haplotypes. RESULTS: The mean age at diagnosis was 49 ± 14 years (range 18 – 80 years) in men and 50 ± 13 years (range 22 – 88 years) in women. The correlations of total blood lymphocyte counts with sex, age, transferrin saturation, and serum ferritin concentration at diagnosis, and units of blood removed by phlebotomy to achieve iron depletion were not significant at the 0.05 level. Univariate analyses revealed significant associations between total blood lymphocyte counts and presence of the HLA-A*01, -B*08, and -B*14 alleles, and the A*01-B*08 haplotype. Presence of the A*01 allele, B*08 allele, or A*01-B*08 haplotype were associated with a lower total blood lymphocyte count, whereas presence of the B*14 allele was associated with a greater total blood lymphocyte count. There was an inverse association of total blood lymphocyte count with units of phlebotomy to achieve iron depletion, serum ferritin concentration, and with presence of the A*01-B*08 haplotype. CONCLUSION: We conclude that there is a significant inverse relationship of total blood lymphocyte counts and severity of iron overload in hemochromatosis probands with HFE C282Y homozygosity. The presence of the HLA-A*01 allele or the -B*08 allele was also associated with significantly lower total blood lymphocyte counts, whereas presence of the -B*14 allele was associated with significantly higher total blood lymphocyte counts. In univariate and multivariate analyses, total blood lymphocyte counts were significantly lower in probands with the HLA-A*01-B*08 haplotype than in probands without this haplotype

A Potential Role for Shed Soluble Major Histocompatibility Class I Molecules as Modulators of Neurite Outgrowth

The neurobiological activities of classical major histocompatibility class I (MHCI) molecules are just beginning to be explored. To further examine MHCI's actions during the formation of neuronal connections, we cultured embryonic mouse retina explants a short distance from wildtype thalamic explants, or thalami from transgenic mice (termed “NSE-Db”) whose neurons express higher levels of MHCI. While retina neurites extended to form connections with wildtype thalami, we were surprised to find that retina neurite outgrowth was very stunted in regions proximal to NSE-Db thalamic explants, suggesting that a diffusible factor from these thalami inhibited retina neurite outgrowth. It has been long known that MHCI-expressing cells release soluble forms of MHCI (sMHCI) due to the shedding of intact MHCI molecules, as well as the alternative exon splicing of its heavy chain or the action proteases which cleave off it's transmembrane anchor. We show that the diffusible inhibitory factor from the NSE-Db thalami is sMHCI. We also show that COS cells programmed to express murine MHCI release sMHCI that inhibits neurite outgrowth from nearby neurons in vitro. The neuroinhibitory effect of sMHCI could be blocked by lowering cAMP levels, suggesting that the neuronal MHCI receptor's signaling mechanism involves a cyclic nucleotide-dependent pathway. Our results suggest that MHCI may not only have neurobiological activity in its membrane-bound form, it may also influence local neurons as a soluble molecule. We discuss the involvement of complement proteins in generating sMHCI and new theoretical models of MHCI's biological activities in the nervous system

HIV-1 Env associates with HLA-C free-chains at the cell membrane modulating viral infectivity

HLA-C has been demonstrated to associate with HIV-1 envelope glycoprotein (Env). Virions lacking HLA-C have reduced infectivity and increased susceptibility to neutralizing antibodies. Like all others MHC-I molecules, HLA-C requires \u3b22-microglobulin (\u3b22m) for appropriate folding and expression on the cell membrane but this association is weaker, thus generating HLA-C free-chains on the cell surface. In this study, we deepen the understanding of HLA-C and Env association by showing that HIV-1 specifically increases the amount of HLA-C free chains, not bound to \u3b22m, on the membrane of infected cells. The association between Env and HLA-C takes place at the cell membrane requiring \u3b22m to occur. We report that the enhanced infectivity conferred to HIV-1 by HLA-C specifically involves HLA-C free chain molecules that have been correctly assembled with \u3b22m. HIV-1 Env-pseudotyped viruses produced in the absence of \u3b22m are less infectious than those produced in the presence of \u3b22m. We hypothesize that the conformation and surface expression of HLA-C molecules could be a discriminant for the association with Env. Binding stability to \u3b22m may confer to HLA-C the ability to preferentially act either as a conventional immune-competent molecule or as an accessory molecule involved in HIV-1 infectivity

Defining family business: a closer look at definitional heterogeneity

Researchers have used a myriad of different definitions in seeking to explain the heterogeneity of family firms and their unique behavior; however, no widely-accepted definition exists today. Definitional clarity in any field is essential to provide (a) the basis for the analysis of performance both spatially and temporally and (b) the foundation upon which theories, frameworks and models are developed. We provide a comprehensive analysis of prior research and identify and classify 82 definitions of family business. We then review and evaluate five key theoretical perspectives in family business to identify how these have shaped and informed the definitions employed in the field and duly explain family firm heterogeneity. Finally, we provide a conceptual diagram to inform the choice of definition in different research settings