Effects of interferon-γ on cell differentiation and cytokine production of a human monoblast cell line,U937

The U937 cell, a human monoblast cell line, has been used as a model to study the function of human monocytes. We investigated the effects of interferon- (IFN-) on Superoxide anion (O ₂ ^– ) production, cell surface antigens, and cytokine production of U937 cells. IFN- treatment enhanced O ₂ ^– production of fMLP or PMA-stimulated U937 cells. IFN- increased the ratio of CD23 and CD 11b positive cells. The fluorescence intensity of CD14 and CD25 was enhanced by IFN- treatment. U937 cells produced IL-1, IL-1, IL-6, and TNF- by lipopolysaccharide (LPS) stimulation. IFN- treatment enhanced TNF- production, but decreased IL-6 production. These results suggest that IFN- differentiates U937 cells to monocytelike cells and it regulates the production systems of IL-6 and TNF- separately in U937 cells.     

Relationships among cell morphology,intrinsic cell stiffness and cell–substrate interactions

Cell modulus (stiffness) is a critical cell property that is important in normal cell functions and increasingly associated with disease states, yet most methods to characterize modulus may skew results. Here we show strong evidence indicating that the fundamental nature of free energies associated with cell/substrate interactions regulates adherent cell morphology and can be used to deduce cell modulus. These results are based on a mathematical model of biophysics and confirmed by the measured morphology of normal and cancerous liver cells adhered on a substrate. Cells select their final morphology by minimizing the total free energy in the cell/substrate system. The key mechanism by which substrate stiffness influences cell morphology is the energy tradeoff between the stabilizing influence of the cell-substrate interfacial adhesive energy and the destabilizing influence of the total elastic energies in the system. Using these findings, we establish a noninvasive methodology to determine the intrinsic modulus of cells by observing global changes in cell morphology in response to substrate stiffness. We also highlight the importance of selecting a relevant morphological index, cell roundness, that reflects the interchange between forms of energy governing cell morphology. Thus, cell-substrate interactions can be rationalized by the underlying biophysics, and cell modulus is easily measured.     

Molecular pathology of papillary, follicular and Hürthle cell carcinomas of the thyroid

In the review some of the molecular alterations that can relate to the neoplastic development of well differentiated thyroid carcinomas (follicular and papillary carcinoma) and of the so-called Hürthle cell carcinomas are described. Diagnostic and/or prognostic significance of the most recent findings is discussed.     

NF-κB in control of regulatory T cell development,identity, and function

Journal of Molecular Medicine - Regulatory T cells (Treg cells) act as a major rheostat regulating the strength of immune responses, enabling tolerance of harmless foreign antigens, and preventing...     

Altered expression of desmocollin 3, desmoglein 3, and β-catenin in oral squamous cell carcinoma: correlation with lymph node metastasis and cell proliferation

Desmocollin 3 (Dsc3) and desmoglein 3 (Dsg3) are both transmembrane glycoproteins that belong to the cadherin family of calcium-dependent cell adhesion molecules. β-Catenin is a member of the cadherin–catenin complex that mediates homotypic cell–cell adhesion and is also an important molecule in the wnt signaling pathway. In this study, we examined the simultaneous expression level of Dsc3, Dsg3, and β-catenin in oral squamous cell carcinomas (OSCCs) and normal oral epithelia using immunohistochemistry. There was a significant correlation (p < 0.05) among the following variables in OSCCs: reduced or loss of expression of Dsc3, Dsg3, and β-catenin compared to normal oral epithelium, reduced or loss of expression of Dsc3 and histological grade (moderately or poorly differentiated), and reduced or loss of expression of β-catenin and lymph node metastasis. Furthermore, a positive correlation was found between reduced or loss of β-catenin staining and reduced or loss of Dsc3 staining in lymph node metastatic cancer tissue (r = 0.734, p < 0.05). These results suggest an abnormal expression of Dsc3, Dsg3, and β-catenin induced in the progression of oral carcinomas and that the Dsc3 expression level might be related to the regulation of β-catenin in lymph node metastasis and cell proliferation in OSCCs.     

Expression of E-Cadherin, β-Catenin,and CD-44v6 cell adhesion molecules in Primary Tumors and Metastases of Colorectal Adenocarcinoma

Immunohistochemical analysis of the expression of E-cadherin, β-catenin, and CD-44v6 proteins was carried out for evaluating the metastatic potential of colorectal cancer cells. Specific features of expression, distribution, and interactions of adhesive molecules in primary tumors of the large intestine and their metastases in the liver and lymph nodes were studied. Reduction and complete absence of E-cadherin expression were much more often observed in patients with colorectal cancer with metastases in the liver than in patients without metastases. Cytoplasmic immunoreactivity and nuclear translocation of β-catenin were increased in more than 80% cases with colorectal adenocarcinoma with metastases. These changes in the expression of E-cadherin and β-catenin in tumor cells can be regarded as factors of unfavorable prognosis of colorectal cancer. No significant relationship between expression of CD-44v6 protein and metastatic potential of cancer cells was detected.__________Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 139, No. 6, pp. 675–679, June, 2005     

T cell receptor binding kinetics and special role of Vα in T cell development and activation

The kinetics of the interaction between T cell receptor (TCR) and major histocompatibility complex (MHC) has an important role in determining thymocyte-positive and-negative selection in the thymus, as well as in T cell activation. The α chain of the TCR is the major player in determining how the TCR fits onto the MHC ligand, and thus has a major role in determining whether a T cell develops as class I or class II restricted. In this article, we summarize recent data from our laboratory and otherson the role of poly-morphism in the Vα combining site in determining MHC class restriction, and on kinetic parameters in thymocyte selection.     

Roles of linear ubiquitinylation,a crucial regulator of NF-κB and cell death,in the immune system

Linear ubiquitinylation, a newly identified post-translational modification, is catalyzed by the linear ubiquitin assembly complex (LUBAC), which is composed of three different subunits, HOIL-1L (heme-oxidized IRP2 ligase 1L), HOIP (HOIL-1 interacting protein), and SHARPIN (SHANK-associated RH domain-interacting protein). LUBAC plays a critical role in the activation of nuclear factor-κB (NF-κB) signaling triggered by a variety of stimuli, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and pathogen-derived components, and in the protection from cell death. Loss of function of SHARPIN in mice triggers chronic inflammation in multiple organs including the skin, as well as immunodeficiency. In humans, mutations in the gene encoding HOIL-1L cause chronic hyperinflammation and immunodeficiency, which are both associated with decreased levels of LUBAC. The linear ubiquitinylation activity of LUBAC is indispensable for B-cell function in mice, and hyperactivation of LUBAC is associated with oncogenesis in certain forms of B-cell lymphoma. In this review, the current understanding of the biochemistry of LUBAC-mediated linear ubiquitinylation and its involvement in the immune system are discussed.     

Impaired T cell receptor signaling and development of T cell–mediated autoimmune arthritis

Mutations of the genes encoding T-cell receptor (TCR)-proximal signaling molecules, such as ZAP-70, can be causative of immunological diseases ranging from T-cell immunodeficiency to T-cell–mediated autoimmune disease. For example, SKG mice, which carry a hypomorphic point mutation of the Zap-70 gene, spontaneously develop T-cell–mediated autoimmune arthritis immunopathologically similar to human rheumatoid arthritis (RA). The Zap-70 mutation alters the sensitivity of developing T cells to thymic positive/negative selection by self-peptides/MHC complexes, shifting self-reactive TCR repertoire to include a dominant arthritogenic specificity and also affecting thymic development and function of autoimmune suppressive regulatory T (Treg) cells. Polyclonal self-reactive T cells, including potentially arthritogenic T cells, thus produced by the thymus recognize self-peptide/MHC complexes on antigen-presenting cells (APCs) in the periphery and stimulate them to produce cytokines including IL-6 to drive the arthritogenic T cells to differentiate into arthritogenic T-helper 17 (Th17) cells. Insufficient Treg suppression or activation of APCs via microbial and other environmental stimuli evokes arthritis by activating granulocyte-macrophage colony-stimulating factor-secreting effector Th17 cells, mediating chronic bone-destructive joint inflammation by activating myeloid cells, innate lymphoid cells, and synoviocytes in the joint. These findings obtained from the study of SKG mouse arthritis are instrumental in understanding how arthritogenic T cells are produced, become activated, and differentiate into effector T cells mediating arthritis, and may help devising therapeutic measures targeting autoimmune pathogenic Th17 cells or autoimmune-suppressing Treg cells to treat and prevent RA.     

siRNA inhibition of aspartyl-asparaginyl β-hydroxylase expression impairs cell motility, Notch signaling, and fetal growth

Aspartyl-asparaginyl-β-hydroxylase (AAH) regulates cell motility and invasiveness by enhancing Notch signaling. Invasive trophoblastic cells, which mediate placentation, normally express high levels of AAH. Previously, we showed that ethanol-impaired placentation is associated with reduced AAH expression. The present study determines the degree to which inhibition of AAH expression is sufficient to impair functions required for placentation.Immortalized, first trimester-derived, human trophoblastic cells (HTR-8/SVneo) were transfected with siRNA targeting AAH (siRNA-AAH) or no specific sequences (siRNA-Scr) using the Amaxa electroporation system. Directional motility was measured using an ATP luminescence-based assay. For in vivo studies, we microinjected siRNA-AAH or siRNA-Scr directly into the implantation sites (mesometrial triangle) of gestation-day-17, Long Evans pregnant rats, and harvested placentas 24 h later for histologic and molecular studies.siRNA-AAH transfection reduced AAH expression and directional motility in HTR-8/SVneo cells. In vivo delivery of siRNA-AAH reduced AAH expression and mean number of invasive trophoblastic cells at the implantation site. These adverse effects of siRNA-AAH were associated with impaired fetal growth and significantly reduced expression of Notch-signaling network genes.AAH is an important, positive regulator of trophoblastic cell motility, and inhibition of AAH in vivo leads to impaired implantation and fetal growth, and alters Notch-signaling mechanisms, similar to the effects of chronic ethanol exposure.     

The role of integrin α2 in cell and matrix therapy that improves perfusion,viability and function of infarcted myocardium

Injectable delivery matrices hold promise in enhancing engraftment and the overall efficacy of cardiac cell therapies; however, the mechanisms responsible remain largely unknown. Here we studied the interaction of a collagen matrix with circulating angiogenic cells (CACs) in a mouse myocardial infarction model. CACs + matrix treatment enhanced CAC engraftment, and improved myocardial perfusion, viability and function compared to cells or matrix alone. Integrin-linked kinase (ILK) was up-regulated in matrix-cultured CACs. Integrin α2β1 blocking prevented ILK up-regulation, significantly reduced the adhesion, proliferation, and paracrine properties of matrix-cultured CACs, and negated the benefits of CACs + matrix therapy in vivo. Furthermore, integrin α5 was essential for the angiogenic potential of CACs on matrix. These findings indicate that the synergistic therapeutic effect of CACs + matrix therapy in MI requires the matrix to enhance CAC function via α2β1 and α5 integrin signaling mechanisms, rather than simply delivering the cells.     

T cell allorecognition and MHC restriction--A case of Jekyll and Hyde?

A great paradox in cellular immunology is how T cell allorecognition exists at high frequencies (up to 10%) despite the stringent requirements of discriminating 'self' from 'non-self' imposed by MHC restriction. Thus, in tissue transplantation, a substantial proportion of the recipient's T cells will have the ability to recognize the graft and instigate an immune response against the transplanted tissue, ultimately resulting in graft rejection--a manifestation of T cell alloreactivity. Transplantation of human organs and lymphoid cells as treatment for otherwise life-threatening diseases has become a more routine medical procedure making this problem of great importance. Immunologists have gained important insights into the mechanisms of T cell alloreactivity from cytotoxic T cell assays, affinity-avidity studies, and crystal structures of peptide-MHC (pMHC) molecules and T cell receptors (TCRs) both alone and in complex. Despite the clinical significance of alloreactivity, the crystal structure of an alloreactive human TCR in complex with both cognate pMHC and an allogeneic pMHC complex has yet to be determined. This review highlights some of the important findings from studies characterizing the way in which alloreactive T cell receptors and pMHC molecules interact in an attempt to resolve this great irony of the cellular immune response.     

Differential expression of β1, β3 and β4 integrins in sarcomas of the small,round, blue cell category

Integrins are a large and complex family of membrane spanning heterodimeric cell surface glycoproteins mediating cell/cell and cell/matrix interactions. Small, round, blue cell sarcomas (SRBCS) are a group of poorly differentiated tumours of various and in part uncertain histogenesis displaying similar cytomorphology. Among them are rhabdomyosarcomas (RMS), ganglioneuroblastomas [(G)NB], primitive peripheral neuroectodermal tumours (pPNET) and Ewing's sarcomas (ES). Thirty-two SRBCS were studied immunohistochemically for the distribution of 1, 3 and 4 integrins in situ. We found complex and to some extent differential patterns of 1, 3 and 4 integrin subunit expression in different types of SRBCS: all of the sarcomas studied were consistently 1⁺, 4^–, 2^–. Four of nine RMS were completely negative for all other integrin subunits studied while one RMS was 5⁺ throughout and three RMS were focally 5⁺. Three RMS expressed the 6 and v chains. In contrast to RMS, pPNET and ES, all of which were 1^–, 3^–, (G)NB were 3⁺ and frequently co-expressed 1. The eight pPNET and seven ES studied showed a similarily restricted integrin profile that was limited to the expression of 1 and 5 in nearly all cases. In summary, RMS were 1⁺, 1^–, 3^– and heterogeneously expressed 5 and 6. (G)NB were generally 1⁺, 1⁺, 3⁺, 5^–, 6^–. pPNET and ES were 1⁺, 1^–, 3^–, 5⁺, 6^–. The data illustrate a complex expression pattern of various integrins in SRBCS, a differential expression pattern of some of the integrin subunits among different types of SRBCS and almost identical integrin profiles in pPNET and ES.This paper is dedicated to Prof. Dr. Dres. h.c. Wilhelm Doerr on the occasion of his 80^th birthday     

Vascular smooth muscle cell calcification and SLC20 inorganic phosphate transporters: effects of PDGF, TNF-α, and Pi

Pi transport by vascular smooth muscle cells (VSMC) has been proposed to play an important role in the pathogenesis of vascular calcification. In this study, we have determined the correlation between calcification induced by Pi, platelet-derived growth factor (PDGF)-BB, and tumor necrosis factor-α and Pi transport activity in primary cultures of rat aortic VSMC. These agents induced calcification and increased the expression of Cbfa1, Msx2, and Bmp2 osteogene messenger RNA in rat aortic VSMC, while Pi transport rate was not modified per milligram of protein. Only PDGF increased Pi transport when it was expressed per unit of DNA, as PDGF also increased total cell protein by 100%, while DNA content and number of cells were not modified. PDGF increased the expression of the Pi transporter, Pit-1, but membrane protein biotinylation showed that Pit-1 abundance was not modified in the cell surface. Immunofluorescence revealed that, under basal conditions, Pit-1 is only slightly expressed at the cell membrane, but strongly expressed inside the cell. The intracellular signal colocalizes with endoplasmic reticulum (ER) markers, and PDGF increases Pit-1 expression in the ER but not the cell membrane. In conclusion, Pi transport across the plasma membrane does not correlate directly with calcification, but the expression of Pit-1 in the ER opens new possibilities for the study of the pathogenesis of vascular calcification.     

Mixed germ cell sex cord–stromal tumors of the testis and ovary. Morphological, immunohistochemical, and molecular genetic study of seven cases

We present the morphological, immunohistochemical, and molecular genetic features of three cases of testicular and four cases of ovarian mixed germ cell sex cord–stromal tumors (MGSCT). The germ cells in the testicular MGSCTs morphologically differed from those in classical seminomas by lacking the typical “square off” quality of the nuclei. In contrast to the nuclei in classical seminomas, their size in testicular MGSCTs was smaller and nucleoli were inconspicuous and the cytoplasm was Periodic Acid-Schiff (PAS) negative. Quite on the contrary, the variability in the size of the nuclei of the germ cells in the testicular MGSCTs was more similar to that seen in the germ cells of spermatocytic seminomas. Immunohistochemically, the germ cells of MGSCTs in one case reacted positively with antibody to AE1–AE3 by paranuclear dot-like or rodlike positivity. All three testicular MGSCTs had a negative reaction with the rest of antibodies, including placental alkaline phosphatase (PLAP), OCT4, and c-kit protein. Ovarian MGSCT in our series differed from the testicular lesions in both the germ cell component and the sex cord component. The germ cells in all four ovarian cases had cytomorphological and immunohistochemical features identical to those in classical seminomas/dysgerminomas. They possessed the typical “square off” quality of the nuclei, which were much more blastic, with more mitoses compared with the testicular tumors in our series, and they were PLAP (4/4), OCT4 (4/4) and c-kit protein (3/4) positive immunohistochemically. The cytoplasm of the germ cells in ovarian neoplasms contained PAS positive glycogen. Germ cells in one ovarian MGSCTs showed amplification of 12p. All other germ cells were negative for amplification of 12p. All five successfully analyzed cases showed no mutation in all studied exons and exon–intron junctions in c-kit and PDFGRA genes.