Association of von Willebrand factor (vWF) expression with lymph node metastasis and hemodynamics in papillary thyroid carcinoma.

OBJECTIVE: The purpose of this study was to analyze the relationship between von Willebrand factor (vWF) expression and lymph node metastasis or hemodynamics parameters in PTC. This work will provide a novel biomarker for the diagnosis of papillary thyroid carcinoma (PTC). PATIENTS AND METHODS: A total of 156 PTC patients were divided into metastatic and non-metastatic groups based on the presence or absence of lymph node metastasis. The Adler blood flow grading, color doppler flow imaging (CDFI), and blood flow index (PSV, PI, RI, AT) were measured and analyzed between the two groups. The expression of vWF was examined by immunocytochemical assay and quantitative Real-Time Polymerase Chain Reaction (qRT-PCR). The function of vWF was investigated by methyl thiazolyl tetrazolium (MTT) and the transwell assays. RESULTS: Both metastatic and non-metastatic groups with the major Adler grades as 0-1 had abundant blood flows. There was a significant difference in the rate of lymph node metastasis between Adler 2-3 and Adler 0-1. Moreover, the expression of vWF was found to be associated with lymph node metastasis or Adler blood flow grade in PTC. Significant differences in peak systolic velocity (PSV), systolic acceleration time (AT), and resistance index (RI) were detected in metastatic and non-metastatic groups. In addition, the upregulation of vWF was positively correlated with PSV, RI, and PI in PTC. Functionally, the knockdown of vWF inhibited the development of PTC by suppressing cell proliferation, migration, and invasion. CONCLUSIONS: Abnormal expression of vWF is closely related to lymph node metastasis and hemodynamics parameters in PTC patients. Furthermore, vWF plays an oncogene role in PTC progression.

Antibody preparation and identification of the Cashmere goat c-kit protein in the testes.

The c-kit protein plays a major role in the regulation of germ cell development. Its expression and distribution in rodent testes have been widely reported. However, research regarding c-kit expression in domestic animals is scarce, and the expression pattern and distribution of c-kit in germ cells have not been clearly defined. In this study, a specific antigenic region for goat c-kit was designed, and a c-kit polyclonal antibody was prepared. This antibody was then applied in a study evaluating c-kit expression in Cashmere goat tissues. A Western blot analysis showed that three forms of c-kit were expressed in goat testes: precursor, mature, and soluble c-kit. Fluorescent immunohistochemical analyses showed that c-kit was primarily expressed in the spermatogonia and spermatocytes of goat testes. These results not only clarify the expression and localization of c-kit in the goat testis, but also accelerate further research regarding the function of c-kit in goat spermatogenesis.

Characterization of the interaction between astrocytes and encephalitogenic lymphocytes during the development of experimental autoimmune encephalitomyelitis (EAE) in mice.

The nature of pathogenic mechanisms associated with the development of multiple sclerosis (MS) have long been debated. However, limited research was conducted to define the interplay between infiltrating lymphocytes and resident cells of the central nervous system (CNS). Data presented in this report describe a novel role for astrocyte-mediated alterations to myelin oligodendrocyte glycoprotein (MOG)(35-55) -specific lymphocyte responses, elicited during the development of experimental autoimmune encephalitomyelitis (EAE). In-vitro studies demonstrated that astrocytes inhibited the proliferation and interferon (IFN)-γ, interleukin (IL)-4, IL-17 and transforming growth factor (TGF)-ß secretion levels of MOG(35-55) -specific lymphocytes, an effect that could be ameliorated by astrocyte IL-27 neutralization. However, when astrocytes were pretreated with IFN-γ, they could promote the proliferation and secretion levels of MOG(35-55) -specific lymphocytes, coinciding with apparent expression of major histocompatibility complex (MHC)-II on astrocytes themselves. Quantitative polymerase chain reaction (qPCR) demonstrated that production of IL-27 in the spinal cord was at its highest during the initial phases. Conversely, production of IFN-γ in the spinal cord was highest during the peak phase. Quantitative analysis of MHC-II expression in the spinal cord showed that there was a positive correlation between MHC-II expression and IFN-γ production. In addition, astrocyte MHC-II expression levels correlated positively with IFN-γ production in the spinal cord. These findings suggested that astrocytes might function as both inhibitors and promoters of EAE. Astrocytes prevented MOG(35-55) -specific lymphocyte function by secreting IL-27 during the initial phases of EAE. Then, in the presence of higher IFN-γ levels in the spinal cord, astrocytes were converted into antigen-presenting cells. This conversion might promote the progression of pathological damage and result in a peak of EAE severity.

Reciprocal effect of mesenchymal stem cell on experimental autoimmune encephalomyelitis is mediated by transforming growth factor-beta and interleukin-6.

Mesenchymal stem cells (MSCs) have the ability to suppress T cell proliferation and modulate cytokine production. Recently, MSCs have been shown to ameliorate autoimmune diseases such as experimental autoimmune encephalomyelitis (EAE), but in some cases shown to stimulate lymphocyte proliferation. So far, mechanisms through which MSCs modulate immune reactions are still undefined. In this report we demonstrate that MSCs have the capacity for either stimulating or inhibiting myelin basic protein-specific T lymphocytes in a dose-dependent manner and modulate antigen-stimulated T cells to differentiate into either T helper type 17 or regulatory T cells, respectively, via pathways involving transforming growth factor-beta and interleukin-6. These results may lead better utility of MSCs as a treatment for autoimmune disease.

IL-17 eliminates the therapeutic effects of myelin basic protein-induced nasal tolerance in experimental autoimmune encephalomyelitis by activating IL-6.

Interleukin (IL)-17 is a proinflammatory cytokine primarily secreted by Th17 cells, which are a CD4(+) T-cell subset. Th17 cells and IL-17 are important in the pathogenesis of multiple sclerosis and in its established animal model, experimental autoimmune encephalomyelitis (EAE). However, it is unclear whether IL-17 contributes to EAE immune tolerance. We used the myelin basic protein (MBP) peptide MBP 68-86 to induce nasal tolerance to EAE, and simultaneously interfered with the tolerance by treatment with different doses of IL-17. We found that IL-17 dramatically interfered with MBP 68-86-induced immune tolerance. IL-17 administration increased IL-6 release, skewing T cell differentiation towards Th17 cells and decreasing the number of Treg cells. This led to an imbalance between Treg cells and Th17 cells and spurred the development of EAE.

[Clinical study on therapeutic effects of treatment according to syndrome differentiation of traditional Chinese medicine combined with captopril on severe viral myocarditis complicated heart failure].

OBJECTIVE: To observe the therapeutic effect and mechanism of the treatment according to Syndrome Differentiation of TCM combined with captopril (CAP) on severe viral myocarditis (SVM) complicated heart failure (CHF). METHODS: One hundred and nine patients of SVM complicated CHF were randomly divided into the treated group (n = 72) and the control group (n = 37), the former was treated with TCM combined with CAP, while the latter was treated with dexamethasone and interferon. The TCM prescriptions were made depending on types of diseases by Syndrome Differentiation, i.e. Heart-Spleen deficiency type, Qi-Yin deficiency type and Spleen-Kidney Yang deficiency type. The efficacy of treatment was evaluated by the criteria including NYHA classification, myocardial enzymology, electrocardiogram, cardiac function and motorial toleration measured before and after treatment. RESULTS: The therapeutic effect of the treated group according to NYHA classification was obviously better than that of the control group; the creatine phosphokinase isoenzyme (CPK-MB), aspartate transaminase (AST), lactate dehydrogenase (LDH) content lowered in both groups, but more significantly lowered in the treated group than in the control group (P < 0.05, P < 0.01); the improvement of S-T segment of ECG in the treated group was better than that in the control (P < 0.01); also some parameters of heart function and motorial toleration were bettered in the treated group more significantly (P < 0.01). CONCLUSION: TCM treatment according to Syndrome Differentiation combined with CAP in treating SVM complicated CHF could elevate the clinical efficacy.

Heat shock proteins in human endometrium throughout the menstrual cycle.

Human endometrium is a steroid-sensitive tissue and there is evidence that supports the viewpoint that heat shock proteins (HSP) are implicated in the regulation of steroid function. Therefore, in this study we examined the expression of various members of the heat shock family of proteins in the steroid-responsive human endometrium. Western blot analysis revealed that the expression of HSP90 showed minimal changes throughout the menstrual cycle. When normalized to the amount of HSP90, the expression of HSP27, HSP60 and the constitutive form of heat shock protein 70 (HSC70) increased progressively during the late proliferative and early secretory phases, and diminished in the mid- to late secretory and menstrual phases. In contrast, the inducible form of heat shock protein 70 (HSP70) did not undergo these changes. The cellular and subcellular localizations of these proteins were examined in human endometria by immunohistochemical staining. With the exception of HSP70, which was found primarily in the epithelial cells, the immunoreactivity for other heat shock proteins was found in both the stroma and the epithelium. Immunoreactivity for HSP27 was found in the lymphoid aggregates within endometrial stroma, and both HSP27 and HSP90 were found in endothelial cells. The immunoreactive heat shock proteins were found in the nuclei and/or cytoplasm of cells. However, no consistent nuclear versus cytoplasmic staining emerged, and such localization was irrespective of the site, the cell type or the phase of the menstrual cycle. Our findings show that endometrium has a full complement of heat shock proteins. The menstrual cycle-dependent changes in the amounts of heat shock protein suggest regulation by steroid hormones.

Progressive rise in the expression of interleukin-6 in human endometrium during menstrual cycle is initiated during the implantation window.

In order to be prepared for implantation, human endometrium undergoes a predictable series of proliferative and secretory changes. Cytokines play an important role in regulation of these changes. Therefore, in this study, we immunolocalized the cytokine, interleukin-6 (IL-6), its receptor and the signal transducer gp130 in human endometrium throughout the menstrual cycle. During the entire menstrual cycle, the IL-6 receptor and gp130 were found primarily in the endometrial glands and to a lesser extent in the stroma. The immunoreactivity of these proteins did not change in endometrial cells during the entire menstrual cycle with an exception of reduced immunoreactivity of gp130 in endometrial glands during menstrual phase. Immunostaining showed that immunoreactive IL-6 was weakly expressed in human endometrium during the proliferative phase. Strong immunoreactivity for IL-6 appeared in endometrium during the putative 'implantation window'. Expression was by far most pronounced both in the glandular and surface epithelial cells. The amount of immunoreactive IL-6 in the epithelium progressively increased during the secretory/menstrual phases. During the late secretory phase, only stromal cells in the upper functionalis exhibited immunoreactivity for IL-6. Western blot analysis corroborated the immunohistochemical data. Human endometrial IL-6 consisted of a protein with an apparent mobility of 26 kDa. The immunoreactive band of IL-6 was weak in the proliferative phase. The expression of this protein increased progressively during the secretory/menstrual phases. The findings show a cell-specific pattern of distribution for immunoreactive IL-6 in human endometrium. The menstrual cycle-dependent expression of IL-6 suggests that this cytokine may play a role in changes in endometrium that prepare this tissue for implantation and menstrual shedding.

Menstruation is associated with disordered expression of desmoplakin I/II and cadherin/catenins and conversion of F- to G-actin in endometrial epithelium.

Endometrium is unique since it is the only tissue that undergoes regular cyclic bleedings. Menstrual shedding is associated with the breakdown of endometrium, including the fragmentation of endometrial glands. To gain insight into the underlying basis of fragmentation of the endometrial epithelium during the menstrual phase, we examined the expression of proteins implicated in epithelial cell-cell binding in human endometria throughout the entire menstrual cycle. Western blotting failed to reveal differences in the relative amount of E-cadherin, alpha- or beta-catenin or actin in the menstrual endometria compared with those in the proliferative or secretory phases. However, specific changes in the expression pattern of these proteins as well as desmoplakin I/II were detected by immunohistochemical staining in epithelial cells of menstrual endometria. Desmoplakin I/II, E-cadherin, alpha- and beta-catenins and beta-actin were localized to intercellular borders as well as the luminal and basal regions of glandular epithelium during the proliferative and secretory phases. Immunoreactivity of E-cadherin and alpha-catenin was confined to epithelial cells, whereas beta-catenin and beta-actin were present in epithelial cells, as well as in stroma and endothelial cells. Binding of F-actin to fluorescein isothiocyanate-labelled phalloidin localized this form of actin to the intercellular borders, and the basal and luminal cytoplasm of epithelial cells in proliferative and secretory endometria. Menstrual shedding was associated with disorganization of the site-specific distribution of desmoplakin I/II, E-cadherin and alpha- and beta-catenins.(ABSTRACT TRUNCATED AT 250 WORDS)

Tumour necrosis factor-alpha-mediated dyscohesion of epithelial cells is associated with disordered expression of cadherin/beta-catenin and disassembly of actin filaments.

Tumour necrosis factor (TNF)-alpha induced, in a time- and dose-dependent fashion, dyscohesion (cell-cell dissociation) of the endometrial epithelial cells. TNF-alpha impaired the ability of cells to aggregate and to attain compaction. The cell-cell adherent junction is a specialized region of the plasma membrane where cadherin molecules act as adhesion molecules and actin filaments are densely associated with the plasma membrane through a well-developed plasmalemmal undercoat. Dyscohesion induced by TNF-alpha was associated with the disordered expression of cadherin/beta-catenin at the sites of cell-cell contact. In addition, within the time-frame that dyscohesion was induced, TNF-alpha down-regulated the expression of actin mRNA only at 100 ng/ml without modulating the overall amount of actin protein, its beta-isoform or the amount of ribosylated actin. However, TNF-alpha-mediated dyscohesion of epithelial cells was associated with loss of plasmalemmal undercoat as well as intracytoplasmic aggregates of F-actin and a simultaneous increase in G-actin. The effect of cytochalasin-B, which disrupts actin filaments on cell-cell binding, was less pronounced than the effect of TNF-alpha, suggesting that the effect of this cytokine on dyscohesion is not solely dependent on the disassembly of actin filaments. These findings show that the induction of disordered expression of adhesion molecules, as well as disassembly of actin filaments, are implicated in the dyscohesion induced by TNF-alpha.

TNF-α induces dyscohesion of epithelial cells. Association with disassembly of actin filaments.

TNF-α induced, in a time and dose-dependent fashion, cell-cell dissociation (dyscohesion) of endometrial epithelial cells. Within the time frame that dyscohesion was induced, TNF-α, in a dose-dependent fashion, reduced filamentous (F) actin and resulted in the loss of F-actin from the intercellular boundaries. Loss of F-actin mediated by TNF-α was not due to a reduction in the overall amount of actin or its ß-isoform. Two proteins, Rho and Rho guanine nucleotide dissociation inhibitor (Rho-GDI), have been implicated in the regulation of organization of actin cytoskeleton. The reduced level of F-actin was not associated with altered expression of Rho protein, however, it was associated with an increase in the amount of Rho available for ribosylationin vitro by the C3 exoenzyme of Clostridium botulinum. The amount of Rho-GDI protein did not change after treatment with TNF-α suggesting that elevated expression of this protein is not responsible for the disassembly of actin filaments. These findings show that TNF-α induces dyscohesion. Dyscohesion induced by this cytokine is associated with perturbation of the actin cytoskeleton which may be due to the regulatory role of TNF-α on Rho.

Expression of adhesion molecules in human endometrial vasculature throughout the menstrual cycle.

In the present study, we examined the pattern of expression of human leukocyte antigen (HLA)-DR as well as several adhesion molecules implicated in leukocyte trafficking, including ICAM-1, E-selectin, and VCAM-1 in human endometrium. All of the vessels in endometrium exhibited HLA-DR and ICAM-1 throughout the menstrual cycle. In the proliferative phase, endothelial cells in the functionalis were weak to nonreactive for VCAM-1 and were E-selectin negative (E-selectin-). Endothelial cells of the vessels in the basalis and within myometrium were VCAM positive (VCAM-1+)/E-selectin-. In sharp contrast, in the secretory phase, endothelial cells in the basalis were VCAM-1+/E-selectin+. Surprisingly, endometrial glands, primarily those in the basalis, expressed E-selectin and VCAM-1 during the entire menstrual cycle. Stromal cells were ICAM-1+ and were focally HLA-DR+ around HLA-DR+ lymphoid cells during the entire menstrual cycle and were E-selectin-/VCAM-1- during the proliferative phase. Immunoreactivity for VCAM-1 and E-selectin, however, appeared in the stromal cells in the upper functionalis in the secretory phase. Immunoreactivity for VCAM-1 was the distinguishing feature that separated the lymphoid cells in the aggregates from other nonaggregated lymphoid cells. Recruitment of leukocytes to tissues is in part due to cytokine-regulated expression of specific molecules on endothelial cells. Therefore, we tested the effects of cytokines on the expression of these molecules in endothelial cells derived from microvasculature. ICAM-1 and VCAM-1 were inducible in a dose-dependent fashion in the endothelial cells by interleukin-1 alpha (IL-1 alpha), interferon-gamma (IFN gamma), and tumor necrosis factor-alpha (TNF alpha). Expression of HLA-DR in endothelial cells was inducible by IL-1 alpha and IFN gamma and not by TNF alpha. Expression of E-selectin on endothelial cells was induced only by IL-1 alpha, not by IFN gamma or TNF alpha. Cytokine treatment of endothelial cells significantly enhanced the binding of leukocytes to endothelial cells. The data show a heterogeneity in the vasculature of endometrium with respect to the expression of various adhesion molecules. This heterogeneity is potentially related to the type or amount of cytokine with which endothelial cells are activated. In addition, unique cell- and site-specific expression of adhesion molecules in human endometrium throughout the menstrual cycle may account for the distinct distribution pattern of leukocytes in this tissue.

Passive acquisition of leukocyte proteins is associated with changes in phosphorylation of cellular proteins and cell-cell adhesion properties.

In this report, we show that interaction of neoplastic epithelial cells with vesicles derived from leukocytes results in passive acquisition by tumor cells of a diverse group of leukocyte proteins. Vesicles shed from leukocytes were heterogeneous and exhibited the specific proteins expressed on leukocyte subsets. Accordingly, epithelial cells differentially acquired leukocyte proteins associated with vesicles. Ultrastructural localization demonstrated that acquired proteins were associated with the plasma membranes of the epithelial cells. The binding of tumor cells that passively acquired leukocyte proteins to immobilized intercellular adhesion molecule-1 and to endothelial cells was significantly increased. Furthermore, passive acquisition of proteins on the plasma membranes of epithelial cells was associated with modulation of overall phosphorylation of proteins in the range of 20-65 kd and consisted of both increased as well as decreased phosphorylation of specific protein species in the cells. These findings demonstrate that leukocyte proteins that are shed in association with vesicles passively coat the plasma membranes of target epithelial cells. Passive acquisition of proteins by cells modulates the constitutive properties endowed upon cells by their native plasma membranes and is associated with changes in phosphorylation of cell proteins.

Induction of a polarized micro-environment by human T cells and interferon-gamma in three-dimensional spheroid cultures of human endometrial epithelial cells.

Expression of human leukocyte antigen (HLA)-DR molecules and proliferation of epithelium in human endometrium are polarized. We have suggested that the induction of such a polarized micro-environment is T cell and interferon (IFN)-gamma dependent. The present study was designed to demonstrate the induction of such a micro-environment around T cells and around the source of IFN-gamma. Spheroids reminiscent of endometrial glands were formed by allowing three-dimensional aggregation of endometrial epithelial cells of a cloned HLA-DR negative endometrial carcinoma cell line (ECC1) over agarose. Both HLA-DR expression and inhibition of proliferation were found to be directly dependent on the dose of IFN-gamma that was allowed to diffuse in the agarose beneath the spheroids. To show that the interaction of the epithelial cells with activated T cells also induces HLA-DR molecules in a paracrine fashion in the epithelial cells, ECC1 spheroids were co-cultured with increasing numbers of allogeneic peripheral blood T cells for various time-intervals. T cells bound to the ECC1 cells, and became activated as indicated by the expression of interleukin (IL)-2 receptor and HLA-DR molecules. A focal HLA-DR expression became apparent in the ECC1 cells adjacent to the T cells. As the number of T cells added to spheroid cultures was increased, a concomitant increase in the number of HLA-DR positive ECC1 cells occurred and HLA-DR immunoreactivity was enhanced in each cell. There was a corresponding decrease in the proliferation of the ECC1 cells in T cell-ECC1 spheroid co-cultures. Based on these data, we suggest that activation of T cells is associated with the induction of HLA-DR expression and inhibition of proliferation in a paracrine fashion in the epithelial cells and may be responsible for the creation of a polarized micro-environment in vivo.