p38-MAPK Signals Survival by Phosphorylation of Caspase-8 and Caspase-3 in Human Neutrophils

Neutrophil apoptosis occurs both in the bloodstream and in the tissue and is considered essential for the resolution of an inflammatory process. Here, we show that p38–mitogen-activated protein kinase (MAPK) associates to caspase-8 and caspase-3 during neutrophil apoptosis and that p38-MAPK activity, previously shown to be a survival signal in these primary cells, correlates with the levels of caspase-8 and caspase-3 phosphorylation. In in vitro experiments, immunoprecipitated active p38-MAPK phosphorylated and inhibited the activity of the active p20 subunits of caspase-8 and caspase-3. Phosphopeptide mapping revealed that these phosphorylations occurred on serine-364 and serine-150, respectively. Introduction of mutated (S150A), but not wild-type, TAT-tagged caspase-3 into primary neutrophils made the Fas-induced apoptotic response insensitive to p38-MAPK inhibition. Consequently, p38-MAPK can directly phosphorylate and inhibit the activities of caspase-8 and caspase-3 and thereby hinder neutrophil apoptosis, and, in so doing, regulate the inflammatory response

Dual mechanisms of action of the RNA-binding protein human antigen R explains its regulatory effect on melanoma cell migration

Overexpression of wingless-type MMTV integration site family 5A (WNT5A) plays a significant role in melanoma cancer progression; however, the mechanism(s) involved remains unknown. In breast cancer, the human antigen R (HuR) has been implicated in the regulation of WNT5A expression. Here, we demonstrate that endogenous expression of WNT5A correlates with levels of active HuR in HTB63 and WM852 melanoma cells and that HuR binds to WNT5A messenger RNA in both cell lines. Although the HuR inhibitor MS-444 significantly impaired migration in both melanoma cell lines, it reduced WNT5A expression only in HTB63 cells, as did small interfering RNA knockdown of HuR. Consistent with this finding, MS-444-induced inhibition of HTB63 cell migration was restored by the addition of recombinant WNT5A, whereas MS-444-induced inhibition of WM852 cell migration was restored by the addition of recombinant matrix metalloproteinase-9, another HuR-regulated protein. Clearly, HuR positively regulates melanoma cell migration via at least 2 distinct mechanisms making HuR an attractive therapeutic target for halting melanoma dissemination

OIL CORROSION AND CONDUCTING CU 2 S DEPOSITION IN POWER TRANSFORMER WINDINGS

SUMMARY Copper corrosion and Cu 2 S deposition, has recently led to failures of transformers & shunt reactors during service. Units from several different operators and several OEMs have been affected. Analysis of actual failure cases showed that these transformers were well within established industry standard practice regarding design as well as operation, and used oil that fulfilled international material standards. Sealed units with rubber sack appear primarily to have been affected. Final failure has occurred as a turn-to-turn breakdown in the HV windings, and inspection of the failed windings showed deposits in the winding consisting of Cuprous Sulphide, Cu 2 S, an electrically conducting compound. The deposits varied within the winding, usually with more deposits in the upper part of the windings, but the complex deposition pattern could not be explained simply by the temperature distribution. We have carried out comprehensive corrosion research aiming at a thorough understanding of the phenomenon, and have reproduced the corrosion as well as the deposition of Cu 2 S in our laboratories. The influence of the gas content of the oil, and other parameters have been studied under very well controlled circumstances, and a number of different oils have been evaluated at temperatures ranging from 80 to 150 °C. The main finding is that Cu 2 S deposition occurs only for certain oils, which can cause corrosion and deposition under the typical environment of a sealed power transformer. With such oils deposition has been demonstrated over a wide temperatures range, from as low as 80 °C, and on a variety of solid materials. These oils further show a very strong oxygen dependence of the deposition, which explains much of the observed complex deposition patterns in the failed units. Real failures have all occurred with transformer oils that passed the corrosion tests in the international standards, DIN 51353 and ASTM D1275 respectively. Improved test methods are clearly needed that can take into account the observed oxygen dependence, an important factor present in real transformers. A Covered Conductor Deposition (CCD) test has demonstrated a superior selectivity for identifying the corrosive oils that have caused actual failures in transformers, and is thus a very promising type of test

Wnt-5a mRNA translation is suppressed by the Elav-like protein HuR in human breast epithelial cells

Wnt-5a is a non-transforming Wnt protein. Since Wnt-5a mRNA and protein levels differ within and between tumours, the potential of Wnt-5a as a prognostic factor has been debated. We have previously shown that the lack of Wnt-5a protein is a predictor of shorter disease-free survival in human breast cancer. Recently, however, we also showed that the breast tumours lacking Wnt-5a protein had a high or normal level of Wnt-5a mRNA that might explain the discrepancies in previous studies. We here report that Wnt-5a is regulated at the post-transcriptional level. The regulation was mediated by the Embryonic Lethal Abnormal Vision (ELAV)-like protein HuR, which inhibited translation of Wnt-5a when bound to highly conserved AU-rich sequences in the 3′-untranslated region (3′-UTR) of the Wnt-5a mRNA molecule, as shown by both HA-tagged Wnt-5a- and Luciferase-Wnt-5a-3′-UTR reporter assays. The HuR-dependent inhibition of Wnt-5a was supported by the fact that active HuR is located in the cytoplasm in invasive human breast tumours and that hypoxia-induced activation of HuR inhibits translation of both Luciferase-Wnt-5a-3′-UTR and endogenous Wnt-5a protein. We propose that the lack of Wnt-5a protein expression in invasive human breast tumours is caused by a HuR-mediated suppression of Wnt-5a mRNA translation

Breast cancer associated CD169+ macrophages possess broad immunosuppressive functions but enhance antibody secretion by activated B cells

CD169+ resident macrophages in lymph nodes of breast cancer patients are for unknown reasons associated with a beneficial prognosis. This contrasts CD169+ macrophages present in primary breast tumors (CD169+ TAMs), that correlate with a worse prognosis. We recently showed that these CD169+ TAMs were associated with tertiary lymphoid structures (TLSs) and Tregs in breast cancer. Here, we show that CD169+ TAMs can be monocyte-derived and express a unique mediator profile characterized by type I IFNs, CXCL10, PGE2 and inhibitory co-receptor expression pattern. The CD169+ monocyte-derived macrophages (CD169+ Mo-M) possessed an immunosuppressive function in vitro inhibiting NK, T and B cell proliferation, but enhanced antibody and IL6 secretion in activated B cells. Our findings indicate that CD169+ Mo-M in the primary breast tumor microenvironment are linked to both immunosuppression and TLS functions, with implications for future targeted Mo-M therapy

Early events in T cell development

T cells constitute a heterogenous population of lymphocytes. Their unifying property is that they express surface bound antigen-receptors, called T cell receptors (TCRs). The TCR is a heterodimer composed of either TCRalpha and beta chain proteins (alphabeta TCR) or gamma and delta chain proteins (gammadelta TCR). Depending on which TCR isotype that is expressed, the T cells are divided into alphabeta T cells or gammadelta T cells. In contrast to other hematopoietic cells, T cells develop in an organ called the thymus. To become a mature T cell the thymocytes proceed through a differentiation program that is partially induced by the surrounding thymic epithelial cells. T cell development can be divided into an early and a late stage of differentiation. The purpose of the early stage is to provide the developing thymocytes with functional TCRs. This is mediated by rearrangements at the DNA level of the TCRbeta, gamma and delta genes. If the TCRbeta gene is functionally rearranged, it will pair with a protein called pTalpha to form the preTCR complex that assures rearrangements of the TCRalpha genes and differentiation into the alphabeta T cell lineage. On the other hand, if the TCRgamma and delta chain genes are rearranged functionally the thymocyte will deviate from the mainstream alphabeta T cell pathway to become a mature gammadelta T cell. Whereas conventional alphabeta T cell development is quite well understood today, that of unconventional alphabeta T cells and gammadelta T cells is not. This thesis concerns the early stages of T cell development. With the articles herein, we provide information about the role of the TCR and preTCR in alphabeta versus gammadelta T cell development. We have furthermore examined the expression and transcriptional regulation of the pTalpha gene. Together, these findings lead to a better understanding of the early events in T cell development

Early TCR alpha beta expression promotes maturation of T cells expressing Fc epsilon RI gamma containing TCR/CD3 complexes

In a previous study we presented data indicating that the expanded population of CD4(-)CD8(-) (DN) alphabeta T cells in TCRalpha-chain-transgenic mice was partially if not entirely derived from gammadelta T cell lineage cells. The development of both gammadelta T cells and DN alphabeta T cells is poorly understood; therefore, we thought it would be important to identify the immediate precursors of the transgene-induced DN alphabeta T cells. We have in this report studied the early T cell development in these mice and we show that the transgenic TCRalpha-chain is expressed by precursor thymocytes already at the CD3(-)CD4(-)CD8(-) (triple negative, TN) CD44(+)CD25(-) stage of development. Both by using purified precursor populations in reconstitution experiments and by analyzing fetal thymocyte development, we demonstrated that early TN precursors expressing endogenous TCRbeta-chains matured into DN alphabeta T cells at several stages of development. The genes encoding the gamma-chain of the high affinity receptor for IgE (FcepsilonRIgamma) and the CD3zeta protein were found to be reciprocally expressed in TN thymocytes such that during development the FcepsilonRIgamma expression decreased whereas CD3zeta expression increased. Furthermore, in a fraction of the transgene-induced DN alphabeta T cells the FcepsilonRIgamma protein colocalized with the TCR/CD3 complex. These data suggest that similarly to gammadelta T cells and NKT cells, precursors expressing the TCR early in the common alphabetagammadelta developmental pathway may use the FcepsilonRIgamma protein as a signaling component of the TCR/CD3 complex

The Generation and Identity of Human Myeloid-Derived Suppressor Cells

Myeloid-derived suppressor cells (MDSCs) are cells of myeloid lineage with a potent immunosuppressive capacity. They are present in cancer patients as well as in patients with severe inflammatory conditions and infections. MDSCs exist as two main subtypes, the granulocytic (G-MDSCs) and the monocytic (Mo-MDSCs) type, as defined by their surface phenotype and functions. While the functions of MDSCs have been investigated in depth, the origin of human MDSCs is less characterized and even controversial. In this review, we recapitulate theories on how MDSCs are generated in mice, and whether this knowledge is translatable into human MDSC biology, as well as on problems of defining MDSCs by their immature cell surface phenotype in relation to the plasticity of myeloid cells. Finally, the challenge of pharmacological targeting of MDSCs in the future is envisioned

The presence of tumor associated macrophages in tumor stroma as a prognostic marker for breast cancer patients

Background: Tumor associated macrophages (TAMs) are alternatively activated macrophages that enhance tumor progression by promoting tumor cell invasion, migration and angiogenesis. TAMs have an anti-inflammatory function resembling M2 macrophages. CD163 is regarded as a highly specific monocyte/macrophage marker for M2 macrophages. In this study we evaluated the specificity of using the M2 macrophage marker CD163 as a TAM marker and compared its prognostic value with the more frequently used pan-macrophage marker CD68. We also analyzed the prognostic value of the localization of CD163(+) and CD68(+) myeloid cells in human breast cancer. Methods: The extent of infiltrating CD163+ or CD68+ myeloid cells in tumor nest versus tumor stroma was evaluated by immunohistochemistry in tissue microarrays with tumors from 144 breast cancer cases. Spearman's Rho and chi(2) tests were used to examine the correlations between CD163(+) or CD68(+) myeloid cells and clinicopathological parameters. Kaplan Meier analysis and Cox proportional hazards modeling were used to assess the impact of CD163(+) and CD68(+) myeloid cells in tumor stroma and tumor nest, respectively, on recurrence free survival, breast cancer specific and overall survival. Results: We found that infiltration of CD163(+) and CD68(+) macrophages into tumor stroma, but not into tumor nest, were of clinical relevance. CD163(+) macrophages in tumor stroma positively correlated with higher grade, larger tumor size, Ki67 positivity, estrogen receptor negativity, progesterone receptor negativity, triple-negative/basal-like breast cancer and inversely correlated with luminal A breast cancer. Some CD163+ areas lacked CD68 expression, suggesting that CD163 could be used as a general anti-inflammatory myeloid marker with prognostic impact. CD68(+) macrophages in tumor stroma positively correlated to tumor size and inversely correlated to luminal A breast cancer. More importantly, CD68 in tumor stroma was an independent prognostic factor for reduced breast cancer specific survival. Conclusion: These findings highlight the importance of analyzing the localization rather than merely the presence of TAMs as a prognostic marker for breast cancer patients