Tumor‐associated macrophages (TAMs) depend on ZEB1 for their cancer‐promoting roles

Accumulation of tumor‐associated macrophages (TAMs) associates with malignant progression in cancer. However, the mechanisms that drive the pro‐tumor functions of TAMs are not fully understood. ZEB1 is best known for driving an epithelial‐to‐mesenchymal transition (EMT) in cancer cells to promote tumor progression. However, a role for ZEB1 in macrophages and TAMs has not been studied. Here we describe that TAMs require ZEB1 for their tumor‐promoting and chemotherapy resistance functions in a mouse model of ovarian cancer. Only TAMs that expressed full levels of Zeb1 accelerated tumor growth. Mechanistically, ZEB1 expression in TAMs induced their polarization toward an F4/80^low pro‐tumor phenotype, including direct activation of Ccr2. In turn, expression of ZEB1 by TAMs induced Ccl2, Cd74, and a mesenchymal/stem‐like phenotype in cancer cells. In human ovarian carcinomas, TAM infiltration and CCR2 expression correlated with ZEB1 in tumor cells, where along with CCL2 and CD74 determined poorer prognosis. Importantly, ZEB1 in TAMs was a factor of poorer survival in human ovarian carcinomas. These data establish ZEB1 as a key factor in the tumor microenvironment and for maintaining TAMs’ tumor‐promoting functions.     

CC chemokine receptor 7 expression by effector/memory CD4+ T cells depends on antigen specificity and tissue localization during influenza A virus infection

The lung is an important entry site for respiratory pathogens such as influenza A virus. In order to combat such invading infectious agents, effector/memory T cells home to the lung and other peripheral tissues as well as lymphoid organs. In this process, chemokines and their receptors fulfill important roles in the guidance of T cells into such organs and specialized microenvironments within tissues. In this study, we determined if CD4(+) T cells residing in different lung compartments and draining lymph nodes of influenza A virus-infected and na?ve mice express receptors allowing their recirculation into secondary lymphoid tissues. We found high levels of l-selectin and CC chemokine receptor 7 (CCR7) expression in lung-derived CD4(+) T cells, similar to that detected on T cells in secondary lymphoid organs. Upon influenza A virus infection, the bulk of gamma interferon-positive (IFN-gamma(+)) and IFN-gamma(-) CD4(+) T cells recovered from lung parenchyma retained functional CCR7, whereas virus-specific IFN-gamma-producing T cells were CCR7(-). In contrast, a majority of virus-specific IFN-gamma(+) T cells in the lung draining lymph node were CCR7(+). Independent of infection, CD4(+) T cells obtained from the lung airways exhibited the lowest expression level of l-selectin and CCR7, indicating that T cells at this anatomical site represent the most differentiated effector cell type, lacking the ability to recirculate. Our results suggest that effector/memory T cells that enter inflammatory sites retain functional CCR7 expression, which is lost only upon response to viral antigen and after localization to the final effector site.     

Distinct transthyretin oxidation isoform profile in spinal fluid from patients with Alzheimer’s disease and mild cognitive impairment

Background

Transthyretin (TTR), an abundant protein in cerebrospinal fluid (CSF), contains a free, oxidation-prone cysteine residue that gives rise to TTR isoforms. These isoforms may reflect conditions in vivo. Since increased oxidative stress has been linked to neurodegenerative disorders such as Alzheimer’s disease (AD) it is of interest to characterize CSF-TTR isoform distribution in AD patients and controls. Here, TTR isoforms are profiled directly from CSF by an optimized immunoaffinity-mass spectrometry method in 76 samples from patients with AD (n = 37), mild cognitive impairment (MCI, n = 17)), and normal pressure hydrocephalus (NPH, n = 15), as well as healthy controls (HC, n = 7). Fractions of three specific oxidative modifications (S-cysteinylation, S-cysteinylglycinylation, and S-glutathionylation) were quantitated relative to the total TTR protein. Results were correlated with diagnostic information and with levels of CSF AD biomarkers tau, phosphorylated tau, and amyloid β_1-42 peptide.

Results

Preliminary data highlighted the high risk of artifactual TTR modification due to ex vivo oxidation and thus the samples for this study were all collected using strict and uniform guidelines. The results show that TTR is significantly more modified on Cys(10) in the AD and MCI groups than in controls (NPH and HC) (p ≤ 0.0012). Furthermore, the NPH group, while having normal TTR isoform distribution, had significantly decreased amyloid β peptide but normal tau values. No obvious correlations between levels of routine CSF biomarkers for AD and the degree of TTR modification were found.

Conclusions

AD and MCI patients display a significantly higher fraction of oxidatively modified TTR in CSF than the control groups of NPH patients and HC. Quantitation of CSF-TTR isoforms thus may provide diagnostic information in patients with dementia symptoms but this should be explored in larger studies including prospective studies of MCI patients. The development of methods for simple, robust, and reproducible inhibition of in vitro oxidation during CSF sampling and sample handling is highly warranted. In addition to the diagnostic information the possibility of using TTR as a CSF oxymeter is of potential value in studies monitoring disease activity and developing new drugs for neurodegenerative diseases.     

18?F-Fluoride positron emission tomography/computed tomography for noninvasive in vivo quantification of pathophysiological bone metabolism in experimental murine arthritis

Introduction

Evaluation of disease severity in experimental models of rheumatoid arthritis is inevitably associated with assessment of structural bone damage. A noninvasive imaging technology allowing objective quantification of pathophysiological alterations of bone structure in rodents could substantially extend the methods used to date in preclinical arthritis research for staging of autoimmune disease severity or efficacy of therapeutical intervention. Sodium ¹⁸ F-fluoride (¹⁸ F-NaF) is a bone-seeking tracer well-suited for molecular imaging. Therefore, we systematically examined the use of ¹⁸ F-NaF positron emission tomography/computed tomography (PET/CT) in mice with glucose-6-phosphate isomerase (G6PI)–induced arthritis for quantification of pathological bone metabolism.

Methods

F-fluoride was injected into mice before disease onset and at various time points of progressing experimental arthritis. Radioisotope accumulation in joints in the fore- and hindpaws was analyzed by PET measurements. For validation of bone metabolism quantified by ¹⁸ F-fluoride PET, bone surface parameters of high-resolution μCT measurements were used.

Results

Before clinical arthritis onset, no distinct accumulation of ¹⁸ F-fluoride was detectable in the fore- and hindlimbs of mice immunized with G6PI. In the course of experimental autoimmune disease, ¹⁸ F-fluoride bone uptake was increased at sites of enhanced bone metabolism caused by pathophysiological processes of autoimmune disease. Moreover, ¹⁸ F-fluoride signaling at different stages of G6PI-induced arthritis was significantly correlated with the degree of bone destruction. CT enabled identification of exact localization of ¹⁸ F-fluoride signaling in bone and soft tissue.

Conclusions

The results of this study suggest that small-animal PET/CT using ¹⁸ F-fluoride as a tracer is a feasible method for quantitative assessment of pathophysiological bone metabolism in experimental arthritis. Furthermore, the possibility to perform repeated noninvasive measurements in vivo allows longitudinal study of therapeutical intervention monitoring.     

IL-33 shifts the balance from osteoclast to alternatively activated macrophage differentiation and protects from TNF-alpha-mediated bone loss

IL-33 is a new member of the IL-1 family, which plays a crucial role in inflammatory response, enhancing the differentiation of dendritic cells and alternatively activated macrophages (AAM). Based on the evidence of IL-33 expression in bone, we hypothesized that IL-33 may shift the balance from osteoclast to AAM differentiation and protect from inflammatory bone loss. Using transgenic mice overexpressing human TNF, which develop spontaneous joint inflammation and cartilage destruction, we show that administration of IL-33 or an IL-33R (ST2L) agonistic Ab inhibited cartilage destruction, systemic bone loss, and osteoclast differentiation. Reconstitution of irradiated hTNFtg mice with ST2(-/-) bone marrow led to more bone loss compared with the chimeras with ST2(+/+) bone marrow, demonstrating an important endogenous role of the IL-33/ST2L pathway in bone turnover. The protective effect of IL-33 on bone was accompanied by a significant increase of antiosteoclastogenic cytokines (GM-CSF, IL-4, and IFN-γ) in the serum. In vitro IL-33 directly inhibits mouse and human M-CSF/receptor activator for NF-κB ligand-driven osteoclast differentiation. IL-33 acts directly on murine osteoclast precursors, shifting their differentiation toward CD206(+) AAMs via GM-CSF in an autocrine fashion. Thus, we show in this study that IL-33 is an important bone-protecting cytokine and may be of therapeutic benefit in treating bone resorption.     

Identification of pathogenic fungi in surgical and autopsy specimens by immunofluorescence

Summary A method is presented for the preparation of immune sera and detection by immunofluorescence ofC. immitis, S. schenckii, B. dermatitidis, C. neoformans, andC. albicans in surgical and autopsy material. Formalin fixation does not affect the antigens of the mycotic agents. There are no cross reactions except withC. immitis andC. neoformans, which can be differentiated by the site of the specific fluorescence in each organism.     

Hes1 Increases the Invasion Ability of Colorectal Cancer Cells via the STAT3-MMP14 Pathway

The Notch pathway contributes to self-renewal of tumor-initiating cell and inhibition of normal colonic epithelial cell differentiation. Deregulated expression of Notch1 and Jagged1 is observed in colorectal cancer. Hairy/enhancer of split (HES) family, the most characterized targets of Notch, involved in the development of many cancers. In this study, we explored the role of Hes1 in the tumorigenesis of colorectal cancer. Knocking down Hes1 induced CRC cell senescence and decreased the invasion ability, whereas over-expression of Hes1 increased STAT3 phosphorylation activity and up-regulated MMP14 protein level. We further explored the expression of Hes1 in human colorectal cancer and found high Hes1 mRNA expression is associated with poor prognosis in CRC patients. These findings suggest that Hes1 regulates the invasion ability through the STAT3-MMP14 pathway in CRC cells and high Hes1 expression is a predictor of poor prognosis of CRC.     

EDC3 phosphorylation regulates growth and invasion through controlling P‐body formation and dynamics

Regulation of mRNA stability and translation plays a critical role in determining protein abundance within cells. Processing bodies (P‐bodies) are critical regulators of these processes. Here, we report that the Pim1 and 3 protein kinases bind to the P‐body protein enhancer of mRNA decapping 3 (EDC3) and phosphorylate EDC3 on serine (S)161, thereby modifying P‐body assembly. EDC3 phosphorylation is highly elevated in many tumor types, is reduced upon treatment of cells with kinase inhibitors, and blocks the localization of EDC3 to P‐bodies. Prostate cancer cells harboring an EDC3 S161A mutation show markedly decreased growth, migration, and invasion in tissue culture and in xenograft models. Consistent with these phenotypic changes, the expression of integrin β1 and α6 mRNA and protein is reduced in these mutated cells. These results demonstrate that EDC3 phosphorylation regulates multiple cancer‐relevant functions and suggest that modulation of P‐body activity may represent a new paradigm for cancer treatment.     

Rheumatoid synovial fluid interleukin-17-producing CD4 T cells have abundant tumor necrosis factor-alpha co-expression,but little interleukin-22 and interleukin-23R expression

Introduction  

Th17 cells have been implicated in the pathogenesis of rheumatoid arthritis (RA). The aim of this study was to systematically analyse the phenotype, cytokine profile and frequency of interleukin-17 (IL-17) producing CD4-positive T cells in mononuclear cells isolated from peripheral blood, synovial fluid and synovial tissue of RA patients with established disease, and to correlate cell frequencies with disease activity.     

Effect of Tetracosactid on Post Partum Cyclicity in Cows after Induction of Parturition with PGF2α

Parturition and retention of fetal membranes were induced with PGF_2α in 3 primiparous dairy cows. Starting on day 12 post partum (PP) the cows were treated with 500 μ g i.m. of ACTH-analogue (tetracosactid) every 6 h for 6 times. Changes in plasma concentrations of cortisol, progesterone and 15-ketodihydro-PGF_2α were evaluated immediately after treatment. The effects on the resumption of ovarian activity were evaluated by clinical and ultrasound examinations and by progesterone and 15-ketodihydro-PGF_2α analyses for 56 days after parturition. Treatment was able to induce a statistically significant (p < 0.01) similar increase in cortisol and progesterone after both the 1^st and the 6^th injections, in all cows. No changes in 15-ketodihydro-PGF_2αconcentrations were seen after any of the injections of ACTH-analogue. The first corpus luteum (CL) was seen on day 18 PP (cow A), and 28 (cow B) and in both cases it was followed by a normal ovarian cyclicity. No CL was observed during the whole period of study in cow C. Progesterone profiles confirmed these clinical and ultrasonographic findings. The steroid output, especially progesterone, induced by the ACTH-analogue might be a stimulus for the onset of ovarian cyclicity, since 2 of the 3 animals ovulated earlier than expected. These findings point to the fact that interference with the stress system might have a positive effect on ovarian cyclicity. The different pattern of response does however demand further studies.