The Early Activation Marker CD69 Regulates the Expression of Chemokines and CD4 T Cell Accumulation in Intestine

Migration of naïve and activated lymphocytes is regulated by the expression of various molecules such as chemokine receptors and ligands. CD69, the early activation marker of C-type lectin domain family, is also shown to regulate the lymphocyte migration by affecting their egress from the thymus and secondary lymphoid organs. Here, we aimed to investigate the role of CD69 in accumulation of CD4 T cells in intestine using murine models of inflammatory bowel disease. We found that genetic deletion of CD69 in mice increases the expression of the chemokines CCL-1, CXCL-10 and CCL-19 in CD4(+) T cells and/or CD4(-) cells. Efficient in vitro migration of CD69-deficient CD4 T cells toward the chemokine stimuli was the result of increased expression and/or affinity of chemokine receptors. In vivo CD69(-/-) CD4 T cells accumulate in the intestine in higher numbers than B6 CD4 T cells as observed in competitive homing assay, dextran sodium sulphate (DSS)-induced colitis and antigen-specific transfer colitis. In DSS colitis CD69(-/-) CD4 T cell accumulation in colonic lamina propria (cLP) was associated with increased expression of CCL-1, CXCL-10 and CCL-19 genes. Furthermore, treatment of DSS-administrated CD69(-/-) mice with the mixture of CCL-1, CXCL-10 and CCL-19 neutralizing Abs significantly decreased the histopathological signs of colitis. Transfer of OT-II×CD69(-/-) CD45RB(high) CD4 T cells into RAG(-/-) hosts induced CD4 T cell accumulation in cLP. This study showed CD69 as negative regulator of inflammatory responses in intestine as it decreases the expression of chemotactic receptors and ligands and reduces the accumulation of CD4 T cells in cLP during colitis.

Mining tissue microarray data to uncover combinations of biomarker expression patterns that improve intermediate staging and grading of clear cell renal cell cancer

PURPOSE: Tumor stage and nuclear grade are the most important prognostic parameters of clear cell renal cell carcinoma (ccRCC). The progression risk of ccRCC remains difficult to predict particularly for tumors with organ-confined stage and intermediate differentiation grade. Elucidating molecular pathways deregulated in ccRCC may point to novel prognostic parameters that facilitate planning of therapeutic approaches. EXPERIMENTAL DESIGN: Using tissue microarrays, expression patterns of 15 different proteins were evaluated in over 800 ccRCC patients to analyze pathways reported to be physiologically controlled by the tumor suppressors von Hippel-Lindau protein and phosphatase and tensin homologue (PTEN). Tumor staging and grading were improved by performing variable selection using Cox regression and a recursive bootstrap elimination scheme. RESULTS: Patients with pT2 and pT3 tumors that were p27 and CAIX positive had a better outcome than those with all remaining marker combinations. A prolonged survival among patients with intermediate grade (grade 2) correlated with both nuclear p27 and cytoplasmic PTEN expression, as well as with inactive, nonphosphorylated ribosomal protein S6. By applying graphical log-linear modeling for over 700 ccRCC for which the molecular parameters were available, only a weak conditional dependence existed between the expression of p27, PTEN, CAIX, and p-S6, suggesting that the dysregulation of several independent pathways are crucial for tumor progression. CONCLUSIONS: The use of recursive bootstrap elimination, as well as graphical log-linear modeling for comprehensive tissue microarray (TMA) data analysis allows the unraveling of complex molecular contexts and may improve predictive evaluations for patients with advanced renal cancer.

Biomedical nanoparticles modulate specific CD4+ T cell stimulation by inhibition of antigen processing in dendritic cells

Understanding how nanoparticles may affect immune responses is an essential prerequisite to developing novel clinical applications. To investigate nanoparticle-dependent outcomes on immune responses, dendritic cells (DCs) were treated with model biomedical poly(vinylalcohol)-coated super-paramagnetic iron oxide nanoparticles (PVA-SPIONs). PVA-SPIONs uptake by human monocyte-derived DCs (MDDCs) was analyzed by flow cytometry (FACS) and advanced imaging techniques. Viability, activation, function, and stimulatory capacity of MDDCs were assessed by FACS and an in vitro CD4+ T cell assay. PVA-SPION uptake was dose-dependent, decreased by lipopolysaccharide (LPS)-induced MDDC maturation at higher particle concentrations, and was inhibited by cytochalasin D pre-treatment. PVA-SPIONs did not alter surface marker expression (CD80, CD83, CD86, myeloid/plasmacytoid DC markers) or antigen-uptake, but decreased the capacity of MDDCs to process antigen, stimulate CD4+ T cells, and induce cytokines. The decreased antigen processing and CD4+ T cell stimulation capability of MDDCs following PVA-SPION treatment suggests that MDDCs may revert to a more functionally immature state following particle exposure.

Expression patterns of Bmi-1 and p16 significantly correlate with overall, disease-specific, and recurrence-free survival in oropharyngeal squamous cell carcinoma

BACKGROUND: The objective of this study was to link expression patterns of B-cell-specific Moloney murine leukemia virus integration site 1 (Bmi-1) and p16 to patient outcome (recurrence and survival) in a cohort of 252 patients with oral and oropharyngeal squamous cell cancer (OSCC). METHODS: Expression levels of Bmi-1 and p16 in samples from 252 patients with OSCC were evaluated immunohistochemically using the tissue microarray method. Staining intensity was determined by calculating an intensity reactivity score (IRS). Staining intensity and the localization of expression within tumor cells (nuclear or cytoplasmic) were correlated with overall, disease-specific, and recurrence-free survival. RESULTS: The majority of cancers were localized in the oropharynx (61.1%). In univariate analysis, patients who had OSCC and strong Bmi-1 expression (IRS >10) had worse outcomes compared with patients who had low and moderate Bmi-1 expression (P = .008; hazard ratio [HR], 1.82; 95% confidence interval [CI], 1.167-2.838); this correlation was also observed for atypical cytoplasmic Bmi-1 expression (P = .001; HR, 2.164; 95% CI, 1.389-3.371) and for negative p16 expression (P < .001; HR, 0.292; 95% CI, 0.178-0.477). The combination of both markers, as anticipated, had an even stronger correlation with overall survival (P < .001; HR, 8.485; 95% CI, 4.237-16.994). Multivariate analysis demonstrated significant results for patients with oropharyngeal cancers, but not for patients with oral cavity tumors: Tumor classification (P = .011; HR, 1.838; 95%CI, 1.146-2.947) and the combined marker expression patterns (P < .001; HR, 6.254; 95% CI, 2.869-13.635) were correlated with overall survival, disease-specific survival (tumor classification: P = .002; HR, 2.807; 95% CI, 1.477-5.334; combined markers: P = .002; HR, 5.386; 95% CI, 1.850-15.679), and the combined markers also were correlated with recurrence-free survival (P = .001; HR, 8.943; 95% CI, 2.562-31.220). CONCLUSIONS: Cytoplasmic Bmi-1 expression, an absence of p16 expression, and especially the combination of those 2 predictive markers were correlated negatively with disease-specific and recurrence-free survival in patients with oropharyngeal cancer. Therefore, the current results indicate that these may be applicable as predictive markers in combination with other factors to select patients for more aggressive treatment and follow-up. Cancer 2011;. © 2011 American Cancer Society.

The relapse risk of AML patients undergoing autologous transplantation correlates with the stem cell mobilizing potential

Autologous stem cell transplantation (ASCT) is widely used to consolidate first remission in AML. We determined the significance of circulating CD34+ cells at the day of blood stem cell collection in 78 AML patients. Patients mobilizing more than 60,000 CD34+ cells/ml had shorter overall survival (OS; P=0.0274), shorter time to progression (TTP; P=0.0014), and a higher relapse rate (P=0.0177). High levels of CD34+ cells were an independent marker for shorter OS and TTP in a multivariate analysis. These data suggest that ASCT is associated with unfavorable outcome in AML patients with high levels of mobilized peripheral CD34+ cells.

Time-lapse microscopy and classification of 2D human mesenchymal stem cells based on cell shape picks up myogenic from osteogenic and adipogenic differentiation

Current methods to characterize mesenchymal stem cells (MSCs) are limited to CD marker expression, plastic adherence and their ability to differentiate into adipogenic, osteogenic and chondrogenic precursors. It seems evident that stem cells undergoing differentiation should differ in many aspects, such as morphology and possibly also behaviour; however, such a correlation has not yet been exploited for fate prediction of MSCs. Primary human MSCs from bone marrow were expanded and pelleted to form high-density cultures and were then randomly divided into four groups to differentiate into adipogenic, osteogenic chondrogenic and myogenic progenitor cells. The cells were expanded as heterogeneous and tracked with time-lapse microscopy to record cell shape, using phase-contrast microscopy. The cells were segmented using a custom-made image-processing pipeline. Seven morphological features were extracted for each of the segmented cells. Statistical analysis was performed on the seven-dimensional feature vectors, using a tree-like classification method. Differentiation of cells was monitored with key marker genes and histology. Cells in differentiation media were expressing the key genes for each of the three pathways after 21 days, i.e. adipogenic, osteogenic and chondrogenic, which was also confirmed by histological staining. Time-lapse microscopy data were obtained and contained new evidence that two cell shape features, eccentricity and filopodia (= 'fingers') are highly informative to classify myogenic differentiation from all others. However, no robust classifiers could be identified for the other cell differentiation paths. The results suggest that non-invasive automated time-lapse microscopy could potentially be used to predict the stem cell fate of hMSCs for clinical application, based on morphology for earlier time-points. The classification is challenged by cell density, proliferation and possible unknown donor-specific factors, which affect the performance of morphology-based approaches. Copyright © 2012 John Wiley & Sons, Ltd.

Controlled angiogenesis in the heart by cell-based expression of specific vascular endothelial growth factor levels

Vascular endothelial growth factor (VEGF) can induce normal angiogenesis or the growth of angioma-like vascular tumors depending on the amount secreted by each producing cell because it remains localized in the microenvironment. In order to control the distribution of VEGF expression levels in vivo, we recently developed a high-throughput fluorescence-activated cell sorting (FACS)-based technique to rapidly purify transduced progenitors that homogeneously express a specific VEGF dose from a heterogeneous primary population. Here we tested the hypothesis that cell-based delivery of a controlled VEGF level could induce normal angiogenesis in the heart, while preventing the development of angiomas. Freshly isolated human adipose tissue-derived stem cells (ASC) were transduced with retroviral vectors expressing either rat VEGF linked to a FACS-quantifiable cell-surface marker (a truncated form of CD8) or CD8 alone as control (CTR). VEGF-expressing cells were FACS-purified to generate populations producing either a specific VEGF level (SPEC) or uncontrolled heterogeneous levels (ALL). Fifteen nude rats underwent intramyocardial injection of 10(7) cells. Histology was performed after 4 weeks. Both the SPEC and ALL cells produced a similar total amount of VEGF, and both cell types induced a 50%-60% increase in both total and perfused vessel density compared to CTR cells, despite very limited stable engraftment. However, homogeneous VEGF expression by SPEC cells induced only normal and stable angiogenesis. Conversely, heterogeneous expression of a similar total amount by the ALL cells caused the growth of numerous angioma-like structures. These results suggest that controlled VEGF delivery by FACS-purified ASC may be a promising strategy to achieve safe therapeutic angiogenesis in the heart.

Comparative expression profiling of E. coli and S. aureus inoculated primary mammary gland cells sampled from cows with different genetic predispositions for somatic cell score

BACKGROUND: During the past ten years many quantitative trait loci (QTL) affecting mastitis incidence and mastitis related traits like somatic cell score (SCS) were identified in cattle. However, little is known about the molecular architecture of QTL affecting mastitis susceptibility and the underlying physiological mechanisms and genes causing mastitis susceptibility. Here, a genome-wide expression analysis was conducted to analyze molecular mechanisms of mastitis susceptibility that are affected by a specific QTL for SCS on Bos taurus autosome 18 (BTA18). Thereby, some first insights were sought into the genetically determined mechanisms of mammary gland epithelial cells influencing the course of infection. METHODS: Primary bovine mammary gland epithelial cells (pbMEC) were sampled from the udder parenchyma of cows selected for high and low mastitis susceptibility by applying a marker-assisted selection strategy considering QTL and molecular marker information of a confirmed QTL for SCS in the telomeric region of BTA18. The cells were cultured and subsequently inoculated with heat-inactivated mastitis pathogens Escherichia coli and Staphylococcus aureus, respectively. After 1, 6 and 24 h, the cells were harvested and analyzed using the microarray expression chip technology to identify differences in mRNA expression profiles attributed to genetic predisposition, inoculation and cell culture. RESULTS: Comparative analysis of co-expression profiles clearly showed a faster and stronger response after pathogen challenge in pbMEC from less susceptible animals that inherited the favorable QTL allele 'Q' than in pbMEC from more susceptible animals that inherited the unfavorable QTL allele 'q'. Furthermore, the results highlighted RELB as a functional and positional candidate gene and related non-canonical Nf-kappaB signaling as a functional mechanism affected by the QTL. However, in both groups, inoculation resulted in up-regulation of genes associated with the Ingenuity pathways 'dendritic cell maturation' and 'acute phase response signaling', whereas cell culture affected biological processes involved in 'cellular development'. CONCLUSIONS: The results indicate that the complex expression profiling of pathogen challenged pbMEC sampled from cows inheriting alternative QTL alleles is suitable to study genetically determined molecular mechanisms of mastitis susceptibility in mammary epithelial cells in vitro and to highlight the most likely functional pathways and candidate genes underlying the QTL effect.

The clinical impact of p16 status in fine-needle aspirates of cervical lymph node metastasis of head and neck squamous cell carcinomas

Lymph node involvement is prognostically the most determinant clinical factor for patients with head and neck squamous cell carcinomas (HNSCCs). Ultrasound of the neck and fine-needle aspiration (FNA) cytology is one of the first diagnostic procedures and the most accurate diagnostic staging tool for the neck. Patients with HPV-positive oropharyngeal carcinomas (OPSCC) show a significantly better prognosis when compared with HPV-negative OPSCC. P16 overexpression is accepted as surrogate marker for HPV-positive in OPSCC. These HPV/p16-positive OPSCC are localized either in the palatal tonsils or the base of tongue and frequently present with lymph node metastases. We analyzed the correlation and reliability of p16 expression of the FNA of the lymph node metastasis with the immunohistochemical expression of p16 of the same lymph node metastasis and its corresponding primary tumor, as it could be of importance for determining the localization and different prognosis of the primary tumor. 54 HNSCC patients were evaluated, p16 expression of the primary tumors and their lymph node metastases correlated precisely. In 25 of the 54 HNSCC patients, a FNA of the lymph node metastases was taken before the treatment. The positive cytological and immunohistochemical p16 staining correlated exactly. Of the 17 histologically p16-negative lymph node metastases 15 FNA were p16-negative, whereas two samples were p16-positive. In our view, a cytological p16 analysis of cervical lymph node metastasis can facilitate the correct localization of the primary tumor and discriminate reliably HPV-positive OPSCC from HPV-negative HNSCC with their significantly diverse prognosis.

Dysferlin is a new marker for leaky brain blood vessels in multiple sclerosis

Dysferlin is a muscle protein involved in cell membrane repair and its deficiency is associated with muscular dystrophy. We describe that dysferlin is also expressed in leaky endothelial cells. In the normal central nervous system (CNS), dysferlin is only present in endothelial cells of circumventricular organs. In the inflamed CNS of patients with multiple sclerosis (MS) or in animals with experimental autoimmune encephalomyelitis, dysferlin reactivity is induced in endothelial cells and the expression is associated with vascular leakage of serum proteins. In MS, dysferlin expression in endothelial cells is not restricted to vessels with inflammatory cuffs but is also present in noninflamed vessels. In addition, many blood vessels with perivascular inflammatory infiltrates lack dysferlin expression in inactive lesions or in the normal-appearing white matter. In vitro, dysferlin can be induced in endothelial cells by stimulation with tumor necrosis factor-alpha. Hence, dysferlin is not only a marker for leaky brain vessels, but also reveals dissociation of perivascular inflammatory infiltrates and blood-brain barrier disturbance in multiple sclerosis.

Single-cell analysis divides bovine monocyte-derived dendritic cells into subsets expressing either high or low levels of inducible nitric oxide synthase

Dendritic cells (DC) are important cells at the interface between innate and adaptive immunity. DC have a key role in antigen processing and presentation to T cells. Effector functions of DC related to innate immunity have not been explored extensively. We show that bovine monocyte-derived DC (mDC) express inducible nitric oxide synthase (iNOS) mRNA and protein and produce NO upon triggering with interferon-gamma (IFN-gamma) and heat-killed Listeria monocytogenes (HKLM). An immunocytochemical analysis revealed that a sizeable subset (20-60%) copiously expresses iNOS (iNOShi) upon IFN-gamma/HKLM triggering, whereas the other subset expressed low levels of iNOS (iNOSlo). Monocyte-derived macrophages (mMphi) are more homogeneous with regard to iNOS expression. The number of cells within the iNOSlo mDC subset is considerably larger than the number of dead cells or cells unresponsive to IFN-gamma/HKLM. The large majority of cells translocated p65 to the nucleus upon triggering by IFN-gamma/HKLM. A contamination of mDC with iNOS-expressing mMphi was excluded as follows. (i) Cell surface marker analysis suggested that mDC were relatively homogeneous, and no evidence for a contaminating subset expressing macrophage markers (e.g. high levels of CD14) was obtained. (ii) iNOS expression was stronger in iNOShi mDC than in mMphi. The use of maturation-promoting stimuli revealed only subtle phenotypic differences between immature and mature DC in cattle. Nevertheless, these stimuli promoted development of considerably fewer iNOShi mDC upon triggering with IFN-gamma/HKLM. Immunocytochemical results showed that although a significant proportion of cells expressed iNOS only or TNF only upon triggering with IFN-gamma/HKLM, a significant number of cells expressed both iNOS and TNF, suggesting that TNF and iNOS producing (TIP) DC are present within bovine mDC populations obtained in vitro.

Prostate specific antigen expression does not necessarily correlate with prostate cancer cell growth

PURPOSE: The antiproliferative effects of pharmacological agents used for androgen ablative therapy in prostate cancer, including goserelin, bicalutamide and cyproterone acetate (Fluka Chemie, Buchs, Switzerland), were tested in vitro. It was determined whether they affected prostate specific antigen mRNA and protein expression independent of growth inhibition. MATERIALS AND METHODS: Goserelin, bicalutamide (AstraZeneca, Zug, Switzerland) and cyproterone acetate were added to prostate specific antigen expressing, androgen dependent LNCaP and androgen independent C4-2 cell line (Urocor, Oklahoma City, Oklahoma) cultures. Proliferation was determined by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide assay (Roche, Mannheim, Germany). Prostate specific antigen mRNA expression was assessed by quantitative real-time polymerase chain reaction. Secreted prostate specific antigen protein levels were quantified by microparticle enzyme-immunoassay. RESULTS: Goserelin inhibited cell growth and prostate specific antigen protein secretion in LNCaP and C4-2 cells. Prostate specific antigen mRNA expression was not decreased. Bicalutamide did not affect cell growth or prostate specific antigen mRNA expression in LNCaP or C4-2 cells, although it significantly decreased prostate specific antigen protein secretion in LNCaP and to a lesser extent in C4-2 cells. Cyproterone acetate decreased the growth of C4-2 but not of LNCaP cells. It did not affect prostate specific antigen mRNA or protein expression in either cell line. CONCLUSIONS: Prostate specific antigen expression does not necessarily correlate with cell growth. Without a substantial effect on cell growth bicalutamide lowers prostate specific antigen synthesis, whereas cyproterone acetate decreases cell growth with no effect on prostate specific antigen secretion. Prostate specific antigen expression may be influenced by growth inhibition but also by altered mRNA and protein levels depending on the agent, its concentration and the cell line evaluated. For interpreting clinical trials prostate specific antigen is not necessarily a surrogate end point marker for a treatment effect on prostate cancer cell growth.

Spindle cell oncocytoma of the adenohypophysis: report of a case with a 16-year follow-up

Spindle cell oncocytoma (SCO) is a recently described, rare neoplasm of the anterior pituitary. Clinically and radiologically simulating a non-functioning macroadenoma, its eponymous fusiform cells display a non-epithelial phenotype with conspicuous cytoplasmic accumulation of mitochondria. We report a case of SCO retrospectively identified in a biopsy specimen 16 years after transsphenoidal operation of a 48-year-old woman. Presenting symptoms were adynamia and transient decrease of visual acuity. Neuroimaging showed an isointense, enhancing, sellar-centered mass 1.8 cm in diameter without evidence of invasive growth. No postoperative adjuvant therapy was administered. The patient was left with panhypopituitarism, yet no recurrence was seen during follow-up. Initially diagnosed as a null cell adenoma of oncocytic type, repeat immunohistochemistry showed the characteristic coexpression of S100 protein, vimentin, and epithelial membrane antigen. Oncocytic granula stained intensely with antimitochondrial antibody 113-1, and were negative with the lysosomal marker CD68. Anterior pituitary hormones tested negative, and there was no evidence of neuroendocrine differentiation using antibodies to synaptophysin and chromogranin. Few cells stained for glial fibrillary acidic protein (GFAP). SCO has been proposed to represent a neoplasm of folliculo-stellate cells (FSCs). While the dynamic properties of the latter are incompletely characterized, and indeed no specific marker allows for their identification, overlapping features of SCO with look alikes, in particular pituicytoma, point to FSCs being a potential adult stem cell. The favorable outcome of the present case further argues for SCO to be considered a low-grade neoplasm. Moderate tumor size, lack of invasiveness, and low proliferation rate are likely predictors of benign behavior.

Daptomycin produces an enhanced bactericidal activity compared to ceftriaxone, measured by [3H]choline release in the cerebrospinal fluid, in experimental meningitis due to a penicillin-resistant pneumococcal strain without lysing its cell wall

Daptomycin monotherapy was superior to ceftriaxone monotherapy and was highly efficacious in experimental pneumococcal meningitis, sterilizing the cerebrospinal fluid (CSF) of three of three rabbits after 4 to 6 h. With daptomycin therapy only a negligible release of [(3)H]choline as marker of cell wall lysis was detectable in the CSF, peaking around 250 cpm/min after 4 h, compared to a peak of around 2,400 cpm/min after 4 to 6 h for the ceftriaxone-treated rabbits.