The chemokines CCL11, CCL20, CCL21, and CCL24 are preferentially expressed in polarized human secondary lymphoid follicles

Chemokines regulate cellular trafficking to and from lymphoid follicles. Here, the distribution pattern of four CCL chemokines is defined by in situ hybridization in human lymphoid follicles from tonsils and lymph nodes (LNs) of newborns and adults. Cells expressing CCL11 (eotaxin) and CCL20 (Exodus) were preferentially located within follicles, while cells expressing CCL21 (secondary lymphoid-tissue chemokine) and CCL24 (eotaxin-2) mRNA were almost exclusively found in the perifollicular areas. Hence, the two CCR3-binding chemokines, CCL11 and CCL24, showed a mutually exclusive expression pattern in the intra- and extra-follicular areas, respectively. Chemokine gene expression paralleled follicular maturation: in tonsils, where approximately 80% of follicles are polarized, CCL11 and CCL20 mRNA-positive cells were detected more frequently than in lymph nodes from adults, where about half of follicles are non-polarized. No intrafollicular chemokine expression was detectable in the primary follicles from newborns. Extrafollicular cells expressing CCL21 and CCL24 were again more frequent in tonsils than in LNs from adults. The observed preferential presence of cells expressing CC chemokines in polarized human lymphoid follicles indicates that chemokines are not only instrumental in the induction of follicle formation, but may also be involved in their further differentiation.

Evidence that the novel receptor FGFRL1 signals indirectly via FGFR1

Fibroblast growth factor (FGF) receptor-like protein 1 (FGFRL1) is a recently discovered member of the FGF receptor (FGFR) family. Similar to the classical FGFRs, it contains three extracellular immunoglobulin-like domains and interacts with FGF ligands. However, in contrast to the classical receptors, it does not contain any intracellular tyrosine kinase domain and consequently cannot signal by transphosphorylation. In mouse kidneys, FgfrL1 is expressed primarily at embryonic stages E14-E15 in regions where nascent nephrons develop. In this study, we used whole-mount in situ hybridization to show the spatial pattern of five different Fgfrs in the developing mouse kidney. We compared the expression pattern of FgfrL1 with that of other Fgfrs. The expression pattern of FgfrL1 closely resembled that of Fgfr1, but clearly differed from that of Fgfr2‑Fgfr4. It is therefore conceivable that FgfrL1 signals indirectly via Fgfr1. The mechanisms by which FgfrL1 affects the activity of Fgfr1 remain to be elucidated.

miR-21 and its target gene CCL20 are both highly overexpressed in the microenvironment of colorectal tumors: significance of their regulation

Recently, we reported a functional interaction between miR-21 and its identified chemokine target CCL20 in colorectal cancer (CRC) cell lines. Here, we investigated whether such functional interactions are permitted at the cellular level which would require an inverse correlation of expression and also co-expression of miR-21 and CCL20 in the same cell. Expression profiling was performed using qPCR, and ELISA, in situ hybridization and immunohistochemistry were applied for the presentation of their cellular localization. We demonstrated that miR-21 as well as CCL20 were both significantly upregulated in CRC tissues; thus, showing no antidromic expression pattern. This provided an initial clue that miR-21 and CCL20 may not be expressed in the same cell. In addition, we located miR-21 expression at the cellular level predominantly in stromal cells such as tumor-associated fibroblasts and to a minor degree in immune cells such as macrophages and lymphocytes. Likewise, CCL20 expression was primarily detected in tumor-infiltrating immune cells. Thus, investigating the cellular localization of miR-21 and its target CCL20 revealed that both molecules are expressed predominantly in the microenvironment of CRC tumors.

High affinity amino acid transporters specifically expressed in xylem parenchyma and developing seeds of Arabidopsis

Arabidopsis amino acid transporters (AAPs) show individual temporal and spatial expression patterns. A new amino acid transporter, AAP8 was isolated by reverse transcription-PCR. Growth and transport assays in comparison to AAP1-5 characterize AAP8 and AAP6 as high affinity amino acid transport systems from Arabidopsis. Histochemical promoter-beta-glucuronidase (GUS) studies identified AAP6 expression in xylem parenchyma, cells requiring high affinity transport due to the low amino acid concentration in xylem sap. AAP6 may thus function in uptake of amino acids from xylem. Histochemical analysis of AAP8 revealed stage-dependent expression in siliques and developing seeds. Thus AAP8 is probably responsible for import of organic nitrogen into developing seeds. The only missing transporter of the family AAP7 was nonfunctional in yeast with respect to amino acid transport, and expression was not detectable. Therefore, AAP6 and -8 are the only members of the family able to transport aspartate with physiologically relevant affinity. AAP1, -6 and -8 are the closest AAP paralogs. Although AAP1 and AAP8 originate from a duplicated region on chromosome I, biochemical properties and expression pattern diverged. Overlapping substrate specificities paired with individual properties and expression patterns point to specific functions of each of the AAP genes in nitrogen distribution rather than to mere redundancy.

Tenascins in stem cell niches.

Tenascins are extracellular matrix proteins with distinct spatial and temporal expression during development, tissue homeostasis and disease. Based on their expression patterns and knockout phenotypes an important role of tenascins in tissue formation, cell adhesion modulation, regulation of proliferation and differentiation has been demonstrated. All of these features are of importance in stem cell niches where a precise regulation of growth versus differentiation has to be guaranteed. In this review we summarize the expression and possible functions of tenascins in neural, epithelial and osteogenic stem cell niches during normal development and organ turnover, in the hematopoietic and pro-inflammatory niche as well as in the metastatic niche during cancer progression.

The molecular signature of the stroma response in prostate cancer-induced osteoblastic bone metastasis highlights expansion of hematopoietic and prostate epithelial stem cell niches

The reciprocal interaction between cancer cells and the tissue-specific stroma is critical for primary and metastatic tumor growth progression. Prostate cancer cells colonize preferentially bone (osteotropism), where they alter the physiological balance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption, and elicit prevalently an osteoblastic response (osteoinduction). The molecular cues provided by osteoblasts for the survival and growth of bone metastatic prostate cancer cells are largely unknown. We exploited the sufficient divergence between human and mouse RNA sequences together with redefinition of highly species-specific gene arrays by computer-aided and experimental exclusion of cross-hybridizing oligonucleotide probes. This strategy allowed the dissection of the stroma (mouse) from the cancer cell (human) transcriptome in bone metastasis xenograft models of human osteoinductive prostate cancer cells (VCaP and C4-2B). As a result, we generated the osteoblastic bone metastasis-associated stroma transcriptome (OB-BMST). Subtraction of genes shared by inflammation, wound healing and desmoplastic responses, and by the tissue type-independent stroma responses to a variety of non-osteotropic and osteotropic primary cancers generated a curated gene signature ("Core" OB-BMST) putatively representing the bone marrow/bone-specific stroma response to prostate cancer-induced, osteoblastic bone metastasis. The expression pattern of three representative Core OB-BMST genes (PTN, EPHA3 and FSCN1) seems to confirm the bone specificity of this response. A robust induction of genes involved in osteogenesis and angiogenesis dominates both the OB-BMST and Core OB-BMST. This translates in an amplification of hematopoietic and, remarkably, prostate epithelial stem cell niche components that may function as a self-reinforcing bone metastatic niche providing a growth support specific for osteoinductive prostate cancer cells. The induction of this combinatorial stem cell niche is a novel mechanism that may also explain cancer cell osteotropism and local interference with hematopoiesis (myelophthisis). Accordingly, these stem cell niche components may represent innovative therapeutic targets and/or serum biomarkers in osteoblastic bone metastasis.

Loss of tumor suppressor mir-203 mediates overexpression of LIM and SH3 Protein 1 (LASP1) in high-risk prostate cancer thereby increasing cell proliferation and migration

Several studies have linked overexpression of the LIM and SH3 domain protein 1 (LASP1) to progression of breast, colon, liver, and bladder cancer. However, its expression pattern and role in human prostate cancer (PCa) remained largely undefined. Analysis of published microarray data revealed a significant overexpression of LASP1 in PCa metastases compared to parental primary tumors and normal prostate epithelial cells. Subsequent gene-set enrichment analysis comparing LASP1-high and -low PCa identified an association of LASP1 with genes involved in locomotory behavior and chemokine signaling. These bioinformatic predictions were confirmed in vitro as the inducible short hairpin RNA-mediated LASP1 knockdown impaired migration and proliferation in LNCaP prostate cancer cells. By immunohistochemical staining and semi-quantitative image analysis of whole tissue sections we found an enhanced expression of LASP1 in primary PCa and lymph node metastases over benign prostatic hyperplasia. Strong cytosolic and nuclear LASP1 immunoreactivity correlated with PSA progression. Conversely, qRT-PCR analyses for mir-203, which is a known translational suppressor of LASP1 in matched RNA samples revealed an inverse correlation of LASP1 protein and mir-203 expression. Collectively, our results suggest that loss of mir-203 expression and thus uncontrolled LASP1 overexpression might drive progression of PCa.

Heterogeneity analysis of Metastasis Associated in Colon Cancer 1 (MACC1) for survival prognosis of colorectal cancer patients: a retrospective cohort study.

BACKGROUND Metastasis of colorectal cancer (CRC) is directly linked to patient survival. We previously identified the novel gene Metastasis Associated in Colon Cancer 1 (MACC1) in CRC and demonstrated its importance as metastasis inducer and prognostic biomarker. Here, we investigate the geographic expression pattern of MACC1 in colorectal adenocarcinoma and tumor buds in correlation with clinicopathological and molecular features for improvement of survival prognosis. METHODS We performed geographic MACC1 expression analysis in tumor center, invasive front and tumor buds on whole tissue sections of 187 well-characterized CRCs by immunohistochemistry. MACC1 expression in each geographic zone was analyzed with Mismatch repair (MMR)-status, BRAF/KRAS-mutations and CpG-island methylation. RESULTS MACC1 was significantly overexpressed in tumor tissue as compared to normal mucosa (p < 0.001). Within colorectal adenocarcinomas, a significant increase of MACC1 from tumor center to front (p = 0.0012) was detected. MACC1 was highly overexpressed in 55% tumor budding cells. Independent of geographic location, MACC1 predicted advanced pT and pN-stages, high grade tumor budding, venous and lymphatic invasion (p < 0.05). High MACC1 expression at the invasive front was decisive for prediction of metastasis (p = 0.0223) and poor survival (p = 0.0217). The geographic pattern of MACC1 did not correlate with MMR-status, BRAF/KRAS-mutations or CpG-island methylation. CONCLUSION MACC1 is differentially expressed in CRC. At the invasive front, MACC1 expression predicts best aggressive clinicopathological features, tumor budding, metastasis formation and poor survival outcome.

Two enzymes responsible for the formation of herbivore-induced volatiles of maize, the methyltransferase AAMT1 and the terpene synthase TPS23, are regulated by a similar signal transduction pathway

Herbivore-induced volatiles play an important role in the indirect defense of plants. After herbivore damage, volatiles are released from the plant and can attract herbivore enemies that protect the plant from additional damage. The herbivore-induced volatile blend is complex and usually consists of mono- and sesquiterpenes, aromatic compounds, and indole. Although these classes of compounds are generally produced at different times after herbivore damage, the release of the terpene (E)-β-caryophyllene and the aromatic ester methyl anthranilate appear to be tightly coordinated. We have studied the herbivore induction patterns of two terpene synthases from Zea mays L. (Poaceae), TPS23 and TPS10, as well as S-adenosyl-L-methionine:anthranilic acid carboxyl methyltransferases (AAMT1), which are critical for the production of terpenes and anthranilate compounds, respectively. The transcript levels of tps23 and aamt1 displayed the same kinetics after damage by the larvae of Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae), and showed the same organ-specific and haplotype-specific expression patterns. Despite its close functional relation to TPS23, the terpene synthase TPS10 is not expressed in roots and does not display the haplotype-specific expression pattern. The results indicate that the same JA-mediated signaling cascade maycontrol the production of both the terpene (E)-β-caryophyllene and aromatic ester methyl anthranilate.

Transcript levels of AtMRPs after cadmium treatment: induction of AtMRP3

In yeasts, the ABC-type transporters are involved in vacuolar sequestration of cadmium. In plants, transport experiments with isolated vacuoles indicate that this is also true. In order to know more about the response of AtMRPs, a subclass of Arabidopsis ABC transporters, to cadmium, their expression pattern was analysed using the microchip technology and semi-quantitative reverse transcriptase-polymerase chain reaction. From 15 putative sequences coding for AtMRPs, transcript levels were detected for 14. All were expressed in the roots as well as in the shoots, although at a different level. In 4-week-old Arabidopsis, transcript levels of four AtMRPs were up-regulated after cadmium treatment. In all cases up-regulation was exclusively observed in the roots. The increase of transcript levels was most pronounced for AtMRP3. A more detailed analysis revealed that induction of AtMRP3 could also be observed in the shoot when leaves were cut and cadmium allowed to be taken up in the shoot. In young plantlets, a far higher portion of Cd2+ was translocated in the aerial part compared with adult plants. Consequently, AtMRP3 transcript levels increased in both root and shoot of young plants. This suggests that 7-day-old seedlings do not exhibit such a strict root–shoot barrier as 4-week-old plants. Expression analysis with mutant plants for glutathione and phytochelatin synthesis as well as with compounds producing oxidative stress indicate that induction of AtMRP3 is likely due to the heavy metal itself.

Can widely used cell type markers predict the suitability of immortalized or primary mammary epithelial cell models?

BACKGROUND Mammary cell cultures are convenient tools for in vitro studies of mammary gland biology. However, the heterogeneity of mammary cell types, e.g., glandular milk secretory epithelial or myoepithelial cells, often complicates the interpretation of cell-based data. The present study was undertaken to determine the relevance of bovine primary mammary epithelial cells isolated from American Holstein (bMECUS) or Swiss Holstein-Friesian (bMECCH) cows, and of primary bovine mammary alveolar epithelial cells stably transfected with simian virus-40 (SV-40) large T-antigen (MAC-T) for in vitro analyses. This was evaluated by testing their expression pattern of cytokeratin (CK) 7, 18, 19, vimentin, and α-smooth muscle actin (α-SMA). RESULTS The expression of the listed markers was assessed using real-time quantitative PCR, flow cytometry and immunofluorescence microscopy. Characteristic markers of the mesenchymal (vimentin), myoepithelial (α-SMA) and glandular secretory cells (CKs) showed differential expression among the studied cell cultures, partly depending on the analytical method used. The relative mRNA expression of vimentin, CK7 and CK19, respectively, was lower (P < 0.05) in immortalized than in primary mammary cell cultures. The stain index (based on flow cytometry) of CK7 and CK19 protein was lower (P < 0.05) in MAC-T than in bMECs, while the expression of α-SMA and CK18 showed an inverse pattern. Immunofluorescence microscopy analysis mostly confirmed the mRNA data, while partly disagreed with flow cytometry data (e.g., vimentin level in MAC-T). The differential expression of CK7 and CK19 allowed discriminating between immortal and primary mammary cultures. CONCLUSIONS The expression of the selected widely used cell type markers in primary and immortalized MEC cells did not allow a clear preference between these two cell models for in vitro analyses studying aspects of milk composition. All tested cell models exhibited to a variable degree epithelial and mesenchymal features. Thus, based on their characterization with widely used cell markers, none of these cultures represent an unequivocal alveolar mammary epithelial cell model. For choosing the appropriate in vitro model additional properties such as the expression profile of specific proteins of interest (e.g., transporter proteins) should equally be taken into account.

Loss of Nogo-A-expressing neurons in a rat model of Parkinson's disease

The myelin-associated protein Nogo-A is among the most potent neurite growth inhibitors in the adult CNS. Recently, Nogo-A expression was demonstrated in a number of neuronal subpopulations of the adult and developing CNS but at present, little is known about the expression of Nogo-A in the nigrostriatal system, a brain structure severely affected in Parkinson's disease (PD). The present study sought to characterize the expression pattern of Nogo-A immunoreactive (ir) cells in the adult ventral mesencephalon of control rats and in the 6-hydroxydopamine (6-OHDA) rat model of PD. Immunohistochemical analyses of normal adult rat brain showed a distinct expression of Nogo-A in the ventral mesencephalon, with the highest level in the substantia nigra pars compacta (SNc) where it co-localized with dopaminergic neurons. Analyses conducted 1week and 1 month after unilateral striatal injections of 6-OHDA disclosed a severe loss of the number of Nogo-A-ir cells in the SNc. Notably, at 1week after treatment, more dopaminergic neurons expressing Nogo-A were affected by the 6-OHDA toxicity than Nogo-A-negative dopaminergic neurons. However, at later time points more of the surviving dopaminergic neurons expressed Nogo-A. In the striatum, both small and large Nogo-A-positive cells were detected. The large cells were identified as cholinergic interneurons. Our results suggest yet unidentified functions of Nogo-A in the CNS beyond the inhibition of axonal regeneration and plasticity, and may indicate a role for Nogo-A in PD.

A new eosinophilic esophagitis (EoE)-like disease without tissue eosinophilia found in EoE Families

BACKGROUND Eosinophilic esophagitis (EoE) is a rapidly emerging, chronic inflammatory, genetically impacted disease of the esophagus, defined clinically by symptoms of esophageal dysfunction and, pathologically, by an eosinophil-predominant tissue infiltration. However, in four EoE-families, we have identified patients presenting with EoE-typical and corticosteroid-responsive symptoms, but without tissue eosinophilia. It was the aim of this study to clinically and immunologically characterize these patients with EoE-like disease. METHODS Five patients suffering from an EoE-like disease were evaluated with endoscopic, histologic, functional and quantitative immunohistologic examinations, and mRNA expression determination. RESULTS The frequency of first generation offspring of EoE-like disease patients affected by EoE or EoE-like disease was 40%. Immunofluorescence analysis confirmed an almost complete absence of eosinophils in the esophageal tissues of patients with EoE-like disease, but revealed a considerable T cell infiltration, comparable to EoE. In contrast to EoE, eotaxin-3 mRNA and protein were markedly reduced in EoE-like disease (P < 0.05). The mRNA expression levels of three selected EoE genes (eotaxin-3, MUC4 and CDH26) allowed to discriminate between EoE-like disease, EoE and normal epithelium. CONCLUSIONS Patients suffering from "EoE without eosinophilia" do not fulfill formally the diagnostic criteria for EoE. However, their clinical manifestation, immunohistology and gene-expression pattern, plus the fact that they bequeath EoE to their offspring, suggest a uniform underlying pathogenesis. Conventional EoE, with its prominent eosinophilia, therefore appears to be only one phenotype of a broader "inflammatory dysphagia syndrome" spectrum. In this light, the role of the eosinophils, the definition of EoE, and its diagnostic criteria must likely be reconsidered. This article is protected by copyright. All rights reserved.

The Trypanosoma brucei MitoCarta and its regulation and splicing pattern during development

It has long been known that trypanosomes regulate mitochondrial biogenesis during the life cycle of the parasite; however, the mitochondrial protein inventory (MitoCarta) and its regulation remain unknown. We present a novel computational method for genome-wide prediction of mitochondrial proteins using a support vector machine-based classifier with approximately 90% prediction accuracy. Using this method, we predicted the mitochondrial localization of 468 proteins with high confidence and have experimentally verified the localization of a subset of these proteins. We then applied a recently developed parallel sequencing technology to determine the expression profiles and the splicing patterns of a total of 1065 predicted MitoCarta transcripts during the development of the parasite, and showed that 435 of the transcripts significantly changed their expressions while 630 remain unchanged in any of the three life stages analyzed. Furthermore, we identified 298 alternatively splicing events, a small subset of which could lead to dual localization of the corresponding proteins.

Expression and localization pattern of ABCA1 in diverse human placental primary cells and tissues

The ATP-binding cassette transporter A1 (ABCA1) mediates the transport of cholesterol, phospholipids, and other lipophilic molecules across cellular membranes. Recent data provide evidence that ABCA1 plays an important role in placental function but the exact cellular sites of ABCA1 action in the placenta remain controversial. To clarify this issue, we analyzed the cellular and subcellular localization of ABCA1 with immunocytochemistry, immunofluorescence and subsequent confocal or immunofluorescence microscopy in different types of isolated primary placenta cells: cytotrophoblast cells, amnion epithelial cells, villous macrophages (Hofbauer cells), and mesenchymal cells isolated from chorionic membrane and placental villi. After 12 h of cultivation, primary cytotrophoblast cells showed intensive membrane and cytoplasmic staining for ABCA1. After 24 h, with progressive syncytium formation, ABCA1 staining intensity was markedly reduced and ABCA1 was dispersed in the cytoplasm of the forming syncytial layer. In amnion epithelial cells, placental macrophages and mesenchymal cells, ABCA1 was predominantly localized at the cell membrane and cytoplasmic compartments partially corresponding to the endoplasmic reticulum. In these cell types, the ABCA1 staining intensity was not dependent on the cultivation time. In conclusion, ABCA1 shows marked expression levels in diverse placental cell types. The multitopic localization of ABCA1 in diverse human placental cells not all directly involved in materno-fetal exchange suggests that this protein may not only participate in transplacental lipid transport but could have additional regulatory functions.