Upregulation of toll-like receptors in chronic enteropathies in dogs

Background: Inflammatory bowel disease (IBD) is thought to result from a dysregulated interaction between the host immune system and commensal microflora. Toll-like receptors (TLRs) recognize microbe-associated molecular patterns (MAMPs), but their role in enteropathies in dogs is unknown. Hypothesis: That there is a dysregulation of TLRs recognizing bacterial MAMPs in dogs with IBD. Animals: Sixteen healthy beagles and 12 dogs with steroid-treated (ST) and 23 dogs with food-responsive (FR) diarrhea. Methods: Prospective, observational study. mRNA expression of canine TLR2, 4, and 9 was evaluated by quantitative real-time RT-PCR in duodenal and colonic biopsies obtained before and after standard therapy. Samples from control dogs were taken at necropsy, with additional biopsies of stomach, jejunum, ileum, and mesenteric lymph node in 6 dogs. Results: There were significant differences (P small intestine >/= colon). Before therapy, ST expressed more mRNA than control dogs for all 3 receptors (P < .05). There were no significant differences between pretreatment and posttreatment values, even though 32/35 dogs improved clinically. No associations were found when comparing receptor mRNA expression with either histology or clinical activity scores. Conclusions and Clinical Importance: Bacteria-responsive TLR2, 4, and 9 are upregulated in duodenal and colonic mucosa in IBD. This might lead to increased inflammation through interaction with the commensal flora. The absence of significant changes after therapy despite clinical improvement might point toward the existence of a genetic predisposition to IBD as described in human IBD.

Metalloprotease meprin beta in rat kidney: glomerular localization and differential expression in glomerulonephritis

Meprin (EC 3.4.24.18) is an oligomeric metalloendopeptidase found in microvillar membranes of kidney proximal tubular epithelial cells. Here, we present the first report on the expression of meprin beta in rat glomerular epithelial cells and suggest a potential involvement in experimental glomerular disease. We detected meprin beta in glomeruli of immunostained rat kidney sections on the protein level and by quantitative RT-PCR of laser-capture microdissected glomeruli on the mRNA level. Using immuno-gold staining we identified the membrane of podocyte foot processes as the main site of meprin beta expression. The glomerular meprin beta expression pattern was altered in anti-Thy 1.1 and passive Heymann nephritis (PHN). In addition, the meprin beta staining pattern in the latter was reminiscent of immunostaining with the sheep anti-Fx1A antiserum, commonly used in PHN induction. Using Western blot and immunoprecipitation assays we demonstrated that meprin beta is recognized by Fx1A antiserum and may therefore represent an auto-antigen in PHN. In anti-Thy 1.1 glomerulonephritis we observed a striking redistribution of meprin beta in tubular epithelial cells from the apical to the basolateral side and the cytosol. This might point to an involvement of meprin beta in this form of glomerulonephritis.

Agents used for chemoprevention of prostate cancer may influence PSA secretion independently of cell growth in the LNCaP model of human prostate cancer progression

BACKGROUND: The aim of this study was to evaluate the inhibitory growth effects of different potential chemopreventive agents in vitro and to determine their influence on PSA mRNA and protein expression with an established screening platform. METHODS: LNCaP and C4-2 cells were incubated with genistein, seleno-L-methionine, lycopene, DL-alpha-tocopherol, and trans-beta-carotene at three different concentrations and cell growth was determined by the MTT assay. PSA mRNA expression was assessed by quantitative real-time RT-PCR and secreted PSA protein levels were quantified by the microparticle enzyme immunoassay. RESULTS: Genistein, seleno-l-methionine and lycopene inhibited LNCaP cell growth, and the proliferation of C4-2 cells was suppressed by seleno-L-methionine and lycopene. PSA mRNA expression was downregulated by genistein in LNCaP but not C4-2 cells. No other compound tested altered PSA mRNA expression. PSA protein expression was downregulated by genistein, seleno-L-methionine, DL-alpha-tocopherol in LNCaP cells. In C4-2 cells only genistein significantly reduced the secretion of PSA protein. CONCLUSIONS: In the LNCaP progression model PSA expression depends on the compound, its concentration and on the hormonal dependence of the cell line used and does not necessarily reflect cell growth or death. Before potential substances are evaluated in clinical trials using PSA as a surrogate end point marker, their effect on PSA mRNA and protein expression has to be considered to correctly assess treatment response by PSA.

Eotaxin/CCL11 expression by infiltrating macrophages in rat heart transplants during ongoing acute rejection

Eotaxin/CCL11 chemokine is expressed in different organs, including the heart, but its precise cellular origin in the heart is unknown. Eotaxin is associated with Th2-like responses and exerts its chemotactic effect through the chemokine receptor-3 (CCR3), which is also expressed on mast cells (MC). The aim of our study was to find the cellular origin of eotaxin in the heart, and to assess whether expression is changing during ongoing acute heart transplant rejection, indicating a correlation with mast cell infiltration which we observed in a previous study. In a model of ongoing acute heart transplant rejection in the rat, we found eotaxin mRNA expression within infiltrating macrophages, but not in mast cells, by in situ-hybridization. A five-fold increase in eotaxin protein in rat heart transplants during ongoing acute rejection was measured on day 28 after transplantation, compared to native and isogeneic control hearts. Eotaxin concentrations in donor hearts on day 28 after transplantation were significantly higher compared to recipient hearts, corroborating an origin of eotaxin from cells within the heart, and not from the blood. The quantitative comparison of eotaxin mRNA expression between native hearts, isografts, and allografts, respectively, revealed no statistically significant difference after transplantation, probably due to an overall increase in the housekeeping gene's 18S rRNA during rejection. Quantitative RT-PCR showed an increase in mRNA expression of CCR3, the receptor for eotaxin, during ongoing acute rejection of rat heart allografts. Although a correlation between increasing eotaxin expression by macrophages and mast cell infiltration is suggestive, functional studies will elucidate the role of eotaxin in the process of ongoing acute heart transplant rejection.

Effect of testosterone on E1S-sulfatase activity in non-malignant and cancerous breast cells in vitro

INTRODUCTION: Testosterone (T) is a therapeutic option for women with hypoactive sexual desire disorder. T may have an impact on the mammary gland by altering local estrogen synthesis. The aim of the present study was to measure the effect of T on estrone-sulfate (E1S)-sulfatase (STS) expression, and activity using hormone-dependent BC cells with high and low aggressive potential (BT-474, MCF-7), and HBL-100 as a breast cell line of non-malignant origin. METHODS: Cells were incubated in RPMI 1640 medium containing 5% steroid-depleted fetal calf serum for 3d, and subsequently incubated in absence or presence of T alone, and combined with anastrozole (A) at 10(-8)M, and 10(-6)M at 37 degrees C for either 24h or directly in cell extracts ("direct"). STS protein expression was measured by dot-blot (immunoblotting), and STS, HSD17B1 and HSD17B2 mRNA levels by quantitative RT-PCR. STS activity was evaluated by incubating homogenized breast cells with [(3)H]-E1S and separating the products E1, and E2 by thin layer chromatography. RESULTS: Basal STS mRNA expression did not reveal group differences. However, STS mRNA was decreased by T+A in MCF-7 cells. 17HSDB1 expression was decreased by T+A in BT-474 cells, and 17HSDB2 expression was decreased by A and T+A treatment in MCF-7 cells. Basal and T treated STS protein expression was significantly higher in malignant compared to non-malignant breast cells. However, T did not induce significant intra-cell line differences. Similarly, basal and T treated STS activity was significantly higher in highly malignant compared to non-malignant breast cells. Regardless of cell lines, T slightly decreased STS activity after "direct" incubation, but led to an increase of local estrogen formation after 24h which was attenuated, and partly reversed by A, respectively. CONCLUSIONS: The more aggressive the breast cell line, the higher the local estrogen formation. The transition from normal to malignant seems to be accompanied by an altered autoregulation. The given local endocrine milieu seems to be essential for response to T.

Vascular endothelial growth factors, angiogenesis, and survival in human ileal enterochromaffin cell carcinoids

BACKGROUND AND AIMS: Well-differentiated neuro-endocrine ileal carcinoids are composed of serotonin-producing enterochromaffin (EC) cells. Life expectancy is determined by metastatic spread to the liver because medical treatment options are still very limited. Selective inhibition of angiogenesis or lymphangiogenesis might prevent tumour growth and metastatic spread. We examined the role of the vascular endothelial growth factors (VEGFs) A, B, C, D, and their receptors (VEGFRs) 1, 2, 3 in angiogenesis and lymphangiogenesis of ileal EC cell carcinoids with and without liver metastases. METHODS: The expression of various VEGFs and VEGFRs was determined by quantitative real-time RT-PCR in healthy mucosa, primary tumour, lymph node metastases and liver metastases of 25 patients with ileal EC cell carcinoids. Microvessel density (MVD) was determined by CD-31 staining in primary tumours and lymphatic vessel density (LVD) by LYVE-1 staining. VEGF expression levels, MVD, LVD, and patients' survival time were correlated using logistic regression and Kaplan-Meier survival analysis. RESULTS: VEGF-A was highly expressed with no difference between normal mucosa and tumours. VEGF-B and -D as well as VEGFR-1 and -2 expression levels were significantly increased in the tumours when compared to normal mucosa. Patients with liver metastasis, however, had a significantly lower expression of the factors A, B, and C and the receptors 2 and 3. MVD in primary tumours positively correlated with the expression of VEGF ligands and their receptors, except for VEGF-D. LVD did not correlate with any VEGF ligand or receptor. Interestingly, low expression levels of VEGF-B were associated with poor survival. CONCLUSION: Patients with more aggressive metastatic spreading had relatively decreased expression levels of VEGF ligands and receptors. Thus, anti-angiogenic therapy may not be a suitable target in metastatic ileal EC cell carcinoids.

Impact of beta-galactosidase mutations on the expression of the canine lysosomal multienzyme complex

beta-galactosidase (GLB1) forms a functional lysosomal multienzyme complex with lysosomal protective protein (PPCA) and neuraminidase 1 (NEU1) which is important for its intracellular processing and activity. Mutations in the beta-galactosidase gene cause the lysosomal storage disease G(M1)-gangliosidosis. In order to identify additional molecular changes associated with the presence of beta-galactosidase mutations, the expression of canine lysosomal multienzyme complex components in GLB1(+/+), GLB1(+/-) and GLB1(-/-) fibroblasts was investigated by quantitative RT-PCR, Western blot and enzymatic assays. Quantitative RT-PCR revealed differential regulation of total beta-galactosidase, beta-galactosidase variants and protective protein for beta-galactosidase gene (PPGB) in GLB1(+/-) and GLB1(-/-) compared to GLB1(+/+) fibroblasts. Furthermore, it was shown that PPGB levels gradually increased with the number of mutant beta-galactosidase alleles while no change in the NEU1 expression was observed. This is the first study that simultaneously examine the effect of GLB1(+/+), GLB1(+/-) and GLB1(-/-) genotypes on the expression of lysosomal multienzyme complex components. The findings reveal a possible adaptive process in GLB1 homozygous mutant and heterozygous individuals that could facilitate the design of efficient therapeutic strategies.

Differential association of MUC5AC and CLCA1 expression in small cartilaginous airways of RAO-affected and control horses

REASONS FOR PERFORMING STUDY: Airway mucus accumulation is associated with indoor irritant and allergen exposure in horses with recurrent airway obstruction (RAO). Epidermal growth factor receptor (EGFR) and a chloride channel (calcium activated, family member 1; CLCA1) are key signalling molecules involved in mucin gene expression. OBJECTIVES: We hypothesised that exposure to irritants and aeroallergens would lead to increased expression of the mucin gene eqMUC5AC and increased stored mucosubstance in the airways of RAO-affected horses, associated with increased neutrophils and CLCA1 and EGFR mRNA levels. METHODS: We performed quantitative RT-PCR of eqMUC5AC, CLCA1 and EGFR; volume density measurements of intraepithelial mucosubstances; and cytological differentiation of intraluminal inflammatory cells in small cartilaginous airways from cranial left and right and caudal left and right lung lobes of 5 clinically healthy and 5 RAO-affected horses that had been exposed to indoor stable environment for 5 days before euthanasia. RESULTS: Neutrophils were increased in RAO-affected horses compared to clinically healthy controls. EqMUC5AC mRNA levels were positively correlated with both CLCA1 and EGFR mRNA levels in RAO-affected horses but only with CLCA1 in controls. The relationship between eqMUC5AC and CLCA1 differed in the 2 groups of horses with RAO-affected animals overexpressing CLCA1 in relation to eqMUC5AC. CONCLUSIONS: These data implicate CLCA1 as a signalling molecule in the expression of eqMUC5AC in horses but also suggest differential regulation by CLCA1 and EGFR between horses with RAO and those with milder degrees of airway inflammation.

Tooth eruption: altered gene expression in the dental follicle of patients with cleidocranial dysplasia

OBJECTIVES The dental follicle plays an important role in tooth eruption by providing key regulators of osteogenesis and bone resorption. Patients with cleidocranial dysplasia (CCD) exhibit delayed tooth eruption in combination with increased bone density in the maxilla and mandible, suggesting disturbances in bone remodeling. The aim of this study was to determine the expression of genes relevant for tooth eruption and bone remodeling in the dental follicles of patients with CCD and normal subjects. MATERIAL AND METHODS Thirteen dental follicles were isolated from five unrelated patients with CCD, and fourteen dental follicles were obtained from 10 healthy individuals. All teeth were in the intraosseous phase of eruption. The expression of RANK, RANKL, OPG, and CSF-1 was determined by quantitative RT-PCR. RESULTS In patients with CCD, the mRNA levels of RANK, OPG, and CSF-1 were significantly elevated compared with the control group. Accordingly, the ratios of RANKL/OPG and RANKL/RANK mRNAs were significantly decreased in patients with CCD. CONCLUSION The observed alterations in the expression and ratios of the aforementioned factors in the dental follicle of CCD individuals suggest a disturbed paracrine signaling for bone remodeling that could be responsible for the impaired tooth eruption seen in these patients.

Identification and expression of different dehydrin subclasses involved in the drought response of Trifolium repens

Reverse transcribed RNAs coding for YnKn, YnSKn, SKn, and KS dehydrin types in drought-stressed white clover (Trifolium repens) were identified and characterized. The nucleotide analyses revealed the complex nature of dehydrin-coding sequences, often featured with alternative start and stop codons within the open reading frames, which could be a prerequisite for high variability among the transcripts originating from a single gene. For some dehydrin sequences, the existence of natural antisense transcripts was predicted. The differential distribution of dehydrin homologues in roots and leaves from a single white clover stolon under normal and drought conditions was evaluated by semi-quantitative RT-PCR and immunoblots with antibodies against the conserved K-, Y- and S-segments. The data suggest that different dehydrin classes have distinct roles in the drought stress response and vegetative development, demonstrating some specific characteristic features. Substantial levels of YSK-type proteins with different molecular weights were immunodetected in the non-stressed developing leaves. The acidic SK2 and KS dehydrin transcripts exhibited some developmental gradient in leaves. A strong increase of YK transcripts was documented in the fully expanded leaves and roots of drought-stressed individuals. The immunodetected drought-induced signals imply that Y- and K-segment containing dehydrins could be the major inducible Late Embryogenesis Abundant class 2 proteins (LEA 2) that accumulate predominantly under drought.

RNA-Seq-based analysis of the physiologic cold shock-induced changes in Moraxella catarrhalis gene expression

BACKGROUND Moraxella catarrhalis, a major nasopharyngeal pathogen of the human respiratory tract, is exposed to rapid downshifts of environmental temperature when humans breathe cold air. The prevalence of pharyngeal colonization and respiratory tract infections caused by M. catarrhalis is greatest in winter. We investigated how M. catarrhalis uses the physiologic exposure to cold air to regulate pivotal survival systems that may contribute to M. catarrhalis virulence. RESULTS In this study we used the RNA-seq techniques to quantitatively catalogue the transcriptome of M. catarrhalis exposed to a 26 °C cold shock or to continuous growth at 37 °C. Validation of RNA-seq data using quantitative RT-PCR analysis demonstrated the RNA-seq results to be highly reliable. We observed that a 26 °C cold shock induces the expression of genes that in other bacteria have been related to virulence a strong induction was observed for genes involved in high affinity phosphate transport and iron acquisition, indicating that M. catarrhalis makes a better use of both phosphate and iron resources after exposure to cold shock. We detected the induction of genes involved in nitrogen metabolism, as well as several outer membrane proteins, including ompA, m35-like porin and multidrug efflux pump (acrAB) indicating that M. catarrhalis remodels its membrane components in response to downshift of temperature. Furthermore, we demonstrate that a 26 °C cold shock enhances the induction of genes encoding the type IV pili that are essential for natural transformation, and increases the genetic competence of M. catarrhalis, which may facilitate the rapid spread and acquisition of novel virulence-associated genes. CONCLUSION Cold shock at a physiologically relevant temperature of 26 °C induces in M. catarrhalis a complex of adaptive mechanisms that could convey novel pathogenic functions and may contribute to enhanced colonization and virulence.

Expression of thymic stromal lymphopoietin in canine atopic dermatitis

BACKGROUND In humans, thymic stromal lymphopoietin (TSLP) plays a central role in the development of allergic inflammation, such as atopic dermatitis (AD), but it is unknown whether it is involved in the pathogenesis of canine AD (CAD). HYPOTHESIS/OBJECTIVES Our aim was to characterize canine TSLP and to assess its expression in CAD. METHODS Canine TSLP was identified based on sequence homology with human TSLP and the complementary DNA (cDNA) cloned by RT-PCR. Real-time quantitative RT-PCR was established to assess the expression of canine TSLP in cultured canine keratinocytes and in skin biopsy specimens from lesional and nonlesional skin of 12 dogs with CAD and eight healthy control dogs. RESULTS Partial canine TSLP cDNA was cloned and characterized. It contained four exons that shared 70 and 73% nucleotide identity with human and equine TSLP, respectively, encoding the signal peptide and full-length secreted protein. We found significantly increased TSLP expression in lesional and nonlesional skin of dogs with CAD compared with healthy control dogs (P < 0.05), whereas no difference was measured between lesional and nonlesional samples. In cultured primary canine keratinocytes, we found increased TSLP expression after stimulation with house dust mite allergen extract or Toll-like receptor ligands lipopolysaccharide and poly I:C. CONCLUSIONS AND CLINICAL IMPORTANCE Increased TSLP expression in the skin of dogs with CAD supports an involvement of TSLP in the pathogenesis of CAD similar to that in humans. Further studies should elucidate the function and therapeutic potential of TSLP in CAD.

A specific expression profile of heat-shock proteins and glucose-regulated proteins is associated with response to neoadjuvant chemotherapy in oesophageal adenocarcinomas

BACKGROUND Oesophageal adenocarcinomas often show resistances to chemotherapy (CTX), therefore, it would be of high interest to better understand the mechanisms of resistance. We examined the expression of heat-shock proteins (HSPs) and glucose-regulated proteins (GRPs) in pretherapeutic biopsies of oesophageal adenocarcinomas to assess their potential role in CTX response. METHODS Ninety biopsies of locally advanced adenocarcinomas before platin/5-fluorouracil (FU)-based CTX were investigated by reverse phase protein arrays (RPPAs), immunohistochemistry (IHC) and quantitative RT-PCR. RESULTS CTX response strongly correlated with survival (P=0.001). Two groups of tumours with specific protein expression patterns were identified by RPPA: Group A was characterised by low expression of HSP90, HSP27 and p-HSP27((Ser15, Ser78, Ser82)) and high expression of GRP78, GRP94, HSP70 and HSP60; Group B exhibited the inverse pattern. Tumours of Group A were more likely to respond to CTX, resulting in histopathological tumour regression (P=0.041) and post-therapeutic down-categorisation from cT3 to ypT0-T2 (P=0.040). High HSP60 protein (IHC) and mRNA expression were also associated with tumour down-categorisation (P=0.016 and P=0.004). CONCLUSION Our findings may enhance the understanding of CTX response mechanisms, might be helpful to predict CTX response and might have translational relevance as they highlight the role of potentially targetable cellular stress proteins in the context of CTX response.

Investigation of border disease and bovine virus diarrhoea in sheep from 76 mixed cattle and sheep farms in eastern Switzerland

The purpose of this study was to examine the occurrence of sheep persistently infected with Border disease virus (BDV) on 76 mixed cattle and sheep farms and whether seroconversion to BDV infection occurred in cattle of these farms. Seroprevalence of BDV and bovine viral disease virus (BVDV) infection in sheep was also investigated. Quantitative RT-PCR for pestivirus detection and an ELISA to detect pestivirus antibodies were used in 2'384 and 2'291 ovine blood samples, respectively. Another 27 seropositive sheep from ten flocks underwent serum neutralization testing to differentiate between BDV and BVDV antibodies. A BDV titre that was at least four times higher than the BVDV titre was interpreted as the result of BDV infection. Titres against BVDV were interpreted in an analogous fashion. All examined sheep were pestivirus-negative, 310 sheep were seropositive, 119 had an indeterminate titre and 1'862 were seronegative. The flock seroprevalence ranged from 0.0 to 73.9 %. Three of the 27 flocks that underwent serum neutralization testing were interpreted as BDV-infected because of 6 sheep with higher BDV titres, and 6 flocks were interpreted as BVDV-infected because of 14 sheep with higher BVDV titres.

Clinical impact of programmed cell death ligand 1 expression in colorectal cancer

BACKGROUND Programmed cell death 1 (PD-1) receptor triggering by PD ligand 1 (PD-L1) inhibits T cell activation. PD-L1 expression was detected in different malignancies and associated with poor prognosis. Therapeutic antibodies inhibiting PD-1/PD-L1 interaction have been developed. MATERIALS AND METHODS A tissue microarray (n=1491) including healthy colon mucosa and clinically annotated colorectal cancer (CRC) specimens was stained with two PD-L1 specific antibody preparations. Surgically excised CRC specimens were enzymatically digested and analysed for cluster of differentiation 8 (CD8) and PD-1 expression. RESULTS Strong PD-L1 expression was observed in 37% of mismatch repair (MMR)-proficient and in 29% of MMR-deficient CRC. In MMR-proficient CRC strong PD-L1 expression correlated with infiltration by CD8(+) lymphocytes (P=0.0001) which did not express PD-1. In univariate analysis, strong PD-L1 expression in MMR-proficient CRC was significantly associated with early T stage, absence of lymph node metastases, lower tumour grade, absence of vascular invasion and significantly improved survival in training (P=0.0001) and validation (P=0.03) sets. A similar trend (P=0.052) was also detectable in multivariate analysis including age, sex, T stage, N stage, tumour grade, vascular invasion, invasive margin and MMR status. Interestingly, programmed death receptor ligand 1 (PDL-1) and interferon (IFN)-γ gene expression, as detected by quantitative reverse transcriptase polymerase chain reaction (RT-PCR) in fresh frozen CRC specimens (n=42) were found to be significantly associated (r=0.33, P=0.03). CONCLUSION PD-L1 expression is paradoxically associated with improved survival in MMR-proficient CRC.