A comparative analysis of phenotype expression in human osteoblasts from heterotopic ossification and normal bone

BACKGROUND AND AIMS: Heterotopic ossification (HO) is a pathological bone formation process in which ectopic bone is formed in soft tissue. The formation of bone depends on the expression of the osteoblast phenotype. Earlier studies have shown conflicting results on the expression of phenotype markers of cells originating from HO and normal bone. The hypothesis of the present study is that cells from HO show an altered expression of osteoblast-specific phenotype markers compared to normal osteoblasts. The aims of the study were to further characterize the expression of osteoblast phenotypemarkers and to provide a comparison with other study results. PATIENTS AND METHODS: Using an in vitro technique, reverse transcription polymerase chain reaction (RT-PCR), real-time PCR and immunohistochemistry, we compared the phenotype gene expression (type I collagen, alkaline phosphatase, Cbfa-1, osteocalcin) of osteoblasts from resected HO and normal bone (iliac crest). RESULTS: Cells from HO expressed the osteoblast phenotype (type I collagen, alkaline phosphatase) but were characterized by a depleted osteocalcin expression. The expression of Cbfa-1 (osteocalcin transcription gene) showed a large variety in our study. Preoperative radiotherapy had no effect on phenotype expression in cells from HO. CONCLUSION: Our results provide a characterization of cells originating from HO and support the thesis of an impaired osteoblast differentiation underlying the formation of HO. The transcription axis from Cbfa-1 to osteocalcin could be involved in the pathogenesis of HO.

Analysis of gene order data supports vertical inheritance of the leukotoxin operon and genome rearrangements in the 5' flanking region in genus Mannheimia

BACKGROUND: The Mannheimia subclades belong to the same bacterial genus, but have taken divergent paths toward their distinct lifestyles. For example, M. haemolytica + M. glucosida are potential pathogens of the respiratory tract in the mammalian suborder Ruminantia, whereas M. ruminalis, the supposed sister group, lives as a commensal in the ovine rumen. We have tested the hypothesis that vertical inheritance of the leukotoxin (lktCABD) operon has occurred from the last common ancestor of genus Mannheimia to any ancestor of the diverging subclades by exploring gene order data. RESULTS: We examined the gene order in the 5' flanking region of the leukotoxin operon and found that the 5' flanking gene strings, hslVU-lapB-artJ-lktC and xylAB-lktC, are peculiar to M. haemolytica + M. glucosida and M. granulomatis, respectively, whereas the gene string hslVU-lapB-lktC is present in M. ruminalis, the supposed sister group of M. haemolytica + M. glucosida, and in the most ancient subclade M. varigena. In M. granulomatis, we found remnants of the gene string hslVU-lapB-lktC in the xylB-lktC intergenic region. CONCLUSION: These observations indicate that the gene string hslVU-lapB-lktC is more ancient than the hslVU-lapB-artJ-lktC and xylAB-lktC gene strings. The presence of (remnants of) the ancient gene string hslVU-lapB-lktC among any subclades within genus Mannheimia supports that it has been vertically inherited from the last common ancestor of genus Mannheimia to any ancestor of the diverging subclades, thus reaffirming the hypothesis of vertical inheritance of the leukotoxin operon. The presence of individual 5' flanking regions in M. haemolytica + M. glucosida and M. granulomatis reflects later genome rearrangements within each subclade. The evolution of the novel 5' flanking region in M. haemolytica + M. glucosida resulted in transcriptional coupling between the divergently arranged artJ and lkt promoters. We propose that the chimeric promoter have led to high level expression of the leukotoxin operon which could explain the increased potential of certain M. haemolytica + M. glucosida strains to cause a particular type of infection.

Performance of gene-expression profiling test score variability to predict future clinical events in heart transplant recipients.

BACKGROUND A single non-invasive gene expression profiling (GEP) test (AlloMap®) is often used to discriminate if a heart transplant recipient is at a low risk of acute cellular rejection at time of testing. In a randomized trial, use of the test (a GEP score from 0-40) has been shown to be non-inferior to a routine endomyocardial biopsy for surveillance after heart transplantation in selected low-risk patients with respect to clinical outcomes. Recently, it was suggested that the within-patient variability of consecutive GEP scores may be used to independently predict future clinical events; however, future studies were recommended. Here we performed an analysis of an independent patient population to determine the prognostic utility of within-patient variability of GEP scores in predicting future clinical events. METHODS We defined the GEP score variability as the standard deviation of four GEP scores collected ≥315 days post-transplantation. Of the 737 patients from the Cardiac Allograft Rejection Gene Expression Observational (CARGO) II trial, 36 were assigned to the composite event group (death, re-transplantation or graft failure ≥315 days post-transplantation and within 3 years of the final GEP test) and 55 were assigned to the control group (non-event patients). In this case-controlled study, the performance of GEP score variability to predict future events was evaluated by the area under the receiver operator characteristics curve (AUC ROC). The negative predictive values (NPV) and positive predictive values (PPV) including 95 % confidence intervals (CI) of GEP score variability were calculated. RESULTS The estimated prevalence of events was 17 %. Events occurred at a median of 391 (inter-quartile range 376) days after the final GEP test. The GEP variability AUC ROC for the prediction of a composite event was 0.72 (95 % CI 0.6-0.8). The NPV for GEP score variability of 0.6 was 97 % (95 % CI 91.4-100.0); the PPV for GEP score variability of 1.5 was 35.4 % (95 % CI 13.5-75.8). CONCLUSION In heart transplant recipients, a GEP score variability may be used to predict the probability that a composite event will occur within 3 years after the last GEP score. TRIAL REGISTRATION Clinicaltrials.gov identifier NCT00761787.

2C-Cas9: a versatile tool for clonal analysis of gene function

CRISPR/Cas9-mediated targeted mutagenesis allows efficient generation of loss-of-function alleles in zebrafish. To date this technology has been primarily used to generate genetic knockout animals. Nevertheless, the study of the function of certain loci might require tight spatiotemporal control of gene inactivation. Here, we show that tissue-specific gene disruption can be achieved by driving Cas9 expression with the Gal4/UAS system. Furthermore, by combining the Gal4/UAS and Cre/loxP systems, we establish a versatile tool to genetically label mutant cell clones, enabling their phenotypic analysis. Our technique has the potential to be applied to diverse model organisms, enabling tissue-specific loss-of-function and phenotypic characterization of live and fixed tissues.

Single-cell gene-expression profiling reveals qualitatively distinct CD8 T cells elicited by different gene-based vaccines

CD8 T cells play a key role in mediating protective immunity against selected pathogens after vaccination. Understanding the mechanism of this protection is dependent upon definition of the heterogeneity and complexity of cellular immune responses generated by different vaccines. Here, we identify previously unrecognized subsets of CD8 T cells based upon analysis of gene-expression patterns within single cells and show that they are differentially induced by different vaccines. Three prime-boost vector combinations encoding HIV Env stimulated antigen-specific CD8 T-cell populations of similar magnitude, phenotype, and functionality. Remarkably, however, analysis of single-cell gene-expression profiles enabled discrimination of a majority of central memory (CM) and effector memory (EM) CD8 T cells elicited by the three vaccines. Subsets of T cells could be defined based on their expression of Eomes, Cxcr3, and Ccr7, or Klrk1, Klrg1, and Ccr5 in CM and EM cells, respectively. Of CM cells elicited by DNA prime-recombinant adenoviral (rAd) boost vectors, 67% were Eomes(-) Ccr7(+) Cxcr3(-), in contrast to only 7% and 2% stimulated by rAd5-rAd5 or rAd-LCMV, respectively. Of EM cells elicited by DNA-rAd, 74% were Klrk1(-) Klrg1(-)Ccr5(-) compared with only 26% and 20% for rAd5-rAd5 or rAd5-LCMV. Definition by single-cell gene profiling of specific CM and EM CD8 T-cell subsets that are differentially induced by different gene-based vaccines will facilitate the design and evaluation of vaccines, as well as enable our understanding of mechanisms of protective immunity.

Evolution of gene expression changes in newborn rats after mechanical ventilation with reversible intubation

Mechanical ventilation (MV) is life-saving but potentially harmful for lungs of premature infants. So far, animal models dealt with the acute impact of MV on immature lungs, but less with its delayed effects. We used a newborn rodent model including non-surgical and therefore reversible intubation with moderate ventilation and hypothesized that there might be distinct gene expression patterns after a ventilation-free recovery period compared to acute effects directly after MV. Newborn rat pups were subjected to 8 hr of MV with 60% oxygen (O(2)), 24 hr after injection of lipopolysaccharide (LPS), intended to create a low inflammatory background as often recognized in preterm infants. Animals were separated in controls (CTRL), LPS injection (LPS), or full intervention with LPS and MV with 60% O(2) (LPS + MV + O(2)). Lungs were recovered either directly following (T:0 hr) or 48 hr after MV (T:48 hr). Histologically, signs of ventilator-induced lung injury (VILI) were observed in LPS + MV + O(2) lungs at T:0 hr, while changes appeared similar to those known from patients with chronic lung disease (CLD) with fewer albeit larger gas exchange units, at T:48 hr. At T:0 hr, LPS + MV + O(2) increased gene expression of pro-inflammatory MIP-2. In parallel anti-inflammatory IL-1Ra gene expression was increased in LPS and LPS + MV + O(2) groups. At T:48 hr, pro- and anti-inflammatory genes had returned to their basal expression. MMP-2 gene expression was decreased in LPS and LPS + MV + O(2) groups at T:0 hr, but no longer at T:48 hr. MMP-9 gene expression levels were unchanged directly after MV. However, at T:48 hr, gene and protein expression increased in LPS + MV + O(2) group. In conclusion, this study demonstrates the feasibility of delayed outcome measurements after a ventilation-free period in newborn rats and may help to further understand the time-course of molecular changes following MV. The differences obtained from the two time points could be interpreted as an initial transitory increase of inflammation and a delayed impact of the intervention on structure-related genes.

A field study on characteristics and diversity of gene expression in the liver of dairy cows during the transition period

Metabolic and endocrine adaptations to support milk production during the transition period vary between individual cows. This variation between cows to adapt to lactation may have a genetic basis. The present field study was carried out to determine hepatic adaptations occurring from late pregnancy through early lactation by measuring mRNA abundance of candidate genes in dairy cows on-farm. Additionally, the objective was to observe the diversity in inter-individual variation for the candidate genes that may give indications where individual adaptations at a molecular level can be found. This study was carried out on-farm including 232 dairy cows (parity >3) from 64 farms in Switzerland. Blood and liver samples were collected on d 20+/-7 before parturition, on d 24+/-2, and on d 89+/-4 after parturition. Blood plasma was assayed for concentrations of glucose, nonesterified fatty acids, beta-hydroxybutyrate, cholesterol, triglycerides, urea, albumin, protein, insulin, insulin-like growth factor-1, leptin, 3,5,3'-triiodothyronine, and thyroxine. Liver samples were obtained at the same time points and were measured for mRNA abundance of 26 candidate genes encoding enzymes and nuclear receptors involved in gluconeogenesis, fatty acid beta-oxidation, fatty acid and triglyceride synthesis, ketogenesis, citric acid cycle, cholesterol synthesis, and the urea cycle. The cows in the present study experienced a marked metabolic load in early lactation, as presented by changes in plasma metabolites and hormones, and responded accordingly with upregulation and downregulation of almost all candidate genes involved in metabolic processes in the liver. The observed inter-individual variation for the candidate genes, which was highest for acetyl-CoA-carboxylase and glycerol-3-phosphate dehydrogenase 2, should be further investigated to unravel the regulation at molecular level for optimal adaptive performance in dairy cows.

Gene expression in cortex and hippocampus during acute pneumococcal meningitis

BACKGROUND: Pneumococcal meningitis is associated with high mortality (approximately 30%) and morbidity. Up to 50% of survivors are affected by neurological sequelae due to a wide spectrum of brain injury mainly affecting the cortex and hippocampus. Despite this significant disease burden, the genetic program that regulates the host response leading to brain damage as a consequence of bacterial meningitis is largely unknown.We used an infant rat model of pneumococcal meningitis to assess gene expression profiles in cortex and hippocampus at 22 and 44 hours after infection and in controls at 22 h after mock-infection with saline. To analyze the biological significance of the data generated by Affymetrix DNA microarrays, a bioinformatics pipeline was used combining (i) a literature-profiling algorithm to cluster genes based on the vocabulary of abstracts indexed in MEDLINE (NCBI) and (ii) the self-organizing map (SOM), a clustering technique based on covariance in gene expression kinetics. RESULTS: Among 598 genes differentially regulated (change factor > or = 1.5; p < or = 0.05), 77% were automatically assigned to one of 11 functional groups with 94% accuracy. SOM disclosed six patterns of expression kinetics. Genes associated with growth control/neuroplasticity, signal transduction, cell death/survival, cytoskeleton, and immunity were generally upregulated. In contrast, genes related to neurotransmission and lipid metabolism were transiently downregulated on the whole. The majority of the genes associated with ionic homeostasis, neurotransmission, signal transduction and lipid metabolism were differentially regulated specifically in the hippocampus. Of the cell death/survival genes found to be continuously upregulated only in hippocampus, the majority are pro-apoptotic, while those continuously upregulated only in cortex are anti-apoptotic. CONCLUSION: Temporal and spatial analysis of gene expression in experimental pneumococcal meningitis identified potential targets for therapy.

Immunogenomic analysis of insect bite hypersensitivity in a model horse population.

Equine insect bite hypersensitivity (IBH) is a seasonal IgE-mediated dermatosis caused by bites of insects of the genus Culicoides. A familial predisposition for the disease has been shown but, except for the MHC, the genes involved have not been identified so far. An immunogenomic analysis of IBH was performed in a model population of Old Kladruby horses, all living in the same environment. Clinical signs of IBH were used as phenotypic manifestation of IBH. Furthermore, total serum IgE levels were determined in the sera of these horses and used as an independent phenotypic marker for the immunogenetic analysis. Single nucleotide polymorphisms (SNPs) in candidate immunity-related genes were used for association analyses. Genotypes composed of two to five genes encoding interferon gamma -IFNG, transforming growth factor beta 1 -TGFB1, Janus kinase 2 -JAK2, thymic stromal lymphopoietin -TSLP, and involucrin -IVL were associated with IBH, indicating a role of the genes in the pathogenesis of IBH. These findings were supported by analysis of gene expression in skin biopsies of 15 affected and 15 unaffected horses. Two markers associated with IBH, IFNG and TGFB1, showed differences in mRNA expression in skin biopsies from IBH-affected and non-affected horses (p<0.05). Expression of the gene coding for the CD14 receptor molecule -CD14 was different in skin biopsies at p<0.06. When total IgE levels were treated as binary traits, genotypes of IGHE, ELA-DRA, and IL10/b were associated with this trait. When treated as a continuous trait, total IgE levels were associated with genes IGHE, FCER1A, IL4, IL4R, IL10, IL1RA, and JAK2. This first report on non-MHC genes associated with IBH in horses is thus supported by differences in expression of genes known to play a role in allergy and immunity.

Analysis of protein expression in zebrafish during gonad differentiation by targeted proteomics

The molecular mechanisms governing sex determination and differentiation in the zebrafish (Danio rerio) are not fully understood. To gain more insights into the function of specific genes in these complex processes, the expression of multiple candidates needs to be assessed, preferably on the protein level. Here, we developed a targeted proteomics method based on selected reaction monitoring (SRM) to study the candidate sex-related proteins in zebrafish which were selected based on a global proteomics analysis of adult gonads and representational difference analysis of male and female DNA, as well as on published information on zebrafish and other vertebrates. We employed the developed SRM protocols to acquire time-resolved protein expression profiles during the gonad differentiation period in vas::EGFP transgenic zebrafish. Evidence on protein expression was obtained for the first time for several candidate genes previously studied only on the mRNA level or suggested by bioinformatic predictions. Tuba1b (tubulin alpha 1b), initially included in the study as one of the potential housekeeping proteins, was found to be preferentially expressed in the adult testis with nearly absent expression in the ovary. The revealed changes in protein expression patterns associated with gonad differentiation suggest that several of the examined proteins, especially Ilf2 and Ilf3 (interleukin enhancer-binding factors 2 and 3), Raldh3 (retinaldehyde dehydrogenase type 3), Zgc:195027 (low density lipoprotein-related receptor protein 3) and Sept5a (septin 5a), may play a specific role in the sexual differentiation in zebrafish.

Geographic analysis of RKIP expression and its clinical relevance in colorectal cancer

BACKGROUND This study evaluates the geographic expression pattern of Raf-1 Kinase Inhibitor Protein (RKIP) in colorectal cancer (CRC) in correlation with clinicopathological and molecular features, markers of epithelial-mesenchymal transition (EMT) and survival outcome. METHODS Whole-tissue sections of 220 well-characterised CRCs were immunostained for RKIP. NF-κB and E-Cadherin expression was assessed using a matched multi-punch tissue microarray. Analysis of mismatch repair (MMR) protein expression, B-Raf and KRAS mutations was performed. RKIP expression in normal mucosa, tumour centre, invasion front and tumour buds was each assessed for clinical relevance. RESULTS RKIP was diffusely expressed in normal mucosa and progressively lost towards tumour centre and front (P<0.0001). Only 0.9% of tumour buds were RKIP-positive. In the tumour centre, RKIP deficiency predicted metastatic disease (P=0.0307), vascular invasion (P=0.0506), tumour budding (P=0.0112) and an invasive border configuration (P=0.0084). Loss of RKIP correlated with NF-κB activation (P=0.0002) and loss of E-Cadherin (P<0.0001). Absence of RKIP was more common in MMR-deficient cancers (P=0.0191), while no impact of KRAS and B-Raf mutation was observed. RKIP in the tumour centre was identified as a strong prognostic indicator (HR (95% CI): 2.13 (1.27-3.56); P=0.0042) independently of TNM classification and therapy (P=0.0474). CONCLUSION The clinical relevance of RKIP expression as an independent prognostic factor is restricted to the tumour centre. Loss of RKIP predicts features of EMT and correlates with frequent distant metastasis.