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ABSTRACT: Human mesenchymal stem cells (hMSC) are currently being introduced for cell therapy, yet, antibodies specific for native and differentiated MSCs are required for their identification prior to clinical use. Herein, high quality antibodies against MSC surface proteins were developed by immunizing mice with hMSC, and by using a panel of subsequent screening methods. Flow cytometry analysis revealed that 83.5, 1.1, and 8.5% of primary cultures of hMSC were double positive for STRO-1 and either of DJ 3, 9, and 18, respectively. However, none of the three DJ antibodies allowed enrichment of clonogenic hMSC from BMMNCs as single reagents. Using mass-spectrometric analysis, we identified the antigen recognised by DJ3 as CD44, whereas DJ9 and DJ18 recognized HLA-DRB1 and Collagen VI, respectively. The identified proteins were highly expressed throughout in vitro osteogenic- and adipogenic differentiation. Interestingly, undifferentiated cells revealed a sole cytoplasmic distribution pattern of Collagen VI, which however changed to an extracellular matrix appearance upon osteogenic- and adipogenic differentiation. In relation to this, we found that STRO-1(+/-)/Collagen VI(-) sorted hMSC contained fewer differentiated alkaline phosphatase(+) cells compared to STRO-1(+/-)/Collagen VI(+) hMSC, suggesting that Collagen VI on the cell membrane exclusively defines differentiated MSCs. In conclusion, we have generated a panel of high quality antibodies to be used for characterization of MSCs, and in addition our results may suggest that the DJ18 generated antibody against Collagen VI can be used for negative selection of cultured undifferentiated MSCs.
Molecules and Cells 05/2011; 32(2):133-42. · 2.18 Impact Factor
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ABSTRACT: To investigate if pregnancy associated plasma protein-A (PAPP-A) was present in the vulnerable plaque, and if not, to find alternative hypothesis for the release of PAPP-A.
Vulnerable plaques and control tissues were examined by immunohistochemistry. Volunteers and patients with non-atherosclerotic disease were examined for release of PAPP-A during ischemia and medical treatment. Non-atherosclerotic tissue samples were examined after incubation with heparins.
We were not able to detect PAPP-A in vulnerable plaques. Patients and volunteers experiencing ischemic events without atherosclerotic lesions only had elevated PAPP-A when treated with heparin. When tissue from normal artery wall was incubated with heparin, PAPP-A was eluted. This was not the case for non-arterial tissue samples.
Elevation of PAPP-A in patients with acute coronary syndromes seems to be caused by heparin induced release of PAPP-A from the arterial wall and not due to excretion from vulnerable plaques.
Clinical biochemistry 03/2011; 44(4):312-8. · 2.02 Impact Factor
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ABSTRACT: Pregnancy associated plasma protein-A (PAPP-A) is a potential new marker for vulnerable plaques in the coronary arteries only examined in stable coronary disease (CAD) in patients undergoing coronary angiography. Here we address the prognostic value of serum PAPP-A in unselected stable CAD patients.
Blood samples were drawn at study entry. Serum PAPP-A values ≥4mIU/L were considered elevated. Mortality and non-fatal myocardial infarction was prospectively registered. The primary outcome was the composite outcome of myocardial infarction and all-cause mortality, secondary outcomes were all-cause mortality and myocardial infarction.
Patients (n=4243) were followed for a median of 2.8 years. In a Cox analysis, elevated PAPP-A was significantly related to the composite outcome of myocardial infarction and death (HR 1.99, 95% CI 1.62-2.45, p<0.0005), all-cause mortality (HR 2.42, 1.92-3.06, p<0.0005), and myocardial infarction (HR 1.40, 1.01-1.94, p=0.046). After Holm's correction, the latter significance disappeared. After adjustment for risk factors and medication at entry, elevated PAPP-A remained significantly related to the composite outcome (HR 1.51, 1.22-1.86, p<0.0005) and all-cause mortality (HR 1.68, 1.32-2.13, p<0.0005).
In patients with stable CAD elevated serum PAPP-A seems promising as aid in identifying patients at high risk for death.
Atherosclerosis 01/2011; 214(1):203-8. · 3.79 Impact Factor
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ABSTRACT: There is a general need for markers of systemic inflammation in acute or chronic diseases, where complement activation is involved. Available methods to monitor complement activation are elaborate and of low sensitivity; they include haemolytic assays (CH50), quantification of fluid phase terminal complex (C5b-C9) and quantification of complement split products by precipitation-in-gel techniques (e.g. C3d). We have developed a mouse monoclonal antibody (mAb) that is able to detect fluid phase C3c without interference from other products generated from the complement component C3. The C3c specific mAb was tested in different ELISA combinations with various types of in vitro activated sera and with plasma or serum samples from factor I deficient patients. The specificity of the mAb was evaluated in immunoprecipitation techniques and by analysis of eluted fragments of C3 after immunoaffinity chromatography. The C3c mAb was confirmed to be C3c specific, as it showed no cross-reactivity with native (un-cleaved) C3, with C3b, iC3b, or with C3d. Also, no significant reaction was observed with C3 fragments in factor I deficient sera or plasma. This antibody forms the basis for the generation of a robust ELISA that allows for a quick and reliable evaluation of complement activation and consumption as a marker for inflammatory processes. We established the C3c plasma range in 100 healthy Danish blood donors with a mean of 3.47 μg/ml and a range of 2.12-4.92 μg/ml. We believe that such an antibody might be of potential value in the assessment of in vivo complement activity during the inflammatory processes.
Journal of immunological methods 10/2010; 362(1-2):142-50. · 2.35 Impact Factor
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ABSTRACT: Delta like 1 homolog (Dlk1) exists in both transmembrane and soluble molecular forms, and is implicated in cellular growth and plays multiple roles in development, tissue regeneration, and cancer. Thus, DLK1 levels are critical for cell function, and abnormal DLK1 expression can be lethal; however, little is known about the underlying mechanisms. We here report that miR-15a modulates DLK1 levels in preadipocytes thus providing a mechanism for DLK1 regulation that further links it to cell cycle arrest and cancer since miR-15a is deregulated in these processes. In preadipocytes, miR-15a increases with cell density, and peaks at the same stage where membrane DLK1(M) and soluble DLK1(S) are found at maximum levels. Remarkably, miR-15a represses the amount of all Dlk1 variants at the mRNA level but also the level of DLK1(M) protein while it increases the amount of DLK1(S) supporting a direct repression of DLK1 and a parallel effect on the protease that cleaves off the DLK1 from the membrane. In agreement with previous studies, we found that miR-15a represses cell numbers, but additionally, we report that miR-15a also increases cell size. Conversely, anti-miR-15a treatment decreases cell size while increasing cell numbers, scenarios that were completely rescued by addition of purified DLK1(S). Our data thus imply that miR-15a regulates cell size and proliferation by fine-tuning Dlk1 among others, and further emphasize miR-15a and DLK1 levels to play important roles in growth signaling networks.
Experimental Cell Research 04/2010; 316(10):1681-91. · 3.58 Impact Factor
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ABSTRACT: Full-thickness 5 mm punch biopsy wounds were made lateral to the sacrum in 48 smokers and 30 never smokers. After 1 week, the wounds were excised and fixed. The smokers were then randomized to continuous smoking or abstinence with a transdermal nicotine patch or a placebo patch. The sequence of wounding and excision was repeated after 4, 8, and 12 weeks. All excised tissue was stained with hematoxylin-eosin and immunohistochemically for macrophages (CD68), procollagen 1 N-terminal propeptide (PINP) in fibroblasts, and endothelial cells (CD31). The cellularity was assessed and scored by two independent histopathologists, and for the analysis, proportional odds models and random effect models for repeated measurements were applied. Macrophages and PINP-stained fibroblasts were reduced in the smokers' wounds (0.28 [0.14-0.58] [OR, 95%CI]; p=0.01 and 0.37[0.19-0.70]; p<0.01, respectively, when compared with never smokers' wounds). Inflammation scores were marginally affected. Following smoking cessation, inflammatory cell infiltration and macrophages in the wounds increased. PINP-stained fibroblasts were unaffected. Neovascularization was not affected by smoking or abstinence. Wound inflammation and fibroblast proliferation were attenuated in smokers, suggesting delayed healing. Abstinence from smoking restores inflammation, but does not affect proliferation. These findings suggest a pathophysiologic mechanism for postoperative wound infection and dehiscence in smokers and why smoking cessation appears to reduce wound infection but not dehiscence.
Wound Repair and Regeneration 02/2010; 18(2):186-92. · 2.91 Impact Factor
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ABSTRACT: Erythrocytes (E) from a cross-sectional group of 22 outpatients with systemic lupus erythematosus (SLE) and/or mixed connective tissue disease (MCTD), the majority without active disease (n = 14), were analyzed for CR1 antigen expression and capacity to bind complement opsonized, radiolabeled immune complexes (IC). Furthermore, E-bound C3 fragments and the plasma C3d concentration were determined. E-bound C3b/iC3b fragments were not elevated in patients with SLE, whereas E from 11 out of 22 SLE patients had increased C3d levels which correlated with the plasma C3d concentration (Rs 0.73, p < 0.001). E-fixed C3d fragments did not affect the binding of Mab or preopsonized IC to E-CR1 and were not correlated with disease activity or medical treatment. Antigen expression of E-CR1 measured by ELISA or agglutination showed positive correlation with the IC binding capacity of E-CR1 (Rs 0.92 and 0.72 respectively, p < 001). The IC binding capacity of E-CR1 from SLE patients was significantly reduced (p > 0.005), whereas the antigen expression of CR1 (ELISA) on E from the patients did not differ from that of E from healthy donors (p > 0.1). E-CR1 antigen was measured by Mab reacting with an epitope outside the IC-binding site of E-CR1. E-CR1 antigen expression or IC binding showed no correlation either with disease activity or prednisolone treatment. However, 4 og 5 patients with MCTD and 4 of 5 patients receiving Imurel® were found to have low E-CR1 expression and capacity to bind IC. Thus, measurement of antigenic E-CR1 in a cross-sectional group of SLE outpatients by use of Mab reacting with an epitope outside the ligand-binding region of CR1 did not reveal a significantly reduced CR1 expression. However, an assay for CR 1 -mediated IC binding showed a clearly reduced E-CR1 function.
Apmis 08/2009; 98(7‐12):637 - 644. · 1.99 Impact Factor
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ABSTRACT: Delta like 1 (DLK1) has been proposed to act as a regulator of cell fate determination and is linked to the development of various tissues including skeletal muscle. Herein we further investigated DLK1 expression during skeletal muscle remodeling. Although practically absent in normal adult muscle, DLK1 was upregulated in all human myopathies analyzed, including Duchenne- and Becker muscular dystrophies. Substantial numbers of DLK1(+) satellite cells were observed in normal neonatal and Duchenne muscle, and furthermore, myogenic DLK1(+) cells were identified during muscle regeneration in animal models in which the peak expression of Dlk1 mRNA and protein coincided with that of myoblast differentiation and fusion. In addition to perivascular DLK1(+) cells, interstitial DLK1(+) cells were numerous in regenerating muscle, and in agreement with colocalization studies of DLK1 and CD90/DDR2, qPCR of fluorescence-activated cell sorting DLK1(+) and DLK1(-) cells revealed that the majority of DLK1(+) cells isolated at day 7 of regeneration had a fibroblast-like phenotype. The existence of different DLK1(+) populations was confirmed in cultures of primary derived myogenic cells, in which large flat nonmyogenic DLK1(+) cells and small spindle-shaped cells coexpressing DLK1 and muscle-specific markers were observed. Myogenic differentiation was achieved when sorted DLK1(+) cells were cocultured together with primary myoblasts revealing a myogenic potential that was 10% of the DLK1(-) population. Transplantation of DLK1(+) cells into lacerated muscle did, however, not give rise to DLK1(+) cell-derived myofibers. We suggest that the DLK1(+) subpopulations identified herein each may contribute at different levels/time points to the processes involved in muscle development and remodeling.
Stem Cells 05/2009; 27(4):898-908. · 7.78 Impact Factor
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ABSTRACT: ABSTRACT Split products of complement component 3 (C3) and complement component 4 (C4) derived from activation of the alternative and classical complement pathways were measured in untreated outpatients, 20 with Crohn's disease and 19 with rheumatoid arthritis. Elevated levels of the d split product of C4 (C4d) were observed in 12 of 19 patients with rheumatoid arthritis and in 9 of 20 patients with Crohn's disease. Levels of the d split product of C3 (C3d) were increased in 14 of 19 patients with rheumatoid arthritis and in 6 of 20 Crohn's disease patients. The median values of C4d and C3d were significantly increased in both groups of patients. C3d concentrations correlated positively with C4d levels (rs=0.51–0.56, p<0.005). The complement activation was not reflected in reduced plasma levels of native C3 and C4. The data indicate activation of the classical complement pathway in both rheumatoid arthritis and Crohn's disease.
Journal of Internal Medicine 04/2009; 223(6):557 - 560. · 5.48 Impact Factor
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ABSTRACT: Pregnancy-associated plasma protein A (PAPP-A) is expressed in eroded and ruptured atheromatous plaques, and circulating levels are elevated in acute coronary syndromes (ACS). Our objective was to investigate release patterns of PAPP-A in ACS and whether they differ among different types of ACS.
In 40 patients, PAPP-A concentrations were measured in serially collected samples assessed by a novel ELISA technique. The patients were grouped according to type of ACS.
Release patterns for ST elevation myocardial infarction (STEMI) patients who underwent primary percutaneous coronary intervention (pPCI) showed a single substantial PAPP-A increase shortly after pPCI, followed by an abrupt return to normal levels without secondary peaks. STEMI, high-risk and low-risk non-ST elevation myocardial infarction/unstable angina pectoris (NSTEMI/UAP) patients without pPCI showed highly variable patterns with primary peaks followed by secondary PAPP-A increases. All patients with elevated PAPP-A levels reached the upper reference level within 24 h. There was a significant difference in median peak levels between STEMI (23.2 mIU/L) and low-risk ACS patients (6.35 mIU/L) (p = 0.004) and between high-risk (median = 15.3 mIU/L) and low-risk ACS patients (p = 0.01). Among high-risk ACS patients, NSTEMI patients had significantly higher peak levels than UAP patients (p = 0.003).
PAPP-A serum levels increase above normal values within 24 h after onset of symptoms in ACS. There are significant differences in PAPP-A peak levels and release patterns across the spectrum of ACS patients.
Scandinavian journal of clinical and laboratory investigation 11/2008; 69(1):121-7. · 1.38 Impact Factor
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ABSTRACT: Previously, we showed high-level, long-term liver replacement after transplantation of unfractionated embryonic day (ED) 14 fetal liver stem/progenitor cells (FLSPC). However, for clinical applications, it will be essential to transplant highly enriched cells, while maintaining high repopulation potential.
Dlk-1, a member of the delta-like family of cell surface transmembrane proteins, is highly expressed in human and rodent fetal liver. Dlk-1(+) cells, isolated from ED14 fetal liver using immunomagnetic beads, were examined for their hepatic gene expression profile and characteristic properties in vitro and their proliferative and differentiation potential in vivo after transplantation into normal adult rat liver.
Rat ED14 FLSPC were purified to 95% homogeneity and exhibited cell culture and gene expression characteristics expected for hepatic stem/progenitor cells. Rat ED14 FLSPC are alpha-fetoprotein(+)/cytokeratin-19(+) or alpha-fetoprotein(+)/cytokeratin-19(-) and contain all of the normal liver repopulation capacity found in fetal liver. Hematopoietic stem cells, a major component in crude fetal liver cell preparations that engraft in other organs, such as bone marrow, spleen, and lung, are totally removed by Dlk-1 selection, and Dlk-1 purified FLSPC repopulate only the liver.
This is the first study reporting purification of hepatic stem/progenitor cells from fetal liver that are fully capable of repopulating the normal adult liver. This represents a major advance toward developing protocols that will be essential for clinical application of liver cell transplantation therapy.
Gastroenterology 03/2008; 134(3):823-32. · 11.68 Impact Factor
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ABSTRACT: Elevated serum levels of biochemical markers of bone turnover and YKL-40 in patients with metastatic prostate cancer (PC) at the time of diagnosis are associated to poor prognosis. In this study, we evaluated the value of these biomarkers in monitoring the patients during hormonal treatment.
Serum procollagen type I N-terminal propeptide (PINP), bone-specific alkaline phosphatase (BAP), CTX-I, and YKL-40 were determined by ELISA in a longitudinal study of 106 patients with metastatic PC during treatment with total androgen ablation or parenteral estrogen. Serum samples were collected with 3 months interval. Median observation time was 4.9 years (range, 3.6-6.2). A total of 78 patients died (64 within 7 months following the last blood sampling).
After 6 months treatment, serum PINP, BAP, and YKL-40 decreased (P < 0.0001), but not serum CTX-I compared with baseline values. Univariate Cox analysis showed that serum PINP at 6 months [log transformed and treated as a continuous variable; hazard ratio (HR), 2.2; P < 0.0001], serum BAP (HR, 1.8; P < 0.0001), and serum CTX-I (HR, 2.4; P < 0.0001), but not serum YKL-40 (HR, 1.4; P = 0.16) were associated with survival. Multivariate Cox analysis including the biomarkers 6 months after the start of treatment showed that Soloway score (HR, 3.9; P = 0.013), WHO tumor grade (HR, 3.9; P = 0.004), and serum PINP (HR, 2.2; P < 0.0001) were independent prognostic variables of survival. Scoring the biomarkers during treatment as time-dependent covariates in univariate Cox regression analysis showed that increases in serum PINP (HR, 2.0; P < 0.0001), BAP (HR, 2.1; P < 0.0001), and YKL-40 (HR, 2.1; P < 0.0001) were predictors of early death.
Serial monitoring of serum PINP, BAP, CTX-I, and YKL-40 in metastatic PC patients during hormonal treatment provided information of prognosis.
Clinical Cancer Research 07/2007; 13(11):3244-9. · 7.74 Impact Factor
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ABSTRACT: PAPP-A has become the principal biochemical serum marker in first trimester screening for Down syndrome, the original data being based on results of a radioimmunoassay (RIA). Recent observations using sandwich ELISA technology have proposed PAPP-A as a potential marker in patients with acute coronary syndrome (ACS). The aims of the present study were to demonstrate (i) the importance of antibody specificity, (ii) the potential pitfalls in changing assay technology, (iii) the importance of strict definition of technology, and (iv) the application of a well-defined assay technology on sera from patients with ACS.
Candidate monoclonal antibodies (Mab) were identified by immunohistochemistry, Western blot and the absence of positive signals (ELISA) with normal, non-pregnant serum as antigen source. The ELISA technology was standardized against the original PAPP-A RIA and the WHO reference preparation (WHO 78/610). Results different from those obtained by the original RIA led to ELISA modifications with respect to dilution buffer and enzymatic digestion of the Mab.
The first generation ELISA revealed serum measurements from a pool of non-pregnant (n=103) individuals which, compared to the RIA, seemed to be false positive. The false positive reaction was abolished by addition of bovine serum (BS) to the dilution buffer. Subsequent analysis of individual sera (n=103) indicated that 7/103 were still false positive. This reaction was eliminated by introduction of F(ab')(2)-fragment of the indicator antibody. This modified ELISA revealed that serum PAPP-A levels in ACS were statistically significantly higher than in controls (p<0.001). Moreover, serum PAPP-A in ACS patients with ST-segment elevation (STEMI) were higher (p<0.001) compared to patients without ST-segment elevation (NSTEMI). Immunohistochemical analysis failed to identify PAPP-A in the atherosclerotic plaques.
Clinical Biochemistry 04/2007; 40(7):478-84. · 2.08 Impact Factor
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ABSTRACT: To examine the prognostic value of markers of bone metabolism (serum PINP, BAP, and CTX-I) and serum YKL-40 in metastatic prostate carcinoma (PC).
The biomarkers were determined by ELISAs in 153 metastatic PC patients before treatment with parenteral estrogen or total androgen ablation. The median follow-up was 4.9 years. One hundred fifteen patients died.
The biomarkers were increased in the patients compared to controls (P < 0.001), and related to performance status and Soloway score (except YKL-40), but not to T-category and WHO tumor grade. PINP was elevated in 87%, BAP (55%), CTX-I (33%), and YKL-40 (43%). Univariate analysis showed an association to survival: PINP (HR = 1.6, P < 0.0001), BAP (HR = 1.4, P < 0.0001), CTX-I (HR = 1.7, P < 0.0001), and YKL-40 (HR = 1.4, P = 0.004). In multivariate Cox analysis performance status, WHO grade, Soloway score, PINP, and YKL-40 were independently predictive factors.
High serum PINP, BAP, CTX-I, and YKL-40 are associated with poor outcome of metastatic PC patients.
The Prostate 05/2006; 66(5):503-13. · 3.48 Impact Factor
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ABSTRACT: Individuals genetically deficient of properdin are more susceptible to meningococcal disease. Likewise low concentration or decreased biological activity of mannose-binding lectin (MBL) is associated with higher incidence of bacterial infections during childhood. In this study we report our findings in a Danish family with a remarkably high incidence of meningococcal meningitis-in total four cases, one of them fatal.
Properdin and MBL were quantified by ELISA and the properdin gene was screened for sequence variations using denaturing high-performance liquid chromatography (DHPLC) and subsequent sequencing of abnormal patterns. The MBL gene was genotyped for the three known variant alleles (B, C and D) as well as three promoter polymorphisms (-221Y/X, -550H/L and +4P/Q).
Two out of six males with undetectable properdin activity had meningitis. They had also low MBL serum levels or carried an MBL variant allele, whereas high MBL concentrations were measured in three out of four properdin deficient males--without meningitis. A splice site mutation in exon 10 (c.1487-2A>G) was found in the properdin gene and co segregated with biochemically measured properdin deficiency.
Our results indicate that a combined deficiency of both properdin and MBL increases the risk of infection with Neisseria meningitidis and stress the importance of epistatic genetic interactions in disease susceptibility.
Molecular Immunology 03/2006; 43(5):473-9. · 2.90 Impact Factor
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ABSTRACT: The present paper describes the primary structure, glycosylation and tissue localization of fetal antigen 1 (FA1) isolated from second-trimester human amniotic fluid. FA1 is a single-chained, heterogeneous glycoprotein of 225–262 amino acid residues. FA1 has six well conserved epidermal-growth-factor motifs and contains up to ten O -glycosylation and N-glycosylation sites, six of which are differentially glycosylated. Alignment to the translated sequences of Mus. musculus dlk and human dlk revealed 86% and 99% identity, respectively, to a 259-amino-acid residue overlap, and this high similarity extends with minor corrections to the human adrenal-specific mRNA, pG2 as well. Immunohistochemical analysis demonstrated the presence of FA1 in 10 out of 14 lung tumors containing neuroendocrine elements, and in the placental villi where FA1 was exclusively seen in stromal cells in close contact to the vascular structure. In the pancreas, FA1 co-localized with insulin in the insulin secretory granules of the β cells within the islets of Langerhans.Our findings suggest that FA1 is synthesized as a membrane anchored protein and released into the circulation after enzymic cleavage, and that circulating FA1 represents the post-translationally modified gene product of human dlk which, in turn, is identical to human adrenal-specific mRNA pG2.
European Journal of Biochemistry. 03/2005; 225(1):83 - 92.
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ABSTRACT: No studies to date have reported activation of satellite cells in vivo in human muscle after a single bout of high intensity exercise. In this investigation, eight individuals performed a single bout of high intensity exercise with one leg, the contralateral leg being the control. A significant increase in mononuclear cells staining for the neural cell adhesion molecule (N-CAM) and fetal antigen 1 (FA1) were observed within the exercised human vastus lateralis muscle on days 4 and 8 post exercise. In addition, a significant increase in the concentration of the FA1 protein was determined in intramuscular dialysate samples taken from the vastus lateralis muscle of the exercising leg (day 0: 1.89 +/- 0.82 ng ml(-1); day 2: 1.68 +/- 0.37 ng ml(-1); day 4: 3.26 +/- 1.29 ng ml(-1), P < 0.05 versus basal; day 8: 4.68 +/- 2.06 ng ml(-1), P < 0.05 versus basal and control). No change was noted in the control leg. Despite this increase in N-CAM- and FA1-positive mononuclear cells, an increased expression of myogenin and the neonatal isoform of the myosin heavy chain (MHCn) was not observed. Interestingly, myofibre lesions resulting from extensive damage to the proteins within the myofibre, particularly desmin or dystrophin, were not observed, and hence did not appear to induce the expression of either N-CAM or FA1. We therefore propose that satellite cells can be induced to re-enter the cell growth cycle after a single bout of unaccustomed high intensity exercise. However, a single bout of exercise is not sufficient for the satellite cell to undergo terminal differentiation.
The Journal of Physiology 08/2004; 558(Pt 1):333-40. · 4.72 Impact Factor
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ABSTRACT: Increases in procollagen processing within skeletal muscle have previously been reported in small animal models only. While indirect measurements in humans have suggested an increase procollagen processing, no intra-skeletal muscle measurements have confirmed these findings. In this study, eight young healthy male subjects performed a single bout of unaccustomed high intensity eccentric exercise on one leg, with the contralateral leg being the control. A significant increase in the muscle interstitial concentration of the N-terminal propeptide of procollagen type I (PINP) was observed (day 0: 1.96 +/- 0.44 ng ml(-1), day 2: 1.94 +/- 0.32 ng ml(-1), day 4: 3.90 +/- 1.03 ng ml(-1), day 8: 7.23 +/- 2.34 ng ml(-1)*, *P < 0.05 vs. basal and control) with no change being noted in the control leg. By day 2 post-exercise, an increase in the histological immunoreactivity of PINP and the N-terminal propeptide of procollagen type III (PIIINP) was also shown in the exercising leg only. Further, from day 2 post-exercise, immunoreactivity for tenascin C and reactive macrophages (CD68+ cells) was seen within the perimysial and endomysial connective tissue of the exercising leg only, indicating a high mechanical load and inflammation. This study shows that following a single bout of high intensity eccentric exercise there is an increase in procollagen processing within skeletal muscle in humans.
Matrix Biology 07/2004; 23(4):259-64. · 3.30 Impact Factor
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ABSTRACT: Hepatic regeneration from toxic or surgical injury to the adult mammalian liver, endorses different cellular responses within the hepatic lineage. The molecular mechanisms determining commitment of a cell population at a specific lineage level to participate in liver repair as well as the fate of its progeny in the hostile environment created by the injury are not well defined. Based on the role of the Notch/Delta/Jagged system in cell fate specification and recent reports linking Notch signaling with normal bile duct formation in mouse and human liver, we examined the expression of Notch1, Notch2, Notch3, Delta1, Delta3, Jagged1, and Jagged2, and delta-like protein/preadipocyte factor 1/fetal antigen 1 (dlk) in four well-defined experimental rat models of liver injury and regeneration. Although Delta3 and Jagged2 were undetectable by reverse transcriptase-polymerase chain reaction and Northern blot, we observed the most significant up-regulation of all other transcripts in the 2-acetylaminofluorene-70% hepatectomy (AAF/PHx) model, in which liver mass is restored by proliferation and differentiation of transit-amplifying ductular (oval) cells. The most profound change was observed for dlk. Accordingly, immunohistochemical analyses in the AAF/PHx model showed a specific expression of dlk in atypical ductular structures composed of oval cells. Delta-like protein was not observed in proliferating hepatocytes or bile duct cells after partial hepatectomy or ligation of the common bile duct whereas clusters of dlk immunoreactive oval cells were found in both the retrorsine and the AAF/PHx models. Finally, we used dlk to isolate alpha-fetoprotein-positive cells from fetal and adult regenerating rat liver by a novel antibody panning technique.
American Journal Of Pathology 05/2004; 164(4):1347-59. · 4.89 Impact Factor
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ABSTRACT: This report describes an assay for comparison of epitope specificity in groups of monoclonal antibodies against a given antigen. The only prerequisite is the biotin-labeled antigen. One of the monoclonal antibodies is captured onto a plastic surface via a rabbit anti-mouse Ig, and the other preincubated with biotinylated antigen. When the two antibodies react with the same epitope subsequent binding of the biotin-labeled antigen is abolished (inhibition). In the cases where no inhibition was observed, the two antibodies were considered to react with distinct, independent epitopes. The obvious advantages using this assay, are that it can be performed directly on culture supernatants in the early phase of monoclonal antibody production, and also works for antigens with repetitive epitopes. Moreover, the bonus effect, i.e., a signal in excess of the reference signal when sets of monoclonal antibodies with different epitope specificity are compared, gives a relative measure of affinity.
Journal of Immunoassay and Immunochemistry 02/2004; 25(2):147-57. · 0.69 Impact Factor
