Non-Invasive Molecular Imaging of Fibrosis Using a Collagen-Targeted Peptidomimetic of the Platelet Collagen Receptor Glycoprotein VI

Background

Fibrosis, which is characterized by the pathological accumulation of collagen, is recognized as an important feature of many chronic diseases, and as such, constitutes an enormous health burden. We need non-invasive specific methods for the early diagnosis and follow-up of fibrosis in various disorders. Collagen targeting molecules are therefore of interest for potential in vivo imaging of fibrosis. In this study, we developed a collagen-specific probe using a new approach that takes advantage of the inherent specificity of Glycoprotein VI (GPVI), the main platelet receptor for collagens I and III.

Methodology/Principal Findings

An anti-GPVI antibody that neutralizes collagen-binding was used to screen a bacterial random peptide library. A cyclic motif was identified, and the corresponding peptide (designated collagelin) was synthesized. Solid-phase binding assays and histochemical analysis showed that collagelin specifically bound to collagen (Kd 10⁻⁷ M) in vitro, and labelled collagen fibers ex vivo on sections of rat aorta and rat tail. Collagelin is therefore a new specific probe for collagen. The suitability of collagelin as an in vivo probe was tested in a rat model of healed myocardial infarctions (MI). Injecting Tc-99m-labelled collagelin and scintigraphic imaging showed that uptake of the probe occurred in the cardiac area of rats with MI, but not in controls. Post mortem autoradiography and histological analysis of heart sections showed that the labeled areas coincided with fibrosis. Scintigraphic molecular imaging with collagelin provides high resolution, and good contrast between the fibrotic scars and healthy tissues. The capacity of collagelin to image fibrosis in vivo was confirmed in a mouse model of lung fibrosis.

Conclusion/Significance

Collagelin is a new collagen-targeting agent which may be useful for non-invasive detection of fibrosis in a broad spectrum of diseases.     

Pathotype and Antibiotic Resistance Gene Distributions of Escherichia coli Isolates from Broiler Chickens Raised on Antimicrobial-Supplemented Diets

The impact of feed supplementation with bambermycin, monensin, narasin, virginiamycin, chlortetracycline, penicillin, salinomycin, and bacitracin on the distribution of Escherichia coli pathotypes in broiler chickens was investigated using an E. coli virulence DNA microarray. Among 256 E. coli isolates examined, 59 (23%) were classified as potentially extraintestinal pathogenic E. coli (ExPEC), while 197 (77%) were considered commensal. Except for chlortetracycline treatment, the pathotype distribution was not significantly different among treatments (P > 0.05). Within the 59 ExPEC isolates, 44 (75%) were determined to be potentially avian pathogenic E. coli (APEC), with the remaining 15 (25%) considered potentially “other” ExPEC isolates. The distribution within phylogenetic groups showed that 52 (88%) of the ExPEC isolates belonged to groups B2 and D, with the majority of APEC isolates classified as group D and most commensal isolates (170, 86%) as group A or B1. Indirect assessment of the presence of the virulence plasmid pAPEC-O2-ColV showed a strong association of the plasmid with APEC isolates. Among the 256 isolates, 224 (88%) possessed at least one antimicrobial resistance gene, with nearly half (107, 42%) showing multiple resistance genes. The majority of resistance genes were distributed among commensal isolates. Considering that the simultaneous detection of antimicrobial resistance tet(A), sulI, and bla_TEM genes and the integron class I indicated a potential presence of the resistance pAPEC-O2-R plasmid, the results revealed that 35 (14%) of the isolates, all commensals, possessed this multigene resistance plasmid. The virulence plasmid was never found in combination with the antimicrobial resistance plasmid. The presence of the ColV plasmid or the combination of iss and tsh genes in the majority of APEC isolates supports the notion that when found together, the plasmid, iss, and tsh serve as good markers for APEC. These data indicate that different resistant E. coli pathotypes can be found in broiler chickens and that the distribution of such pathotypes and certain virulence determinants could be modulated by antimicrobial agent feed supplementation.Several classes of antimicrobial agents, such as glycolipids (bambermycin), cyclic peptides (bacitracin), ionophores (monensin and salinomycin), streptogramins (virginiamycin), and β-lactams (penicillin), are widely used as food additives in modern animal husbandry to prevent infections and promote growth (6). Increasing antimicrobial resistance in animals and its potential threat to human health led to the ban of bacitracin, spiramycin, tylosin, and virginiamycin as feeding additives by the European Union in 1999 (7, 46). Although this precautionary measure is still controversial because of being seen as having a negligible impact on human health, negative consequences for animal health and welfare, including economic losses for farmers, were subsequently observed in Europe (7). In stark contrast, however, the ban has been beneficial in reducing the total quantity of antibiotics administered to food animals (7, 47). Under good production conditions and correct use of antibiotics, poultry production is reported to be competitive (14, 47, 48) and even beneficial in reducing antimicrobial resistance in important food animal reservoirs and thus the potential threat to public health (48).Escherichia coli is generally considered a commensal member of the normal gastrointestinal microflora in humans and animals, yet some strains are known to cause serious morbidity and mortality. The expression of various virulence factors, which affect cellular processes, can result in different clinical diseases, such as cystitis, pyelonephritis, sepsis/meningitis, and gastroenteritis. The possession of different virulence gene subsets can further define the E. coli pathotype (31). The extraintestinal pathogenic E. coli (ExPEC) strains are epidemiologically and phylogenetically distinct from both intestinal pathogenic and commensal strains (43). In North America, annually, several million cases of urinary tract infections, abdominal infections, pelvic infections, pneumonia, meningitis, and sepsis are caused by ExPEC (42). In poultry production, avian pathogenic E. coli (APEC) is responsible for significant economic losses. APEC strains induce extraintestinal diseases such as air sacculitis, colibacillosis, polysorositis, and septicemia in birds (9, 21, 22, 31, 35, 45). Although no specific set of virulence factors has been clearly linked to APEC strains, most identified virulence factors are similar to those frequently associated with ExPEC (36).Bearing in mind that the avian intestinal environment has been considered a reservoir of E. coli having zoonotic potential (15) and the possible contamination of poultry products with such bacteria during slaughter, the impact of antimicrobial feeding additives on the distribution and dissemination of bacterial pathotypes and antibiotic resistance needs to be explored to address human, animal, and environmental health concerns. To this end, an E. coli DNA virulence microarray previously employed to assess the genotypes (virulence and antibiotic resistance genes) of E. coli strains isolated from different environmental ecosystems and from the chicken intestinal tract (1, 10, 19, 20, 33) was used. The aim of the present trial was to investigate the distributions of pathotypes and of virulence and antibiotic resistance genes in E. coli isolates from broilers fed with antimicrobial supplementation diets including bambermycin, penicillin, salinomycin, bacitracin, chlortetracycline, virginiamycin, monensin, and narasin.     

Regulation of ENaC and CFTR expression with K+ channel modulators and effect on fluid absorption across alveolar epithelial cells

In a recent study (Leroy C, Dagenais A, Berthiaume Y, and Brochiero E. Am J Physiol Lung Cell Mol Physiol 286: L1027-L1037, 2004), we identified an ATP-sensitive K(+) (K(ATP)) channel in alveolar epithelial cells, formed by inwardly rectifying K(+) channel Kir6.1/sulfonylurea receptor (SUR)2B subunits. We found that short applications of K(ATP), voltage-dependent K(+) channel KvLQT1, and calcium-activated K(+) (K(Ca)) channel modulators modified Na(+) and Cl(-) currents in alveolar monolayers. In addition, it was shown previously that a K(ATP) opener increased alveolar liquid clearance in human lungs by a mechanism possibly related to epithelial sodium channels (ENaC). We therefore hypothesized that prolonged treatment with K(+) channel modulators could induce a sustained regulation of ENaC activity and/or expression. Alveolar monolayers were treated for 24 h with inhibitors of K(ATP), KvLQT1, and K(Ca) channels identified by PCR. Glibenclamide and clofilium (K(ATP) and KvLQT1 inhibitors) strongly reduced basal transepithelial current, amiloride-sensitive Na(+) current, and forskolin-activated Cl(-) currents, whereas pinacidil, a K(ATP) activator, increased them. Interestingly, K(+) inhibitors or membrane depolarization (induced by valinomycin in high-K(+) medium) decreased alpha-, beta-, and gamma-ENaC and CFTR mRNA. alpha-ENaC and CFTR proteins also declined after glibenclamide or clofilium treatment. Conversely, pinacidil augmented ENaC and CFTR mRNAs and proteins. Since alveolar fluid transport was found to be driven, at least in part, by Na(+) transport through ENaC, we tested the impact of K(+) channel modulators on fluid absorption across alveolar monolayers. We found that glibenclamide and clofilium reduced fluid absorption to a level similar to that seen in the presence of amiloride, whereas pinacidil slightly enhanced it. Long-term regulation of ENaC and CFTR expression by K(+) channel activity could benefit patients with pulmonary diseases affecting ion transport and fluid clearance.     

Cytogenetic characterization of <Emphasis Type="Italic">Hydrangea involucrata</Emphasis> Sieb. and <Emphasis Type="Italic">H. aspera</Emphasis> D. Don complex (Hydrangeaceae)<Emphasis Type="Italic">:</Emphasis> genetic,evolutional, and taxonomic implications

The subsection Asperae of genus Hydrangea L. (Hydrangeaceae) has been investigated for three reasons: several ambiguous classifications concerning Hydrangea aspera have been published, unexpected differences in genome size among seven accessions have been reported Cerbah et al. (Theor Appl Genet 103:45–51, 2001), and two atypical chromosome numbers (2n = 30 for Hydrangea involucrata and 2n = 34 for H. aspera) have been found when all other species of the genus present 2n = 36. Therefore, these two species and four subspecies of Hydrangea in all 29 accessions were analyzed for their genome size, chromosome number, and karyotype features. This investigation includes flow cytometric measurements of nuclear DNA content and bases composition (GC%), fluorochrome banding for detection of GC- and AT-rich DNA regions, and fluorescent in situ hybridisation (FISH) for chromosome mapping of 5 S and 18 S-5.8 S-26 S rDNA genes. In the H. aspera complex, the genome size ranged from 2.98 (subsp. sargentiana) to 4.67 pg/2C (subsp. aspera), an exceptional intraspecific variation of 1.57-fold. The mean base composition was 40.5% GC. Our report establishes the first karyotype for the species H. involucrata, and for the subspecies of H. aspera which indeed present different formulae, offering an element of discrimination. FISH and fluorochrome banding revealed the important differentiation between these two species (H. involucrata and H. aspera) and among four subspecies of the H. aspera complex. Our results are in agreement with the Chinese classification that places the groups Kawakami and Villosa as two different species: Hydrangea villosa Rehder and Hydrangea kawakami Hayata. This knowledge can contribute to effective germplasm management and horticultural use.     

Change in bone mineral density as a function of age in women and men and association with the use of antiresorptive agents

Background

Measurement of bone mineral density is the most common method of diagnosing and assessing osteoporosis. We sought to estimate the average rate of change in bone mineral density as a function of age among Canadians aged 25–85, stratified by sex and use of antiresorptive agents.

Methods

We examined a longitudinal cohort of 9423 participants. We measured the bone mineral density in the lumbar spine, total hip and femoral neck at baseline in 1995–1997, and at 3-year (participants aged 40–60 years only) and 5-year follow-up visits. We used the measurements to compute individual rates of change.

Results

Bone loss in all 3 skeletal sites began among women at age 40–44. Bone loss was particularly rapid in the total hip and was greatest among women aged 50–54 who were transitioning from premenopause to postmenopause, with a change from baseline of –6.8% (95% confidence interval [CI] –7.5% to –4.9%) over 5 years. The rate of decline, particularly in the total hip, increased again among women older than 70 years. Bone loss in all 3 skeletal sites began at an earlier age (25–39) among men than among women. The rate of decline of bone density in the total hip was nearly constant among men 35 and older and then increased among men older than 65. Use of antiresorptive agents was associated with attenuated bone loss in both sexes among participants aged 50–79.

Interpretation

The period of accelerated loss of bone mineral density in the hip bones occurring among women and men older than 65 may be an important contributor to the increased incidence of hip fracture among patients in that age group. The extent of bone loss that we observed in both sexes indicates that, in the absence of additional risk factors or therapy, repeat testing of bone mineral density to diagnose osteoporosis could be delayed to every 5 years.Low bone mineral density is one of the most important risk factors for fracture.^1,2,3–7 Treatment with antiresorptive agents has been widely used for several decades, and the results of randomized controlled trials have shown that at least part of their efficacy is associated with their capacity to increase or stabilize bone density.⁴ Although clinical guidelines recommend measurement of bone density, among other important risk factors, when assessing a patient''s risk for fracture,^3,8,9 there is no international consensus on the optimal age at which to begin measurement, or on the frequency of measurement.¹⁰ The Canadian guidelines recommend it for patients aged 65 and older, even in the absence of risk factors or treatment, and suggest a frequency of every 2–3 years.⁸ Furthermore, it has been suggested that the rate of decline rather than a single measurement of bone density may better identify patients with an elevated risk for fracture.¹¹ Consequently, determining changes in bone density over time may provide clues on the pathophysiology of fractures and provide more accurate estimates of the optimal timing for repeat measurement.Previous studies of change in bone mineral density as a function of age have had a number of limitations. Many were cross-sectional; had small samples, limited age ranges or differing inclusion and exclusion criteria; and most excluded men.^12–20 The third National Health and Nutrition Examination Survey,²¹ a large cross-sectional study based in the United States included women and men aged 20 years and older but excluded only those who were pregnant or who had a fracture in both hips. It reported that, based on a single measurement of bone density in the hip, age-dependent bone loss in the hips begins early (20–40 years) and continues in both sexes throughout life. Cross-sectional data from the ongoing Canadian Multicentre Osteoporosis Study suggested that, although this finding may hold true for the femoral neck, which consists of both cortical and trabecular bone, it is not true for the largely trabecular lumbar spine.²² Furthermore, the use of cross-sectional data to estimate changes over time has fundamental limitations: the effect of age cannot be separated from the effect of birth cohort and survivorship, and estimates are based on between-group differences rather than changes in an individual participant.The use of longitudinal data would allow examination of the rate of change of bone mineral density over time with and without antiresorptive therapy. We sought to assess the average rate of change in bone density as a function of age among Canadians aged 25–85, stratified by sex and use of antiresorptive agents.     

Age-dependent increase in hydrogen peroxide production by cardiac monoamine oxidase A in rats

Oxidative stress is one of the factors involved in age-related impairment of cardiac function. In the present study, we investigated the role of the catecholamine-degrading enzyme monoamine oxidase (MAO) in H(2)O(2) production in the hearts of young, adult, and old rats. MAO-dependent H(2)O(2) production, measured by a chemiluminescence-based assay, increased with age, reaching the maximum in 24-mo-old rats (7.5-fold increase vs. 1-mo-old rats). The following observations indicate that the age-dependent increase in H(2)O(2) generation was fully related to the MAO-A isoform: 1) at all the ages tested, chemiluminescence production was inhibited by the MAO-A inhibitor clorgyline but not by the MAO-B inhibitor RO-19 6327; 2) enzyme assay, Western blot, and semiquantitative RT-PCR analysis showed an age-dependent increase in cardiac MAO-A activity, immunodetection, and mRNA expression, respectively; and 3) the MAO-B isoform was undetectable by enzyme assay and Western blot analysis. These results suggest that MAO-A could be a major source of H(2)O(2) in the aging heart.     

Adriamycin induced resistance of sensitive K 562 cells to natural killer lymphocyte attack

Summary The effect of Adriamycin (ADM) on eryhtroleukaemia K 562 cell susceptibility to human natural killer (NK) cell activity has been studied. When cultivated for 3 days in the presence of 10 to 40 nM ADM, K 562 cells decreased their susceptibility to NK-mediated lysis in a dose-dependent fashion. At a concentration of 40 nM, previously found to induce optimal differentiation-associated properties in K 562 cells, the induced resistance to NK-mediated lysis increased progressively from day 1 to day 3 of culture. ADM treatment did not induce K 562 cells to release factors which interfered with NK activity since supernatants from ADM-treated K 562 cell cultures caused no significant modification in the NK lytic process. Binding to NK of ADM-treated K 562 cells was unaffected since treated and untreated cells had identical capacities in a conjugate-forming cell assay or adsorption of NK cells on target cell monolayers. In cold target competition assays ADM-treated K 562 cells acted as more effective competitors than untreated K 562 cells. These observations imply that the reduced killing of the ADM-treated K 562 cells was independent of target-NK cell recognition, and suggest that ADM treatment could allow malignant cells to escape NK surveillance.     

Sensitivity of Na+-coupled d-glucose uptake,Mg2+-ATPase and sucrase to perturbations of the fluidity of brush-border membrane vesicles induced by n-aliphatic alcohols

The aim of our work is to show the importance of the role of hydrophobic bonds in maintaining Mg²⁺-ATPase or sucrase activity and Na⁺-coupled d-glucose uptake normal for the brush border of rat enterocytes. The activity of the two enzymes and the d-glucose uptake were therefore measured under the action of $\text{n-}\text{aliphatic alcohols}$ and related to the fluidity determined by ESR. Three concentrations were used for the first eight alcohols, those of octanol being about 1500-times lower than those of methanol. For each alcohol the d-glucose uptake and the fluidity were linear functions of the logarithm of the concentration, the linear regressions being practically parallel and equidistant. The concentrations ($\text{C}$) of the eight alcohols inhibiting the d-glucose uptake by 80% were similar to those increasing the membrane fluidity by 3%. The linear relationship which existed in both cases between log 1 / $\text{C}$ and log $\text{P, P}$ being octanol / water partition coefficients of the alcohols, was evidence of great sensitivity to the hydrophobic effect of the alcohols. Only the first alcohols, however, produced any notable inhibition of Mg²⁺-ATPase and sucrase. Hydrophobic bonds are thus shown to have little influence in maintaining the activity of Mg²⁺-ATPase and sucrase, but they modulate the Na⁺-coupled d-glucose uptake.     

Sensitivity of Na-coupled d-glucose uptake, Mg-ATPase and sucrase to perturbations of the fluidity of brush-border membrane vesicles induced by n-aliphatic alcohols

The aim of our work is to show the importance of the role of hydrophobic bonds in maintaining Mg²⁺-ATPase or sucrase activity and Na⁺-coupled d-glucose uptake normal for the brush border of rat enterocytes. The activity of the two enzymes and the d-glucose uptake were therefore measured under the action of n-aliphatic alcohols and related to the fluidity determined by ESR. Three concentrations were used for the first eight alcohols, those of octanol being about 1500-times lower than those of methanol. For each alcohol the d-glucose uptake and the fluidity were linear functions of the logarithm of the concentration, the linear regressions being practically parallel and equidistant. The concentrations (C) of the eight alcohols inhibiting the d-glucose uptake by 80% were similar to those increasing the membrane fluidity by 3%. The linear relationship which existed in both cases between log 1 / C and log P, P being octanol / water partition coefficients of the alcohols, was evidence of great sensitivity to the hydrophobic effect of the alcohols. Only the first alcohols, however, produced any notable inhibition of Mg²⁺-ATPase and sucrase. Hydrophobic bonds are thus shown to have little influence in maintaining the activity of Mg²⁺-ATPase and sucrase, but they modulate the Na⁺-coupled d-glucose uptake.     

The combined effects of blue light and dilution rate on lipid class and fatty acid composition of <Emphasis Type="Italic">Tisochrysis lutea</Emphasis>

The lipid class and the fatty acid compositions of microalgae highly influence bivalve larval and post-larval development. Light is an essential environmental factor for microalgal culture, and quantity and quality of light may induce changes in the biochemical composition of the algae. The objective of this study was to investigate the effect of light spectrum (blue vs. white light) on lipid class and fatty acid compositions of Tisochrysis lutea cultured in a chemostat. Two different dilution rates (D) were assayed for each light spectrum: 0.2 and 0.7 day^?1. Triacylglycerol (TAG), sterol, and hydrocarbon (HC) content increased sharply at low D. The proportion of alkenones was significantly reduced under blue light. Polyunsaturated fatty acids (PUFA), and particularly n-3 PUFA, content in phospholipids (PL) increased under blue light compared to white light at low D. Thus, blue light raised 22:6(n-3) levels in total lipids of T. lutea at low D. The cultivation of T. lutea in a chemostat at low D under blue light may improve nutritional value as feed for bivalve larvae by modifying the PUFA profile, especially increasing 22:6(n-3).