Objective: We earlier proposed that erythrophagocytosis and iron metabolism by macrophages may co... more Objective: We earlier proposed that erythrophagocytosis and iron metabolism by macrophages may contribute to iron-driven oxidative stress in atherogenesis. Recent studies indicate the macrophage hemoglobin scavenger receptor (HbSR/CD163) is a key molecule in the process of removing hemoglobin released from ruptured erythrocytes. In this study we aimed to investigate erythrophagocytosis and its relation to ferritin accumulation in human atherosclerotic lesions and if CD163 is involved in ferritin induction in human atheroma lesions. Methods: Normal human arterial segments from mammary (n = 4), carotid (n = 4), coronary arteries (n = 5), and atherosclerotic coronary (n = 5), and carotid arteries (n = 16) were collected for Fe histochemistry, immunohistochemistry of CD68, hemoglobin, CD163 and ferritin, and computerized image analysis. Results: The lesion-dependent accumulation of ferritin and hemoglobin were seen in atherosclerotic carotid and coronary arteries. The immunoreactivity of hemoglobin was significantly corresponding to the same regions of ferritin immunoreactivity on serial sections of carotid and coronary atherosclerotic arteries. The staining intensity of hemoglobin and ferritin also significantly correlated. Hemoglobin deposition is often associated with micro vessels surrounding the lipid core areas in advanced lesions, where there was mainly CD68 positive macrophages. CD163 expression was noted in both early and advanced lesions. Accumulation of tissue iron and ferritin also frequently occur at vascular rich regions and shoulder areas of the advanced atheroma, both of which were significantly correlated. Conclusion: Erythrophagocytosis and hemoglobin catabolism by macrophages contribute to iron deposition and ferritin induction in human atheroma. The involvement of CD163 during ferritin induction may play an important role in modulating inflammatory processes in atherogenesis.
Variation on chromosome 9p21 is associated with risk of coronary artery disease (CAD). This genom... more Variation on chromosome 9p21 is associated with risk of coronary artery disease (CAD). This genomic region contains the CDKN2A and CDKN2B genes which encode the cell cycle regulators p16 INK4a , p14 ARF and p15 INK4b and the ANRIL gene which encodes a non-coding RNA. Vascular smooth muscle cell (VSMC) proliferation plays an important role in the pathogenesis of atherosclerosis which causes CAD. We ascertained whether 9p21 genotype had an influence on CDKN2A/CDKN2B/ANRIL expression levels in VSMCs, VSMC proliferation and VSMC content in atherosclerotic plaques. Immunohistochemical examination showed that VSMCs in atherosclerotic lesions expressed p16 INK4a , p14 ARF and p15 INK4b. Analyses of primary cultures of VSMCs showed that the 9p21 risk genotype was associated with reduced expression of p16 INK4a , p15 INK4b and ANRIL (P 5 1.2 3 10 25 , 1.4 3 10 22 and 3.1 3 10 29) and with increased VSMC proliferation (P 5 1.6 3 10 22). Immunohistochemical analyses of atherosclerotic plaques revealed an association of the risk genotype with reduced p15 INK4b levels in VSMCs (P 5 3.7 3 10 22) and higher VSMC content (P 5 5.6 3 10 24) in plaques. The results of this study indicate that the 9p21 variation has an impact on CDKN2A and CDKN2B expression in VSMCs and influences VMSC proliferation, which likely represents an important mechanism for the association between this genetic locus and susceptibility to CAD.
The interaction of cationic proteins with IgG, IgA and IgM were investigated by solid phase radio... more The interaction of cationic proteins with IgG, IgA and IgM were investigated by solid phase radioimmunoassay. All these immunoglobulins showed avid binding, IgM giving the strongest reaction, followed by IgA and then IgG. Fc fragments of IgG gave binding, but F(ab')2 fragments from the three main Ig classes did not, showing that the Fc region is the active part of the molecule. The effects of changes of ionic strength and pH are compatible with the interaction being ionic, and are similar to those seen between immunoglobulins and both Clq and cationic ion exchange gels. The addition of other serum proteins resulted in marked inhibition of the interaction. These phenomena are likely to have fundamental significance for the understanding of interactions of immunoglobulins in vivo and in vitro.
Considerable evidence indicates that microvascular changes observed in psoriasis are a result of ... more Considerable evidence indicates that microvascular changes observed in psoriasis are a result of vascular proliferation. A critical step in the sequence of events leading to neovascularization involves interactions between endothelial cells and extracellular matrix proteins mediated in part by the integrin family of adhesion molecules. A number of endothelial integrins have been shown to participate in neovascularization, including members of the beta 1, beta 3, and beta 4 subfamilies. To investigate the role of these integrins in psoriasis, specimens of lesional and nonlesional skin were taken from 10 patients with active, untreated plaque disease. Vascular endothelium was labeled with monoclonal antibodies specific for alpha 2, alpha 5, alpha 6, beta 1, av beta 3, and beta 4 integrins. The use of image analysis permitted quantification of immunoperoxidase staining and comparison of endothelial labeling in lesional and nonlesional skin. There was a significant increase in endothelial staining of av beta 3 integrin in lesional compared with nonlesional skin, both in superficial and deep vasculature. In contrast, there was a significant decrease in endothelial beta 4 staining in lesional compared with nonlesional superficial dermal vessels, alpha 2, alpha 5, alpha 6, and beta 1 staining showed no significant difference between the two groups. These results demonstrate an important role of av beta 3 and beta 4 integrins in the microvascular changes of psoriatic lesions.
Controlled drug delivery and gene expression is required for a large variety of applications incl... more Controlled drug delivery and gene expression is required for a large variety of applications including cancer therapy, wound healing, cell migration, cell modification, cell-analysis, reproductive and regenerative medicine. Controlled delivery of precise amounts of drugs to a single cell is especially interesting for cell and tissue engineering as well as therapeutics and has until now required the application of micro-pipettes, precisely placed dispersed drug delivery vehicles, or injections close to or into the cell. Here we present surface bound microchamber arrays able to store small hydrophilic molecules for prolonged times in subaqueous conditions supporting spatiotemporal near infrared laser mediated release. The micro-chambers (MCs) are composed of biocompatible and biodegradable polylactic acid (PLA). Biocompatible gold nanoparticles are employed as light harvesting agents to facilitate photothermal MC opening. The degree of photothermal heating is determined by numerical simulations utilizing optical properties of the MC, and confirmed by Brownian motion measurements of laser-irradiated micro-particles exhibiting similar optical properties like the MCs. The amount of bioactive small molecular cargo (doxycycline) from local release is determined by fluorescence spectroscopy and gene expression in isolated C2C12 cells via enhanced green fluorescent protein (EGFP) biosynthesis.
High frequency alternating magnetic fields (AMF) have been widely used as a non-invasive method t... more High frequency alternating magnetic fields (AMF) have been widely used as a non-invasive method to induce local hyperthermia for antitumor treatment and to efficiently trigger drug release from various carriers. However, few studies have exploited the potential of targeted drug delivery to healthy cells or tissue and the use of low frequency AMF (LF-AMF) for intracellular triggered release. To achieve this goal, doxycycline was delivered with the layer-by-layer (LbL) assembled magnetic microcapsules, and AMF with low frequency (50 Hz) was applied. The low frequency AMF had little effect on morphology of microcapsules, which upon exposure for 360 min caused no significant damage and had the advantage of minimizing heating effects. Nonetheless, microcapsule permeability increased as a function of exposure time when assessed using FITC-dextran (70 kDa) with the number of permeable microcapsules increased from 13.5% (20 min) to 52.8% (360 min). Increased permeability also enhanced in vitro doxycycline release in genetically engineered myoblast cells where EGFP expression is regulated by the tetracycline system, while targeted EGFP expression was observed by magnetically navigating the microcapsules to a site of interest. Upon LF-AMF exposure of 30 min, no cytotoxicity was observed, but intracellular doxycycline release was promoted and enhanced EGFP expression as demonstrated by EGFP fluorescence intensity measurement. This study reveals the possibility of targeted drug delivery and using LF-AMF as a non-cytotoxic intracellular trigger of drug release from microcapsules without alteration in cell viability.
Immunohistochemistry of human atherosclerotic arteries demonstrates expression of the intercellul... more Immunohistochemistry of human atherosclerotic arteries demonstrates expression of the intercellular adhesion molecule-1 (ICAM-1) on endothelial cells, macrophages, and smooth muscle cells of the plaques. Normal arterial endothelial cells and intimal smooth muscle outside plaques give weaker or negative reactions; these differ from the strong endothelial expression in small vessels. Quantitative color-image analysis of the endothelial layer shows increased expression of ICAM-1 in all subtypes of atherosclerotic lesions, except fibrous plaques. Endothelial expression of ICAM-1 may be involved in the recruitment of monocytes to the lesion, as suggested by its role in the entry of leukocytes, including monocytes, into foci of inflammation. Collaboration with other mechanisms, particularly chemoattractant factors, may be important for this effect. ICAM-1 enhanced monocyte recruitment is a potential mechanism for the growth of an atherosclerotic plaque.
Background Genome-wide association studies have identifi ed ADAMTS7 as a risk locus for coronary ... more Background Genome-wide association studies have identifi ed ADAMTS7 as a risk locus for coronary artery disease and myocardial infarction. Functional studies suggest that ADAMTS7 might promote cellular processes in atherosclerosis. We sought to examine whether carriers of a loss-of-function genetic variant exhibit favourable characteristics on plaque histology, angiographic coronary artery disease severity, and cardiovascular outcomes. Methods The single-nucleotide polymorphism rs3825807 was used as a marker for ADAMTS7. Human coronary atherosclerotic plaques (n=50) were genotyped, and characterised with immunohistochemical analysis. Burden of angiographic coronary artery disease was assessed by angiographic severity scores in two independent populationbased cohorts-the Southampton Atherosclerosis Study (SAS, n=1359) in the UK, and the prospective Emory Genebank study (Emory GB, n=2684) in the USA. Follow-up data were collected in Emory GB over a median of 7 years. Findings Human coronary atherosclerotic plaques with the loss-of-function G allele exhibited thinner fi brous cap (p=0•017) and lower percentage area of α-actin (smooth muscle cell marker) in the intima (p=0•029). After adjustment for age and sex, the G allele was associated with less coronary artery disease in both SAS (odds ratio 0•82, 95% CI 0•67-0•99), and Emory GB (0•84, 0•74-0•95). Angiographic burden was further characterised with the Gensini Score (GS) and Sullivan Extent Score (SES). ADAMTS genotypes were associated with all of the angiographic severity scores in both SAS (GS p=0•026, SES p=0•029) and Emory GB (GS p<0•0001, SES p<0•0001). Outcome analysis showed lower incidence of revascularisation for G allele carriers (hazard ratio 0•77, 95% CI 0•60-0•98), but not all cause mortality (1•12, 0•92-1•35). Interpretation Carriers of the ADAMTS7 loss-of-function allele G had reduced atherosclerotic plaque progression, as demonstrated by thinner cap and smooth muscle migration. In addition there was less severe angiographic coronary artery disease in the SAS and Emory GB cohorts, as well as lower incidence of revascularisation procedure, further supporting the role of this protease in promoting atherosclerosis. Funding None. Contributors KC, RP, XP, RNP, IAS, and SH conceived the study, collected and analysed the data, and wrote the abstract. SY and AQ conceived the study, obtained funding, and supervised the study. Declaration of interests We declare no competing interests.
Publisher Summary The treatment of formalin fixed paraffin embedded sections with enzymes is esta... more Publisher Summary The treatment of formalin fixed paraffin embedded sections with enzymes is established as a means of restoring antigenicity for immunohistochemistry. The mechanism probably depends on the removal from antigenic sites of blocking proteins that have been cross-linked by formalin. With polyclonal antibodies trypsin and pronase have been the principal enzymes used, but there have been few reports to date of the effects of enzymes on the staining properties of monoclonal antibodies (MCs). The present study investigates the effects of a range of enzymes on five MCs that have been found useful for diagnostic immunohistochemistry with formalin fixed sections. As with polyclonal antibodies, enhancement of the immunohistochemical staining of formalin fixed sections could be obtained with MCs by enzyme treatment. Some enzymes were capable of destroying antigenicity, and the pattern of effect was unpredictable and variable from one MC to another.
Nanoengineered vehicles have the potential to deliver cargo drugs directly to disease sites, but ... more Nanoengineered vehicles have the potential to deliver cargo drugs directly to disease sites, but can potentially be cleared by immune system cells or lymphatic drainage. In this study we explore the use of magnetism to hold responsive particles at a delivery site, by incorporation of superparamagnetic iron oxide nanoparticles (SPIONs) into layer-by-layer (LbL) microcapsules. Microcapsules with SPIONs were rapidly phagocytosed by cells but did not trigger cellular ROS synthesis within 24 hours of delivery nor affect cell viability. In a non-directional cell migration assay, SPION containing microcapsules significantly inhibited movement of phagocytosing cells when placed in a magnetic field. Similarly, under flow conditions, a magnetic field retained SPION containing microcapsules at a physiologic wall shear stress of 0.751 dyne cm −2. Even when the SPION content was reduced to 20%, the majority of microcapsules were still retained. Dexamethasone microcrystals were synthesised by solvent evaporation and underwent LbL encapsulation with inclusion of a SPION layer. Despite a lower iron to volume content of these structures compared to microcapsules, they were also retained under shear stress conditions and displayed prolonged release of active drug, beyond 30 hours, measured using a glucocorticoid sensitive reporter cell line generated in this study. Our observations suggest use of SPIONs for magnetic retention of LbL structures is both feasible and biocompatible and has potential application for improved local drug delivery. † Electronic supplementary information (ESI) available. See
Expansion of the dermal microvasculature is a prominent feature of psoriasis. Although the pathog... more Expansion of the dermal microvasculature is a prominent feature of psoriasis. Although the pathogenetic process resulting in vascular morphological changes remains unclear, considerable evidence suggests the involvement of angiogenesis. To assess the degree and site of psoriatic microvascular expansion, immunohistochemical studies were performed on paired lesional and non-lesional specimens from 10 patients with active, untreated plaque psoriasis. Five-micron thick sections were labelled with monoclonal antibody JC/70A specific for the endothelial marker CD31, and vascular quantification was achieved using hue-saturation-intensity image analysis. Assessment of vasculature in the papillary dermis (superficial plexus) demonstrated a fourfold increase in endothelial surface area of lesional compared with non-lesional skin (P &lt; 0.01), while there was no significant increase in vasculature of the upper reticular dermis. Subsequently, 18-micron thick sections were double-labelled with MIB-1 antibody to the nuclear proliferation marker Ki-67 and JC/70A. Endothelial cell proliferation was identified in the vertical limbs of capillary loops in eight out of 10 lesional biopsies and in no non-lesional biopsies. The endothelial MIB-1 labelling index was 3.1% of total endothelial cells of the superficial plexus. These findings confirm endothelial proliferation underlying psoriatic microvascular expansion, and indicate that this process is limited to a specific site in the dermal capillary bed.
Objective: We earlier proposed that erythrophagocytosis and iron metabolism by macrophages may co... more Objective: We earlier proposed that erythrophagocytosis and iron metabolism by macrophages may contribute to iron-driven oxidative stress in atherogenesis. Recent studies indicate the macrophage hemoglobin scavenger receptor (HbSR/CD163) is a key molecule in the process of removing hemoglobin released from ruptured erythrocytes. In this study we aimed to investigate erythrophagocytosis and its relation to ferritin accumulation in human atherosclerotic lesions and if CD163 is involved in ferritin induction in human atheroma lesions. Methods: Normal human arterial segments from mammary (n = 4), carotid (n = 4), coronary arteries (n = 5), and atherosclerotic coronary (n = 5), and carotid arteries (n = 16) were collected for Fe histochemistry, immunohistochemistry of CD68, hemoglobin, CD163 and ferritin, and computerized image analysis. Results: The lesion-dependent accumulation of ferritin and hemoglobin were seen in atherosclerotic carotid and coronary arteries. The immunoreactivity of hemoglobin was significantly corresponding to the same regions of ferritin immunoreactivity on serial sections of carotid and coronary atherosclerotic arteries. The staining intensity of hemoglobin and ferritin also significantly correlated. Hemoglobin deposition is often associated with micro vessels surrounding the lipid core areas in advanced lesions, where there was mainly CD68 positive macrophages. CD163 expression was noted in both early and advanced lesions. Accumulation of tissue iron and ferritin also frequently occur at vascular rich regions and shoulder areas of the advanced atheroma, both of which were significantly correlated. Conclusion: Erythrophagocytosis and hemoglobin catabolism by macrophages contribute to iron deposition and ferritin induction in human atheroma. The involvement of CD163 during ferritin induction may play an important role in modulating inflammatory processes in atherogenesis.
Variation on chromosome 9p21 is associated with risk of coronary artery disease (CAD). This genom... more Variation on chromosome 9p21 is associated with risk of coronary artery disease (CAD). This genomic region contains the CDKN2A and CDKN2B genes which encode the cell cycle regulators p16 INK4a , p14 ARF and p15 INK4b and the ANRIL gene which encodes a non-coding RNA. Vascular smooth muscle cell (VSMC) proliferation plays an important role in the pathogenesis of atherosclerosis which causes CAD. We ascertained whether 9p21 genotype had an influence on CDKN2A/CDKN2B/ANRIL expression levels in VSMCs, VSMC proliferation and VSMC content in atherosclerotic plaques. Immunohistochemical examination showed that VSMCs in atherosclerotic lesions expressed p16 INK4a , p14 ARF and p15 INK4b. Analyses of primary cultures of VSMCs showed that the 9p21 risk genotype was associated with reduced expression of p16 INK4a , p15 INK4b and ANRIL (P 5 1.2 3 10 25 , 1.4 3 10 22 and 3.1 3 10 29) and with increased VSMC proliferation (P 5 1.6 3 10 22). Immunohistochemical analyses of atherosclerotic plaques revealed an association of the risk genotype with reduced p15 INK4b levels in VSMCs (P 5 3.7 3 10 22) and higher VSMC content (P 5 5.6 3 10 24) in plaques. The results of this study indicate that the 9p21 variation has an impact on CDKN2A and CDKN2B expression in VSMCs and influences VMSC proliferation, which likely represents an important mechanism for the association between this genetic locus and susceptibility to CAD.
The interaction of cationic proteins with IgG, IgA and IgM were investigated by solid phase radio... more The interaction of cationic proteins with IgG, IgA and IgM were investigated by solid phase radioimmunoassay. All these immunoglobulins showed avid binding, IgM giving the strongest reaction, followed by IgA and then IgG. Fc fragments of IgG gave binding, but F(ab')2 fragments from the three main Ig classes did not, showing that the Fc region is the active part of the molecule. The effects of changes of ionic strength and pH are compatible with the interaction being ionic, and are similar to those seen between immunoglobulins and both Clq and cationic ion exchange gels. The addition of other serum proteins resulted in marked inhibition of the interaction. These phenomena are likely to have fundamental significance for the understanding of interactions of immunoglobulins in vivo and in vitro.
Considerable evidence indicates that microvascular changes observed in psoriasis are a result of ... more Considerable evidence indicates that microvascular changes observed in psoriasis are a result of vascular proliferation. A critical step in the sequence of events leading to neovascularization involves interactions between endothelial cells and extracellular matrix proteins mediated in part by the integrin family of adhesion molecules. A number of endothelial integrins have been shown to participate in neovascularization, including members of the beta 1, beta 3, and beta 4 subfamilies. To investigate the role of these integrins in psoriasis, specimens of lesional and nonlesional skin were taken from 10 patients with active, untreated plaque disease. Vascular endothelium was labeled with monoclonal antibodies specific for alpha 2, alpha 5, alpha 6, beta 1, av beta 3, and beta 4 integrins. The use of image analysis permitted quantification of immunoperoxidase staining and comparison of endothelial labeling in lesional and nonlesional skin. There was a significant increase in endothelial staining of av beta 3 integrin in lesional compared with nonlesional skin, both in superficial and deep vasculature. In contrast, there was a significant decrease in endothelial beta 4 staining in lesional compared with nonlesional superficial dermal vessels, alpha 2, alpha 5, alpha 6, and beta 1 staining showed no significant difference between the two groups. These results demonstrate an important role of av beta 3 and beta 4 integrins in the microvascular changes of psoriatic lesions.
Controlled drug delivery and gene expression is required for a large variety of applications incl... more Controlled drug delivery and gene expression is required for a large variety of applications including cancer therapy, wound healing, cell migration, cell modification, cell-analysis, reproductive and regenerative medicine. Controlled delivery of precise amounts of drugs to a single cell is especially interesting for cell and tissue engineering as well as therapeutics and has until now required the application of micro-pipettes, precisely placed dispersed drug delivery vehicles, or injections close to or into the cell. Here we present surface bound microchamber arrays able to store small hydrophilic molecules for prolonged times in subaqueous conditions supporting spatiotemporal near infrared laser mediated release. The micro-chambers (MCs) are composed of biocompatible and biodegradable polylactic acid (PLA). Biocompatible gold nanoparticles are employed as light harvesting agents to facilitate photothermal MC opening. The degree of photothermal heating is determined by numerical simulations utilizing optical properties of the MC, and confirmed by Brownian motion measurements of laser-irradiated micro-particles exhibiting similar optical properties like the MCs. The amount of bioactive small molecular cargo (doxycycline) from local release is determined by fluorescence spectroscopy and gene expression in isolated C2C12 cells via enhanced green fluorescent protein (EGFP) biosynthesis.
High frequency alternating magnetic fields (AMF) have been widely used as a non-invasive method t... more High frequency alternating magnetic fields (AMF) have been widely used as a non-invasive method to induce local hyperthermia for antitumor treatment and to efficiently trigger drug release from various carriers. However, few studies have exploited the potential of targeted drug delivery to healthy cells or tissue and the use of low frequency AMF (LF-AMF) for intracellular triggered release. To achieve this goal, doxycycline was delivered with the layer-by-layer (LbL) assembled magnetic microcapsules, and AMF with low frequency (50 Hz) was applied. The low frequency AMF had little effect on morphology of microcapsules, which upon exposure for 360 min caused no significant damage and had the advantage of minimizing heating effects. Nonetheless, microcapsule permeability increased as a function of exposure time when assessed using FITC-dextran (70 kDa) with the number of permeable microcapsules increased from 13.5% (20 min) to 52.8% (360 min). Increased permeability also enhanced in vitro doxycycline release in genetically engineered myoblast cells where EGFP expression is regulated by the tetracycline system, while targeted EGFP expression was observed by magnetically navigating the microcapsules to a site of interest. Upon LF-AMF exposure of 30 min, no cytotoxicity was observed, but intracellular doxycycline release was promoted and enhanced EGFP expression as demonstrated by EGFP fluorescence intensity measurement. This study reveals the possibility of targeted drug delivery and using LF-AMF as a non-cytotoxic intracellular trigger of drug release from microcapsules without alteration in cell viability.
Immunohistochemistry of human atherosclerotic arteries demonstrates expression of the intercellul... more Immunohistochemistry of human atherosclerotic arteries demonstrates expression of the intercellular adhesion molecule-1 (ICAM-1) on endothelial cells, macrophages, and smooth muscle cells of the plaques. Normal arterial endothelial cells and intimal smooth muscle outside plaques give weaker or negative reactions; these differ from the strong endothelial expression in small vessels. Quantitative color-image analysis of the endothelial layer shows increased expression of ICAM-1 in all subtypes of atherosclerotic lesions, except fibrous plaques. Endothelial expression of ICAM-1 may be involved in the recruitment of monocytes to the lesion, as suggested by its role in the entry of leukocytes, including monocytes, into foci of inflammation. Collaboration with other mechanisms, particularly chemoattractant factors, may be important for this effect. ICAM-1 enhanced monocyte recruitment is a potential mechanism for the growth of an atherosclerotic plaque.
Background Genome-wide association studies have identifi ed ADAMTS7 as a risk locus for coronary ... more Background Genome-wide association studies have identifi ed ADAMTS7 as a risk locus for coronary artery disease and myocardial infarction. Functional studies suggest that ADAMTS7 might promote cellular processes in atherosclerosis. We sought to examine whether carriers of a loss-of-function genetic variant exhibit favourable characteristics on plaque histology, angiographic coronary artery disease severity, and cardiovascular outcomes. Methods The single-nucleotide polymorphism rs3825807 was used as a marker for ADAMTS7. Human coronary atherosclerotic plaques (n=50) were genotyped, and characterised with immunohistochemical analysis. Burden of angiographic coronary artery disease was assessed by angiographic severity scores in two independent populationbased cohorts-the Southampton Atherosclerosis Study (SAS, n=1359) in the UK, and the prospective Emory Genebank study (Emory GB, n=2684) in the USA. Follow-up data were collected in Emory GB over a median of 7 years. Findings Human coronary atherosclerotic plaques with the loss-of-function G allele exhibited thinner fi brous cap (p=0•017) and lower percentage area of α-actin (smooth muscle cell marker) in the intima (p=0•029). After adjustment for age and sex, the G allele was associated with less coronary artery disease in both SAS (odds ratio 0•82, 95% CI 0•67-0•99), and Emory GB (0•84, 0•74-0•95). Angiographic burden was further characterised with the Gensini Score (GS) and Sullivan Extent Score (SES). ADAMTS genotypes were associated with all of the angiographic severity scores in both SAS (GS p=0•026, SES p=0•029) and Emory GB (GS p<0•0001, SES p<0•0001). Outcome analysis showed lower incidence of revascularisation for G allele carriers (hazard ratio 0•77, 95% CI 0•60-0•98), but not all cause mortality (1•12, 0•92-1•35). Interpretation Carriers of the ADAMTS7 loss-of-function allele G had reduced atherosclerotic plaque progression, as demonstrated by thinner cap and smooth muscle migration. In addition there was less severe angiographic coronary artery disease in the SAS and Emory GB cohorts, as well as lower incidence of revascularisation procedure, further supporting the role of this protease in promoting atherosclerosis. Funding None. Contributors KC, RP, XP, RNP, IAS, and SH conceived the study, collected and analysed the data, and wrote the abstract. SY and AQ conceived the study, obtained funding, and supervised the study. Declaration of interests We declare no competing interests.
Publisher Summary The treatment of formalin fixed paraffin embedded sections with enzymes is esta... more Publisher Summary The treatment of formalin fixed paraffin embedded sections with enzymes is established as a means of restoring antigenicity for immunohistochemistry. The mechanism probably depends on the removal from antigenic sites of blocking proteins that have been cross-linked by formalin. With polyclonal antibodies trypsin and pronase have been the principal enzymes used, but there have been few reports to date of the effects of enzymes on the staining properties of monoclonal antibodies (MCs). The present study investigates the effects of a range of enzymes on five MCs that have been found useful for diagnostic immunohistochemistry with formalin fixed sections. As with polyclonal antibodies, enhancement of the immunohistochemical staining of formalin fixed sections could be obtained with MCs by enzyme treatment. Some enzymes were capable of destroying antigenicity, and the pattern of effect was unpredictable and variable from one MC to another.
Nanoengineered vehicles have the potential to deliver cargo drugs directly to disease sites, but ... more Nanoengineered vehicles have the potential to deliver cargo drugs directly to disease sites, but can potentially be cleared by immune system cells or lymphatic drainage. In this study we explore the use of magnetism to hold responsive particles at a delivery site, by incorporation of superparamagnetic iron oxide nanoparticles (SPIONs) into layer-by-layer (LbL) microcapsules. Microcapsules with SPIONs were rapidly phagocytosed by cells but did not trigger cellular ROS synthesis within 24 hours of delivery nor affect cell viability. In a non-directional cell migration assay, SPION containing microcapsules significantly inhibited movement of phagocytosing cells when placed in a magnetic field. Similarly, under flow conditions, a magnetic field retained SPION containing microcapsules at a physiologic wall shear stress of 0.751 dyne cm −2. Even when the SPION content was reduced to 20%, the majority of microcapsules were still retained. Dexamethasone microcrystals were synthesised by solvent evaporation and underwent LbL encapsulation with inclusion of a SPION layer. Despite a lower iron to volume content of these structures compared to microcapsules, they were also retained under shear stress conditions and displayed prolonged release of active drug, beyond 30 hours, measured using a glucocorticoid sensitive reporter cell line generated in this study. Our observations suggest use of SPIONs for magnetic retention of LbL structures is both feasible and biocompatible and has potential application for improved local drug delivery. † Electronic supplementary information (ESI) available. See
Expansion of the dermal microvasculature is a prominent feature of psoriasis. Although the pathog... more Expansion of the dermal microvasculature is a prominent feature of psoriasis. Although the pathogenetic process resulting in vascular morphological changes remains unclear, considerable evidence suggests the involvement of angiogenesis. To assess the degree and site of psoriatic microvascular expansion, immunohistochemical studies were performed on paired lesional and non-lesional specimens from 10 patients with active, untreated plaque psoriasis. Five-micron thick sections were labelled with monoclonal antibody JC/70A specific for the endothelial marker CD31, and vascular quantification was achieved using hue-saturation-intensity image analysis. Assessment of vasculature in the papillary dermis (superficial plexus) demonstrated a fourfold increase in endothelial surface area of lesional compared with non-lesional skin (P &lt; 0.01), while there was no significant increase in vasculature of the upper reticular dermis. Subsequently, 18-micron thick sections were double-labelled with MIB-1 antibody to the nuclear proliferation marker Ki-67 and JC/70A. Endothelial cell proliferation was identified in the vertical limbs of capillary loops in eight out of 10 lesional biopsies and in no non-lesional biopsies. The endothelial MIB-1 labelling index was 3.1% of total endothelial cells of the superficial plexus. These findings confirm endothelial proliferation underlying psoriatic microvascular expansion, and indicate that this process is limited to a specific site in the dermal capillary bed.
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Papers by Robin Poston