Circulating Sex-Specific Markers of Plaque Instability in Women and Men With Severe Carotid Atherosclerosis.

BACKGROUND: Sex-specific differences in plaque composition and instability underscore the need to explore circulating markers for better prediction of high-risk plaques. This cross-sectional study aims to (1) investigate differences in lipid, immune, and adipokine circulating profiles between men and women with stable versus unstable plaques and (2) identify circulating markers that can better classify men and women according to plaque instability. METHODS: Preoperative blood samples and plaque specimens were collected from consecutive men and women with carotid artery stenosis ≥50% and who underwent a carotid endarterectomy between 2009 and 2018. Adipokine, lipid, and immune profiling was conducted. Plaque stability was determined by gold-standard histological classifications. Statistical analyses, including χ2, ANOVA, Kruskal-Wallis, and logistic regression, assessed differences in plaque features and blood parameters between men and women with stable and unstable plaques. RESULTS: Of 470 recruited patients (aged 70.8±9.2 years), the final study analyses included 317 men and 143 women (aged 71.0±9.0 years). Men exhibited more unstable plaques (P<0.001), characterized by increased plaque hemorrhage, larger lipid core, and inflammation (P<0.001), along with less favorable circulating profiles. Antagonistic interactions between sex and white blood cell (WBC) counts, basophil-to-WBC ratio, and platelet counts influenced plaque instability. In men, low WBC counts, high monocyte-to-WBC ratio, low basophil-to-WBC ratio, and high LDL-C (low-density lipoprotein cholesterol) levels were associated with greater plaque instability (odds ratio, 0.827 [95% CI, 0.713-0.926], 1.158 [95% CI, 1.027-1.305], 0.495 [95% CI, 0.281-0.871], and 1.564 [95% CI, 1.001-2.443], respectively) and more unstable features (ie, inflammation, foam cells, and neovascularization). In women, a high basophil-to-WBC ratio was associated with greater plaque instability (3.142 [95% CI, 1.220-8.093]), hemorrhage, and thrombosis, while a high molecular weight-to-total adiponectin ratio was associated with decreased instability (0.014 [95% CI, 0.000-0.646]) and inflammation. CONCLUSIONS: Our findings demonstrated sex-specific differences, with women displaying more stable plaque phenotypes and favorable circulating profiles compared with men. This proof-of-concept study was also designed as the key first step in exploring novel sex-specific associations between circulating lipid, immune, and adipokine profiles and carotid plaque instability.

Relationship between circulating adipokines and cholesterol efflux in subjects with severe carotid atherosclerosis.

AIMS: Cholesterol efflux capacity (CEC) as a measure of high-density lipoprotein functionality is independently and inversely associated with increased risk of cardiovascular events and mortality, and advanced plaque morphology. Adipokines, adipose tissue-derived factors, can influence systemic lipoprotein metabolism, and participate in the regulation of vascular function and inflammation. We aimed to investigate the association between CEC and circulating adipokine levels (anti-inflammatory adiponectin, and pro-inflammatory chemerin and resistin) in subjects with severe carotid atherosclerotic disease and evaluate its impact on post-surgical outcomes. METHODS AND RESULTS: This is a cross-sectional study with a 5-year follow-up component. Consecutive patients with severe carotid atherosclerosis scheduled for a carotid endarterectomy were recruited from hospital-based centres in Montreal, Canada (n = 285). Fasting blood samples were collected pre-operatively and used to measure plasma total and high-molecular weight (HMW) adiponectin, chemerin, and resistin, and to perform cholesterol efflux assays in J774 macrophage-like cells. Five-year post-surgery outcomes were obtained through medical chart review. Subjects had a mean age of 70.1 ± 9.4, were 67.0 % male, had various comorbidities (hypercholesterolemia [85.3 %], hypertension [83.5 %], type 2 diabetes [34.5 %], coronary artery disease [38.6 %]), and previously experienced cerebrovascular symptomatology (77.9 %). CEC was independently and positively associated with total and HMW adiponectin levels (ß [95 % confidence interval]; 0.216 [0.134-0.298] and 0.107 [0.037-0.176], respectively) but not with chemerin or resistin. Total adiponectin had the greatest association accounting for 8.3 % of the variance in CEC. Interaction regression models demonstrated a significant interaction between adiponectin and chemerin in increasing CEC. Notably, with each unit increase in CEC there was a 93.9 % decrease in the odds of having an ischemic cerebrovascular event 5 years post-surgery (0.061 [0.007-0.561]). CONCLUSIONS: Our findings demonstrated circulating adiponectin to have a strong association with increased CEC in subjects with severe carotid atherosclerosis and high CEC to be associated with more favourable post-surgical outcomes. These findings reflect the importance of adipose tissue health in influencing CEC levels and atherosclerotic cardiovascular disease risk.

Expression of Resistin, Chemerin, and Chemerin's Receptor in the Unstable Carotid Atherosclerotic Plaque.

Background and Purpose: Unstable carotid plaques are a common cause of ischemic strokes. Identifying markers that reflect/contribute to plaque instability has become a prominent focus in cardiovascular research. The adipokines, resistin and chemerin, and ChemR23 (chemerin receptor), may play a role in carotid atherosclerosis, making them potential candidates to assess plaque instability. However, the expression and interrelationship of resistin and chemerin (and ChemR23) protein and mRNA within the carotid atherosclerotic plaque remains elusive. Thus, we investigated herein, the association between plaque mRNA and protein expression of resistin and chemerin (and ChemR23) and carotid plaque instability in humans, and whether sex differences exist in the relationship between these adipokines and plaque instability. Methods: Human carotid plaques were processed for immunohistochemical/mRNA analysis of resistin, chemerin, and ChemR23. Plaque instability was assessed by gold-standard histological classifications. A semi-quantitative scoring system was used to determine the intensity of adipokine expression on macrophages/foam cells, as well as the percentage of inflammatory cells stained positive. Plaque adipokine protein expression was also digitally quantified and mRNA expression was assessed by qRT-PCR. Results: Resistin and chemerin mRNA expression was 80% and 32% lower, respectively, in unstable versus stable plaques (P<0.05), while no difference in ChemR23 mRNA expression was observed. In contrast, greater resistin staining intensity and percentage of cells stained positive were detected in unstable versus stable plaques (P<0.01). Similarly, chemerin and ChemR23 staining intensity and percentage of cells stained were positively associated with plaque instability (P<0.05). No strong sex-specific relationship was observed between adipokines and plaque instability. Conclusions: This study examined the relationship between resistin, chemerin, and ChemR23, and carotid plaque instability, with a specific analysis at the plaque level. We reported a positive association between plaque instability and protein levels of resistin, chemerin, and ChemR23 but a negative association with resistin and chemerin mRNA expression. This suggests these adipokines exert proinflammatory roles in the process of carotid atherosclerosis and may be regulated via a negative feedback regulatory mechanism.

Intensive Statin Therapy Compromises the Adiponectin-AdipoR Pathway in the Human Monocyte-Macrophage Lineage.

Background and Purpose- Statins are widely used for cardiovascular disease prevention through cholesterol-lowering and anti-inflammatory effects. Adiponectin, an anti-inflammatory adipokine, acts via two receptors, AdipoR1 and AdipoR2, to exert atheroprotective effects on the vasculature. We investigated whether statins can modulate the adiponectin-AdipoR pathway in the human monocyte-macrophage lineage. Methods- Monocytes were isolated from the whole blood of patients with severe carotid atherosclerosis (cross-sectional study) or from patients with cardiovascular risk factors (longitudinal study) and assessed for AdipoR1 and AdipoR2 gene expression using quantitative real-time polymerase chain reaction. In vitro, THP-1 (Tamm-Horsfall protein 1) macrophages were treated with increasing atorvastatin or rosuvastatin doses for 24- or 72-hours to determine the effect of statins on AdipoR expression and activity. Macrophage cytokine secretion (IL [interleukin]-1ß, IL-10, IL-6, and TNF [tumor necrosis factor]-α) was assessed by electrochemiluminescence. Results- AdipoR1 and AdipoR2 mRNA expression on circulating monocytes from patients with carotid atherosclerosis, was significantly lower by 1.36- and 1.17-fold, respectively, in statin users versus statin-naïve patients. Specifically, patients on high doses of atorvastatin (40-80 mg) or rosuvastatin (20-40 mg) had significantly lower AdipoR gene expression versus statin-naïve patients. Similarly, in the longitudinal in vivo study, longer atorvastatin/rosuvastatin treatment (≥5 months) in patients with cardiovascular risk factors resulted in lower AdipoR gene expression on circulating monocytes versus prestatin levels. In vitro, higher statin doses and longer exposure resulted in a greater decrease in AdipoR mRNA expression and greater macrophage secretion of pro-inflammatory cytokines, IL-1ß, IL-6, and TNF-α. High statin doses also reduced adiponectin's capacity to suppress intracellular cholesteryl ester levels in oxLDL (oxidized LDL)-loaded macrophages, with rosuvastatin exhibiting higher potency than atorvastatin. Conclusions- Our in vivo and in vitro studies identified a novel pleiotropic property of statins in modulating the adiponectin-AdipoR pathway in the human monocyte-macrophage lineage, where intensive statin therapy compromised the expression and function of adiponectin and its receptors.

New Quantitative Digital Image Analysis Method of Histological Features of Carotid Atherosclerotic Plaques.

OBJECTIVE: Atherosclerosis and its thrombotic complications are major causes of morbidity and mortality worldwide. Plaque stability assessment is considered to be important for both clinical and fundamental applications. The current gold standard method to investigate plaque stability is performed by histological assessment of plaque features using semi-quantitative classifications. However, these assessments can be limited by subjectivity and variability. Thus, the aim was to develop a new digital image analysis method to measure quantitatively individual plaque features that is more precise than existing semi-quantitative methods. METHODS: A quantitative method was developed using Image Pro Primer software. Carotid plaque specimens were obtained from patients who underwent carotid endarterectomy and categorised according to stability (definitely stable, probably stable, probably unstable, definitely unstable) based on the gold standard semi-quantitative method that assesses 10 histological plaque features. Using the new quantitative method, plaque features (n = 15) from each stability grade were then analysed by two independent raters. For the semi-quantitative analysis, quadratic weighted Cohen's kappa was used to test intra- and inter-rater reliability, while for the quantitative analysis, intraclass correlation coefficients (ICCs) were assessed. RESULTS: Intra-rater reliability demonstrated almost perfect agreement between both methods (Cohen's kappa range 0.831-0.969, ICC range 0.848-1.000). However, inter-rater reliability demonstrated mainly fair to moderate agreement (Cohen's kappa range 0.341-0.778) for the semi-quantitative analysis, while the digital image analysis method performed most optimally regarding reproducibility, yielding high ICCs close to 1 (ICC range 0.816-0.999). Using quantitative measurements, a statistically significant proportion of the individual plaque features (p < .05) were re-classified from one grade to another (shift by one) under the semi-quantitative classification. CONCLUSION: A new quantitative digital image analysis was developed for the accurate assessment of histological plaque features, which demonstrated higher precision than the gold standard semi-quantitative methods, as measured by between and within rater analysis. Moreover, quantitative image analysis of histological plaque features provided more detailed insight into plaque morphology and composition.

Decreased Adiponectin-Mediated Signaling Through the AdipoR2 Pathway Is Associated With Carotid Plaque Instability.

BACKGROUND AND PURPOSE: Adiponectin, the most abundantly secreted anti-inflammatory adipokine, protects against all stages of atherosclerotic plaque formation by acting on its receptors, AdipoR1 (adiponectin receptor 1) and AdipoR2 (adiponectin receptor 2). Through binding of AdipoR1, adiponectin leads to the activation of the AMPK (adenosine monophosphate-activated protein kinase) pathway, whereas stimulation of PPAR-α (peroxisome proliferator-activated receptor-α) is attributed to the binding of AdipoR2. However, the role of adiponectin and its receptors in plaque instability remains to be characterized. Thus, we aimed to investigate whether the adiponectin-AdipoR pathway is associated with carotid atherosclerotic plaque instability. METHODS: The instability of plaque specimens obtained from patients who underwent a carotid endarterectomy (n=143) was assessed using gold standard histological classifications. RESULTS: Using immunohistochemistry, we showed that adiponectin and AdipoR1/AdipoR2 are expressed in human carotid plaques and that their expression was localized most abundantly in areas of macrophage and foam cell accumulation. Unstable plaques expressed more adiponectin protein (Western blot, P<0.05) and less AdipoR2 mRNA (2.11-fold decrease, P<0.05) than stable plaques, whereas AdipoR1 expression remained similar between stable and unstable plaques. Beyond AdipoR1/AdipoR2 expression, a graded decrease in PPAR-α protein levels was observed in relation to carotid plaque instability (P<0.001), whereas AMPK phosphorylation was increased (P<0.05). Our in vitro model of plaque instability, involving the induction of foam cells from human THP-1 (Tamm-Horsfall protein 1) macrophages treated with acetylated low-density lipoprotein, supported our in vivo conclusions. CONCLUSIONS: An overall abundance of adiponectin with a decrease in AdipoR2 expression and activity was observed in unstable plaques, suggesting that reduced signaling through the AdipoR2 pathway, and not through AdipoR1, may contribute to plaque instability.

Functional deficits in peripheral nerve mitochondria in rats with paclitaxel- and oxaliplatin-evoked painful peripheral neuropathy.

Cancer chemotherapeutics like paclitaxel and oxaliplatin produce a dose-limiting chronic sensory peripheral neuropathy that is often accompanied by neuropathic pain. The cause of the neuropathy and pain is unknown. In animal models, paclitaxel-evoked and oxaliplatin-evoked painful peripheral neuropathies are accompanied by an increase in the incidence of swollen and vacuolated mitochondria in peripheral nerve axons. It has been proposed that mitochondrial swelling and vacuolation are indicative of a functional impairment and that this results in a chronic axonal energy deficiency that is the cause of the neuropathy's symptoms. However, the significance of mitochondrial swelling and vacuolation is ambiguous and a test of the hypothesis requires a direct assessment of the effects of chemotherapy on mitochondrial function. The results of such an assessment are reported here. Mitochondrial respiration and ATP production were measured in rat sciatic nerve samples taken 1-2 days after and 3-4 weeks after induction of painful peripheral neuropathy with paclitaxel and oxaliplatin. Significant deficits in Complex I-mediated and Complex II-mediated respiration and significant deficits in ATP production were found for both drugs at both time points. In addition, prophylactic treatment with acetyl-l-carnitine, which inhibited the development of paclitaxel-evoked and oxaliplatin-evoked neuropathy, prevented the deficits in mitochondrial function. These results implicate mitotoxicity as a possible cause of chemotherapy-evoked chronic sensory peripheral neuropathy.

Experimental research on the management of combined radiation-burn injury in China.

Combined radiation-burn injury can occur in people exposed to nuclear explosions, nuclear accidents or radiological terrorist attacks. Using different combined radiation-burn injury animal models, the pathological mechanisms underlying combined radiation-burn injury and effective medical countermeasures have been explored for several years in China, mainly at our institute. Targeting key features of combined radiation-burn injury, several countermeasures have been developed. Fluid transfusion and the calcium antagonist verapamil can prevent early shock and improve myocardial function after combined radiation-burn injury. Recombinant human interleukin 4 (rhIL-4) is able to effectively reduce bacterial infection and increase intestinal immunological ability. Chitosan-wrapped human defensin 5 (HD5) and glucagon-like peptide 2 (GLP-2) nanoparticles can increase the average survival time of animals with severe combined radiation-burn injury. After treatment by cervical sympathetic ganglia block (SB), hematopoietic function is promoted and the release of inflammatory cytokines is suppressed. The optimal time for escharectomy and allo-skin grafting is 24 h after injury. Transfusion of irradiated (20 Gy) or stored (4°C, 7 days) blood improves the survival of allo-skin grafting and allo-bone marrow cells. In conclusion, as our understanding of the mechanisms of combined radiation-burn injury has progressed, new countermeasures have been developed for its treatment. Because of the complexity of its pathology and the difficulty in clinical management, further efforts are needed to improve the treatment of this kind of injury.

Protective effect of atorvastatin on radiation-induced vascular endothelial cell injury in vitro.

Vascular endothelial cells are very sensitive to ionizing radiation, and it is important to develop effective prevent agents and measures in radiation exposure protection. In the present study, the protective effects of atorvastatin on irradiated human umbilical vein endothelial cells (HUVEC) and the possible mechanisms were explored. Cultured HUVEC were treated by atorvastatin at a final concentration of 10 µ mol/ml for 10 minutes, and then irradiated at a dose of 2 Gy or 25 Gy. Twenty-four hours after irradiation, apoptosis of HUVEC was monitored by flow cytometry, and the expression of thrombomodulin (TM) and protein C activation in HUVEC was respectively assessed by flow cytometry and spectrophotometry. After treatment with atorvastatin for 24 h, the rate of cell apoptosis decreased by 6% and 16% in cells irradiated with 2 Gy and 25 Gy, respectively. TM expression increased by 77%, 59%, and 61% in untreated cells, 2 Gy irradiation-treated cells, and 25 Gy irradiation-treated cells, respectively. The protein C levels in 2 Gy and 25 Gy irradiation-treated cells were reduced by 23% and 34% when compared with untreated cells, but up-regulated by 79% and 76% when compared with cells which were irradiated and treated with atorvastatin. In conclusion, these data indicate that atorvastatin exerts protective effects on irradiated HUVEC by reducing apoptosis by up-regulating TM expression and enhancing protein C activation in irradiated HUVEC.

Lifelong protection from global cerebral ischemia and reperfusion in long-lived Mclk1(+/)(-) mutants.

To achieve a long life span, animals must be resistant to various injuries as well as avoid or delay lethality from age-dependent diseases. Reduced expression of the mitochondrial enzyme CLK-1/MCLK1 (a.k.a. Coq7), a mitochondrial hydroxylase that is necessary for the biosynthesis of ubiquinone (UQ), extends lifespan in Caenorhabditiselegans and in mice. Here, we show that long-lived Mclk1(+/)(-) mutants have enhanced resistance to neurological damage following global cerebral ischemia-reperfusion (I/R) injury induced by transient bilateral common carotid artery occlusion (BCCAO). Both young ( approximately 100days old) and relatively aged ( approximately 450days old) mutants display increased resistance as indicated by a significant decrease in the amount of degenerating cells observed in forebrain cortex and in hippocampal areas after ischemia and reperfusion. Furthermore, less oxidative damage resulting from the procedure was measured in the brain of young Mclk1(+/)(-) animals. The finding that both young and old mutants are protected indicates that this is a basic phenotype of these mutants and not a secondary consequence of their slow rate of aging. Thus, the partial resistance to I/R injury suggests that Mclk1(+/)(-) mutants have an enhanced recovery potential following age-dependant vascular accidents, which correlates well with their longer survival. By relating this neuroprotective effect to previously reported characteristics of the Mclk1(+/)(-) phenotype, including altered mitochondrial metabolism and increased HIF-1alpha expression, this study establishes these mutants as useful models to analyze the mechanisms underlying tolerance to ischemia, particularly those associated with ischemic preconditioning, as well as to clarify the relation between aging and age-dependent diseases.

Regulation of IL-13 receptor alpha 1 expression and signaling on human tonsillar B-lymphocyte subsets.

BACKGROUND: T(H)2 cytokines play crucial roles in driving human B lymphocytes to produce IgE. However, it is unclear whether IL-4 and IL-13 have parallel or sequential roles in the development of B lymphocytes. OBJECTIVE: We investigated IL-13 receptor (IL-13R) expression and regulation in mature and immature human B cells. METHODS: Purified B cells were isolated from human tonsils. We evaluated IL-13Ralpha1 mRNA expression using real-time PCR and IL-13Ralpha1 and IL-4 receptor (IL-4R) alpha expression using flow cytometry and microscopy. Signal transduction was assessed on the basis of signal transducer and activator of transcription 6 phosphorylation. RESULTS: IL13Ralpha1 mRNA was induced after stimulation with anti-CD40 antibodies, anti-CD40 plus IL-4, or anti-IgM/IgG. Baseline surface IL13Ralpha1 levels were low in unstimulated B cells but increased significantly at 24 hours and were sustained for 5 to 14 days. In contrast, IL4R alpha was constitutively expressed on tonsillar B cells, and levels did not significantly vary after stimulation. B cells activated by CD40 ligation or B-cell receptor cross-linking, but not resting B cells, showed significant increases in signal transducer and activator of transcription 6 phosphorylation in response to IL-13. IL-13Ralpha1 expression was induced on mature and memory B cells, as well as on naive subsets. CONCLUSIONS: There is lower constitutive expression and signaling of IL13Ralpha1 in resting tonsillar B lymphocytes compared with that of IL4R alpha. IL-13 is induced on both immature and mature B lymphocytes. CLINICAL IMPLICATIONS: This implies different roles for IL-4 and IL-13 in B-cell development, which would allow for specific targeting of IL-13 in IgE-mediated diseases.

Effects of serum from rats with combined radiation-burn injury on the growth of hematopoietic progenitor cells.

BACKGROUND: This study aims to observe the effects of blood serum from rats with radiation injury, burn injury, and combined radiation-burn injury on the growth of hematopoietic progenitor cells and to explore the possible mechanisms. METHODS: Serum from rats with radiation injury, burn injury, and combined radiation-burn injury were collected at 3 hours, 12 hours, 24 hours, 48 hours, 72 hours, and 96 hours after injury and then was added to the culture medium to see its effect on the growth of hematopoietic progenitor cells (HPCs) at a final protein concentration of 10 microg/mL. Radioimmunoassay and enzyme-linked immunosorbent assay were employed to measure the level of tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 in each group, and the effect of TNF-alpha and IL-6 on the growth of HPC was also observed. RESULTS: The number of HPCs colonies formed after addition of the serum from rats with burn or combined radiation-burn injuries was significantly higher than that from normal rats at 3 hours, 12 hours, 24 hours, 48 hours, 72 hours, and 96 hours after injury and reached its peak value at 24 hours after injury. However, fewer HPCs colonies were found after the addition of the serum from irradiated rats. At the same time, the levels of TNF-alpha and IL-6 in the serum of burn group and combined radiation-burn injury group were significantly higher than that of normal group, and much higher than that of the irradiation injury group (p < 0.01). Also, TNF-alpha and IL-6 demonstrated promoting effect on the growth of HPC. CONCLUSION: Serum from rats with burn injury and combined radiation-burn injury stimulates the growth of HPCs, while serum from irradiated rats shows inhibitory effects on the growth of HPCs. These effects may lie in the different level of TNF-alpha and IL-6 in the serum of each group.

Regulation of early and delayed radiation responses in rat small intestine by capsaicin-sensitive nerves.

PURPOSE: Mast cells protect against the early manifestations of intestinal radiation toxicity, but promote chronic intestinal wall fibrosis. Intestinal sensory nerves are closely associated with mast cells, both anatomically and functionally, and serve an important role in the regulation of mucosal homeostasis. This study examined the effect of sensory nerve ablation on the intestinal radiation response in an established rat model. METHODS AND MATERIALS: Rats underwent sensory nerve ablation with capsaicin or sham ablation. Two weeks later, a localized segment of ileum was X-irradiated or sham irradiated. Structural, cellular, and molecular changes were examined 2 weeks (early injury) and 26 weeks (chronic injury) after irradiation. The mast cell dependence of the effect of sensory nerve ablation on intestinal radiation injury was assessed using c-kit mutant (Ws/Ws) mast cell-deficient rats. RESULTS: Capsaicin treatment caused a baseline reduction in mucosal mast cell density, crypt cell proliferation, and expression of substance P and calcitonin gene-related peptide, two neuropeptides released by sensory neurons. Sensory nerve ablation strikingly exacerbated early intestinal radiation toxicity (loss of mucosal surface area, inflammation, intestinal wall thickening), but attenuated the development of chronic intestinal radiation fibrosis (collagen I accumulation and transforming growth factor beta immunoreactivity). In mast cell-deficient rats, capsaicin treatment exacerbated postradiation epithelial injury (loss of mucosal surface area), but none of the other aspects of radiation injury were affected by capsaicin treatment. CONCLUSIONS: Ablation of capsaicin-sensitive enteric neurons exacerbates early intestinal radiation toxicity, but attenuates development of chronic fibroproliferative changes. The effect of capsaicin treatment on the intestinal radiation response is partly mast cell dependent.

An autoimmune domain-reduced HCV core gene remains effective in stimulating anti-core cytotoxic T lymphocyte activity.

Chronic hepatitis C virus (HCV) infection cases resistant to conventional therapies might be treated by immunotherapy as cytotoxic T lymphocytes (CTL) are the main mechanism through which viral infections are cleared. The HCV core gene, with the highest homology between HCV types, deleted of its autoimmune-stimulating regions (pseudo-GOR and pseudo-p450), may be an appropriate antigen for targeting HCV-infected cells. Two recombinant adeno-associated virus (rAAV) vectors, carrying either the full length (aa 1-190) or truncated (aa 49-180, deleted of the pseudo-GOR and pseudo-p450 sequences) versions of core, were generated. Both AAV/core (l-190) and AAV/core (49-180) were used to transduce/load dendritic cells (DC) at high levels (88-95%). These two genetically altered DC types then stimulated anti-core CTL. The DC and CTL were analyzed by FACS and for killing efficiency (percent target killing). The rAAV-altered DC displayed higher levels of CD80, CD83, CD86, and CD 1a than control DC. The truncated core (aa 49-180) gene stimulated equivalent and strong killing of synthetic core-positive autologous peripheral blood lymphocyte (PBL) targets to that stimulated by the full length core gene. However, the smaller core (49-180) antigen gene stimulated lower levels of killing of core-negative "self" PBL targets (GOR- and p450-positive) (p = 0.002). These AAV/core: DC-stimulated CTL displayed higher IFN-gamma expression, higher CD8:CD4 ratios, and lower CD56:CD8 ratios than controls. The rAAV-loading derived CD8+ T cells had more CD69+ cells and the CD4+ T populations had fewer CD25+ cells than controls. We conclude that the core (49-180) gene is an effect antigen, but has the advantage of stimulating less self-recognition. Thus, core (49-180) may be useful for further translational immunotherapy studies against HCV.

Modulation of the intestinal response to ionizing radiation by anticoagulant and non-anticoagulant heparins.

Endothelial dysfunction is involved in radiation responses in many normal tissues, including intestine. Endothelium-directed interventions ameliorate intestinal radiation injury (radiation enteropathy) in animal models, and anecdotal reports also suggest a beneficial effect of heparin. This study assessed low molecular weight heparin as an intestinal radiation response modifier. Rats underwent localized small bowel irradiation. Groups of rats were treated with saline, nadroparin (3 mg/kg/d), or a non-anticoagulant heparin (SR80258, 3 mg/kg/d), from 3 days before to 2 weeks after irradiation. The intestinal radiation response was assessed 2 weeks and 6 weeks after irradiation using quantitative histology; morphometry, and cellular and molecular end-points. Compared to vehicle-treated controls, nadroparin significantly exacerbated structural radiation injury, neutrophil infiltration, and TGFbeta and collagen I immunoreactivity levels 2 weeks after irradiation. SR80258 was associated with increased TGFbeta levels, but the other parameters did not reach statistical significance. At 6 weeks, structural, cellular, and molecular injury was similar in the three experimental groups. Heparin, in contrast to antiplatelet agents and direct thrombin inhibitors, does not ameliorate, but exacerbates acute intestinal radiation toxicity. These data underscore the importance of heparin as an inhibitor of physiological anti-inflammatory mechanisms during tissue injury, as well as the non-anticoagulant effects of heparin. Moreover, these data may have implications for the use of heparin during radiation therapy.

[Effects of blood serum from rats with combined radiation-thermal injury on the bone marrow hematopoietic progenitor cells growth].

To observe the effects of blood serum from rats with radiation injury, thermal injury and combined radiation-thermal lesions on growth of hematopoietic progenitor cells and the change of their serum cytokine levels, total body irradiation of rats was performed with 12 Gy gamma ray from a (60)Co source, and 30% total body surface area III degree thermal lesion on the back was inflicted with a 5 kW bromotungsten lamp. The blood serum from these animals was collected at 3, 12, 24, 48, 72 and 96 hours after injury. Then the blood serum was added to the culture medium of erythrocyte progenitor cells (CFU-E, BFU-E) or granulocyte-macrophage progenitor cells (CFU-GM) at final concentration of 10 microg/ml. The results showed that the colony number of CFU-E, BFU-E and CFU-GM formed after addition of the blood serum from rats with thermal or combined radiation-thermal injury was significantly higher than that from normal rats at 3, 12, 24, 48, 72 and 96 hours after injury and reached its peak value at 24 hours after injury (342.8, 261.6 and 228.4% respectively from burned rats, 252.4, 205.1 and 174.2% respectively from rats with combined radiation-thermal injury as compared with that of normal rats). However, a few CFU-E, BFU-E or CFU-GM formation was found after addition of the blood serum from irradiated rats. At the same time, the level of TNF alpha and IL-6 in serum of burn group and combined radiation-thermal injury group was markedly higher than that of normal group, even more higher than that of irradiation injury group (P < 0.01). It is concluded that the blood serum from rats with thermal lesion or combined radiation-thermal injury improves the growth of erythrocyte and granulocyte progenitor cells. On the contrary, the blood serum from the irradiated rats shows the inhibiting effects, definitely related to their serum cytokines changes.

Synthesis of IL-13 by human B lymphocytes: regulation and role in IgE production.

BACKGROUND: Our laboratory has demonstrated previously that human tonsillar B lymphocytes express IL-13 mRNA OBJECTIVE: We sought to investigate IL-13 production by human B cells and the association between B cell-derived IL-13 and IgE secretion. METHODS: Human B lymphocytes were isolated from tonsils and purified by means of rosetting with sheep RBCs or positive or negative selection with magnetic beads. They were stimulated with anti-CD40 antibodies with or without recombinant IL-4. Total mRNA was extracted, and IL-13 mRNA was measured by means of standard RT-PCR or by means of real-time PCR with commercially available primers. B cells were cultured with or without IL-13 neutralizing antibodies, and C epsilon transcripts and supernatant IgE levels were measured. RESULTS: IL-13 mRNA was detected in human B lymphocytes stimulated with anti-CD40 antibodies and IL-4 or IL-2 but not in unstimulated B cells. Real-time PCR demonstrated a 10- to 15-fold increase in IL-13 mRNA, maximizing at 36 hours. IL-13 protein was detected from B lymphocytes on day 3 and accumulated through day 7. The synthesis of IL-13 required both CD40 and IL-4 stimulation. The presence of IL-13 was confirmed by means of intracellular staining of cultured B lymphocytes and antigen-stimulated nasal biopsy specimens from atopic individuals. Addition of IL-13 neutralizing antibodies to purified B-cell cultures inhibited IgE production by up to 80% and diminished IgE (C epsilon) transcripts by 50%. CONCLUSION: Human B lymphocytes express IL-13 mRNA after ligation of CD40 and the addition of cytokines. Human B lymphocytes produce significant IL-13, and neutralization of IL-13 impairs IgE synthesis. IL-13 might be an important autocrine growth factor for IgE-producing B lymphocytes.

Up-regulation and activation of proteinase-activated receptor 2 in early and delayed radiation injury in the rat intestine: influence of biological activators of proteinase-activated receptor 2.

Proteinase-activated receptor 2 (Par2, F2rl1, also designated PAR-2 or PAR2) is prominently expressed in the intestine and has been suggested as a mediator of inflammatory, mitogenic and fibrogenic responses to injury. Mast cell proteinases and pancreatic trypsin, both of which have been shown to affect the intestinal radiation response, are the major biological activators of Par2. Conventional Sprague-Dawley rats, mast cell-deficient rats, and rats in which pancreatic exocrine secretion was blocked pharmacologically by octreotide underwent localized irradiation of a 4-cm loop of small bowel. Radiation injury was assessed 2 weeks after irradiation (early, inflammatory phase) and 26 weeks after irradiation (chronic, fibrotic phase). Par2 expression and activation were assessed by in situ hybridization and immunohistochemistry, using antibodies that distinguished between total (preactivated and activated) Par2 and preactivated Par2. Compared to unirradiated intestine, irradiated intestine exhibited increased Par2 expression, particularly in areas of myofibroblast proliferation and collagen accumulation, after both single-dose and fractionated irradiation. The majority of Par2 expressed in fibrotic areas was activated. Postirradiation Par2 overexpression was greatly attenuated in both mast cell-deficient and octreotide-treated rats. The severity of acute mucosal injury did not affect postirradiation Par2 expression. Mast cells and pancreatic proteinases may exert their fibro-proliferative effects partly through activation of Par2. Par2 may be a potential target for modulating the intestinal radiation response, particularly delayed intestinal wall fibrosis.

Statins increase thrombomodulin expression and function in human endothelial cells by a nitric oxide-dependent mechanism and counteract tumor necrosis factor alpha-induced thrombomodulin downregulation.

Expression of functionally active thrombomodulin (TM) on the luminal surface of endothelial cells is critical for vascular thromboresistance. TM maintains thrombohemorrhagic homeostasis by forming a complex with thrombin, which subsequently loses its procoagulant properties and instead activates protein C. Acquired deficiency of endothelial TM is of particular pathophysiological significance in sepsis and related disorders. We show here that two different 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins), atorvastatin and simvastatin, strongly increase the expression and functional activity of TM in human umbilical vein endothelial cells, human coronary artery endothelial cells, and EA.hy926 endothelial cells. The increase in endothelial TM conferred by statin was prevented by the addition of mevalonic acid, geranylgeranyl-pyrophosphate, and nitric oxide scavenger, and was mimicked by the addition of a specific inhibitor of geranylgeranyl transferase, as well as by nitric oxide donors. Moreover, statin counteracted tumor necrosis factor alpha-induced downregulation of endothelial cell TM. The increase in endothelial cell TM activity in response to statin constitutes a novel pleiotropic (non-lipid-related) effect of these commonly used compounds, and may be of clinical significance in disorders where deficient endothelial TM and protein C activation play a pathophysiological role.

Deficiency of microvascular thrombomodulin and up-regulation of protease-activated receptor-1 in irradiated rat intestine: possible link between endothelial dysfunction and chronic radiation fibrosis.

Microvascular injury is believed to be mechanistically involved in radiation fibrosis, but direct molecular links between endothelial dysfunction and radiation fibrosis have not been established in vivo. We examined radiation-induced changes in endothelial thrombomodulin (TM) and protease-activated receptor-1 (PAR-1) in irradiated intestine, and their relationship to structural, cellular, and molecular aspects of radiation injury. Rat small intestine was locally exposed to fractionated X-radiation. Structural injury was assessed 24 hours and 2, 6, and 26 weeks after the last radiation fraction using quantitative histology and morphometry. TM, neutrophils, transforming growth factor-beta, and collagens I and III were assessed by quantitative immunohistochemistry. PAR-1 protein was localized immunohistochemically, and cells expressing TM or PAR-1 transcript were identified by in situ hybridization. Steady-state PAR-1 mRNA levels in intestinal smooth muscle were determined using laser capture microdissection and competitive reverse transcriptase-polymerase chain reaction. Radiation caused a sustained, dose-dependent decrease in microvascular TM. The number of TM-positive vessels correlated with all parameters of radiation enteropathy and, after adjusting for radiation dose and observation time in a statistical model, remained independently associated with neutrophil infiltration, intestinal wall thickening, and collagen I accumulation. PAR-1 immunoreactivity and transcript increased in vascular and intestinal smooth muscle cells in irradiated intestine. PAR-1 mRNA increased twofold in irradiated intestinal smooth muscle. Intestinal irradiation up-regulates PAR-1 and causes a dose-dependent, sustained deficiency of microvascular TM that is independently associated with the severity of radiation toxicity. Interventions aimed at preserving or restoring endothelial TM or blocking PAR-1 should be explored as strategies to increase the therapeutic ratio in clinical radiation therapy.