Immunostaining For Macrophages Research Articles

Candesartan Attenuates Vasculitis in a Mouse Model of Kawasaki Disease Induced by Candida albicans Water-Soluble Fraction.

The standard treatment for Kawasaki disease is immunoglobulin therapy, but the high frequency of coronary sequelae in immunoglobulin-refractory cases indicates a need for further improvement in treatment. Kawasaki disease-like vasculitis was induced in 5-week-old DBA/2 mice by intraperitoneal administration of 0.5 mg Candida albicans water-soluble fraction (CAWS) daily for 5 days followed by daily administration of candesartan, an angiotensin receptor blocker. The vasculitis suppression effect was confirmed histologically and serologically in mice sacrificed at 28 days after the start of candesartan. The area of inflammatory cell infiltration at the aortic root was 2.4±1.4% in the Control group, 18.1±1.9% in the CAWS group, and 7.1±2.3%, 5.8±1.4%, 7.6±2.4%, and 7.9±5.0% in the CAWS+candesartan 0.125-mg/kg, 0.25-mg/kg, 0.5-mg/kg, and 1.0-mg/kg groups, respectively (p=0.0200, p=0.0122, p=0.0122, and p=0.0200 vs. CAWS, respectively). The low-dose candesartan group also showed significantly reduced inflammatory cell infiltration. A similar trend was confirmed by immunostaining of macrophages and TGFβ receptors. Measurement of the inflammatory cytokines IL-1β, IL-6, and TNF-α confirmed the anti-vasculitis effect of candesartan. Candesartan inhibited vasculitis even at clinical doses used in children, making it a strong future candidate as an additional treatment for immunoglobulin-refractory Kawasaki disease.

Intradiscal inflammatory stimulation induces spinal pain behavior and intervertebral disc degeneration invivo.

Degeneration of the intervertebral disc (IVD) results in a range of symptomatic (i.e., painful) and asymptomatic experiences. Components of the degenerative environment, including structural disruption and inflammatory cytokine production, often correlate with pain severity. However, the role of inflammation in the activation of pain and degenerative changes has been complex to delineate. The most common IVD injury model is puncture; however, it initiates structural damage that is not representative of the natural degenerative cascade. In this study, we utilized invivo injection of lipopolysaccharide (LPS), a pro-inflammatory stimulus, into rat caudal IVDs using 33G needles to induce inflammatory activation without the physical tissue disruption caused by puncture using larger needles. LPS injection increased gene expression of pro-inflammatory cytokines (Tnfa, Il1b) and macrophage markers (Inos, Arg1), supported by immunostaining of macrophages (CD68, CCR7, Arg1) and systemic changes in blood cytokine and chemokine levels. Disruption of the IVD structural integrity after LPS injection was also evident through changes in histological grading, disc height, and ECM biochemistry. Ultimately, intradiscal inflammatory stimulation led to local mechanical hyperalgesia, demonstrating that pain can be initiated by inflammatory stimulation of the IVD. Gene expression of nociceptive markers (Ngf, Bdnf, Cgrp) and immunostaining for neuron ingrowth (PGP9.5) and sensitization (CGRP) in the IVD were also shown, suggesting a mechanism for the pain exhibited. To our knowledge, this rat IVD injury model is the first to demonstrate local pain behavior resulting from inflammatory stimulation of caudal IVDs. Future studies will examine the mechanistic contributions of inflammation in mediating pain.

Enhanced expression of iNOS is associated with aortic valve stenosis in patients with bicuspid aortic valve

Abstract Background A congenital bicuspid aortic valve (BAV) is one of the most common congenital cardiac abnormalities, with a prevalence estimated between 0.5% and 2%. Aortic valve stenosis (AS) is the most common complication in patients with BAV. The mechanisms responsible for the rapid progression of AS in patients with BAV relative to those with a tricuspid aortic valve (TAV) are unknown. A previous study showed that the pathogenesis of AS in patients with BAV is associated with a more aggressive inflammatory process, with increased macrophage infiltration and neo-vascularization when compared with that seen in patients with TAV. A considerable number of human diseases have an inflammatory component, and a key mediator of immune activation and inflammation is inducible nitric oxide synthase (iNOS). Overexpressed iNOS has been implicated in the pathogenesis of numerous conditions, including coronary plaque destabilization. To elucidate the role of iNOS in the aortic valve of patients with AS, we carried out an immunohistochemical study of the presence of iNOS in aortic valve specimens from patients with AS with either TAV or BAV. Methods Frozen aortic valve samples were obtained surgically from patients with AS with TAV (n=34) or BAV (n=34), and stained with antibodies against macrophages, microvessels, iNOS and 4-hydroxy-2-nonenal (4-HNE), an index of lipid peroxidation. The immunoreactivity of macrophages, microvessels, iNOS and 4-HNE was quantified using computer-aided polarimetry. Double immunostaining was also performed to identify cell types that stained positive for iNOS. Results Quantitative analysis demonstrated that macrophage-, iNOS-, and 4-HNE-positive areas, as well as the number of microvessels, were significantly higher in patients with AS with BAV than with TAV (macrophages, P<0.001; microvessels, P<0.05; 4-HNE, P<0.05; iNOS, P<0.001). The iNOS-positive area was positively correlated with the 4-HNE-positive area (R=0.43, P<0.0001). Double immunostaining for both iNOS and macrophages identified iNOS-positive cells as macrophages. Conclusions These findings suggest that accumulation of iNOS in macrophages may increase oxidative stress and contribute to the rapid progression of AS in patients with BAV.

Therapeutic or lifelong training effects on pancreatic morphological and functional parameters in an animal model of aging and obesity

AimsObesity, aging, and physical training are factors influencing pancreatic functional and morphological parameters. Aiming to clarify the impact of the interaction of these factors, we analyzed the effect of therapeutic or lifelong physical training on body adiposity and pancreatic functional and morphological parameters of aged and obese rats. Methods24 male Wistar rats were (initial age = 4 months and final age = 14 months) randomly divided into three aged and obese experimental groups (n = 8/group): untrained, therapeutic trained, and lifelong trained. Body adiposity, plasmatic concentration and pancreatic immunostaining of insulin, markers of tissue inflammation, lipid peroxidation, activity and immunostaining of antioxidant enzymes, and parameters of pancreatic morphology were evaluated. ResultsLifelong physical training improved the body adiposity, plasmatic insulin concentration, and macrophage immunostaining in the pancreas. The animals submitted to therapeutic and lifelong training showed an increase in the density of the pancreatic islets; lower insulin, Nuclear Factor Kappa B (NF-κB), and Transforming Growth Factor beta (TGF-β) immunostaining in the pancreatic parenchyma, as well as lower pancreatic tissue lipid peroxidation, lower fibrosis area, increased catalase and glutathione peroxidase (GPx) activity and increased heme oxygenase-1 (HO-1) immunostaining, with the greatest effect in the lifelong training group. ConclusionLifelong training promoted greater beneficial effects on the pancreatic functional and morphological parameters of aged and obese animals compared to therapeutic exercise.

Immunohistochemical Evidence of Melatonin Protection on Lung Tissue During Chemotherapy

The present study investigated the possible protective effects of melatonin on Bleomycin, Cisplatin and etoposide (BEP) chemotherapy regimens using immunohistochemistry. Forty male Wistar rats were divided into four groups of ten as; group 1 as untreated control; group 2 as BEP group which received the three cycles of 21 days’ regimen each of 0.5¥ dose levels ofBEP (bleomycin 0.75 mg/kg, etoposide 7.5 mg/kg and cisplatin 1.5 mg/kg). Rats in the group 3 (MEL group) received 10 mg/kg/day melatonin once daily. Group 4 received the melatonin (30 min before the BEP injections) and BEP as in groups 2. Proliferating cell nuclear antigen (PCNA) staining was used to detect cell proliferation and caspase-3, caspase-9 and Caspase-8 were detected to investigate apoptosis. PCNA immunostaining in alveolar epithelium, alveolar macrophages and bronchus was weak to moderate in BEP group. However, diffuse and strong caspase immunoreactions for caspase-3, caspase 8- and caspase-9 were detected in the bronchioles epithelium, vascular endothelium, alveolar luminal macrophages in the BEP group. PCNA and caspase immunoreactivities in MEL and Mel + BEP groups were close to the control one. The surface are in the BEP group was significantly reduced as compared to the control one ((P<0.05). However, in MEL and Mel + BEP groups, it was close to the control value (P>0.05). It can be concluded that BEP regimen can affects negatively on lung tissue and melatonin inhibits lung tissue injuries during BEP chemotherapy.

Molecular imaging of fibroblast activation protein after myocardial infarction using the novel radiotracer [68Ga]MHLL1.

Background: Myocardial infarction (MI) evokes an organized remodeling process characterized by the activation and transdifferentiation of quiescent cardiac fibroblasts to generate a stable collagen rich scar. Early fibroblast activation may be amenable to targeted therapy, but is challenging to identify in vivo. We aimed to non-invasively image active fibrosis by targeting the fibroblast activation protein (FAP) expressed by activated (myo)fibroblasts, using a novel positron emission tomography (PET) radioligand [68Ga]MHLL1 after acute MI.Methods: One-step chemical synthesis and manual as well as module-based radiolabeling yielded [68Ga]MHLL1. Binding characteristics were evaluated in murine and human FAP-transfected cells, and stability tested in human serum. Biodistribution in healthy animals was interrogated by dynamic PET imaging, and metabolites were measured in blood and urine. The temporal pattern of FAP expression was determined by serial PET imaging at 7 d and 21 d after coronary artery ligation in mice as percent injected dose per gram (%ID/g). PET measurements were validated by ex vivo autoradiography and immunostaining for FAP and inflammatory macrophages.Results: [68Ga]MHLL1 displayed specific uptake in murine and human FAP-positive cells (p = 0.0208). In healthy mice the tracer exhibited favorable imaging characteristics, with low blood pool retention and dominantly renal clearance. At 7 d after coronary artery ligation, [68Ga]MHLL1 uptake was elevated in the infarct relative to the non-infarcted remote myocardium (1.3 ± 0.3 vs. 1.0 ± 0.2 %ID/g, p < 0.001) which persisted to 21 d after MI (1.3 ± 0.4 vs. 1.1 ± 0.4 %ID/g, p = 0.013). Excess unlabeled compound blocked tracer accumulation in both infarct and non-infarct remote myocardium regions (p < 0.001). Autoradiography and histology confirmed the regional uptake of [68Ga]MHLL1 in the infarct and especially border zone regions, as identified by Masson trichrome collagen staining. Immunostaining further delineated persistent FAP expression at 7 d and 21 d post-MI in the border zone, consistent with tracer distribution in vivo.Conclusion: The simplified synthesis of [68Ga]MHLL1 bears promise for non-invasive characterization of fibroblast activation protein early in remodeling after MI.

Microdistribution of Magnetic Resonance Imaging Contrast Agents in Atherosclerotic Plaques Determined by LA-ICP-MS and SR-\u03bcXRF Imaging

PurposeContrast-enhanced magnetic resonance imaging (MRI) has the potential to replace angiographic evaluation of atherosclerosis. While studies have investigated contrast agent (CA) uptake in atherosclerotic plaques, exact CA spatial distribution on a microscale is elusive. The purpose of this study was to investigate the microdistribution of gadolinium (Gd)- and iron (Fe) oxide-based CA in atherosclerotic plaques of New Zealand White rabbits.ProceduresThe study was performed as a post hoc analysis of archived tissue specimens obtained in a previous in vivo MRI study conducted to investigate signal changes induced by very small superparamagnetic iron oxide nanoparticles (VSOP) and Gd-BOPTA. For analytical discrimination from endogenous Fe, VSOP were doped with europium (Eu) resulting in Eu-VSOP. Formalin-fixed arterial specimens were cut into 5-μm serial sections and analyzed by immunohistochemistry (IHC: Movat’s pentachrome, von Kossa, and Alcian blue (pH 1.0) staining, anti-smooth muscle cell actin (anti-SMA), and anti-rabbit macrophage (anti-RAM-11) immunostaining) and elemental microscopy with laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) and synchrotron radiation μX-ray fluorescence (SR-μXRF) spectroscopy. Elemental distribution maps of Fe, Eu, Gd, sulfur (S), phosphorus (P), and calcium (Ca) were investigated.ResultsIHC characterized atherosclerotic plaque pathomorphology. Elemental microscopy showed S distribution to match the anatomy of arterial vessel wall layers, while P distribution corresponded well with cellular areas. LA-ICP-MS revealed Gd and Fe with a limit of detection of ~ 0.1 nmol/g and ~ 100 nmol/g, respectively. Eu-positive signal identified VSOP presence in the vessel wall and allowed the comparison of Eu-VSOP and endogenous Fe distribution in tissue sections. Extracellular matrix material correlated with Eu signal intensity, Fe concentration, and maximum Gd concentration. Eu-VSOP were confined to endothelium in early lesions but accumulated in cellular areas in advanced plaques. Gd distribution was homogeneous in healthy arteries but inhomogeneous in early and advanced plaques. SR-μXRF scans at 0.5 μm resolution revealed Gd hotspots with increased P and Ca concentrations at the intimomedial interface, and a size distribution ranging from a few micrometers to submicrometers.ConclusionsEu-VSOP and Gd have distinct spatial distributions in atherosclerotic plaques. While Eu-VSOP distribution is more cell-associated and might be used to monitor atherosclerotic plaque progression, Gd distribution indicates arterial calcification and might help in characterizing plaque vulnerability.

Acute kidney injury induced by glycerol is worsened by orchiectomy and attenuated by testosterone replacement

Although several studies have demonstrated that the male gender represents an independent risk factor for renal disease, evidence shows that androgens exert renal protective actions. The findings are controversial and no studies have evaluated the effects of orchiectomy and testosterone replacement on glycerol-induced renal injury. Male Wistar rats were submitted to orchiectomy or sham surgery and divided into four groups: SC, sham control rats injected with NaCl; SG, sham rats injected with glycerol; OG, orchiectomized rats injected with glycerol; OGT, orchiectomized rats injected with glycerol and testosterone. Testosterone was administered daily for 14 days in the OGT group. After 11 days of testosterone replacement in the OGT group, SC rats were submitted to a saline injection, while SG, OG and OGT rats received glycerol. All rats were euthanized three days after injections. OG rats presented higher serum creatinine and urea, and sodium excretion, compared to SC and SG, while testosterone attenuated these changes. Acute tubular necrosis was also mitigated by testosterone. Renal immunostaining for macrophages, lymphocytes and NF-κB was higher in OG compared to SC and SG. In addition, renal interleukin-1β, Caspase 3 and AT1 gene expression was higher in OG rats compared to SG. Testosterone attenuated these alterations, except the NF-κB immunostaining. The renal NO was lower in OG rats compared to SG. Only the OG rats presented decreases in serum NO and renal HO-1, and increased TNF-α, angiotensinogen and AT1 expression compared to SC. We conclude that orchiectomy worsened glycerol-induced kidney injury, while testosterone attenuated this renal damage.

ADAM15 expression is increased in lung CD8+ T cells, macrophages, and bronchial epithelial cells in patients with COPD and is inversely related to airflow obstruction

BackgroundA disintegrin and metalloproteinase domain-15 (ADAM15) is expressed by activated leukocytes, and fibroblasts in vitro. Whether ADAM15 expression is increased in the lungs of COPD patients is not known.MethodsADAM15 gene expression and/or protein levels were measured in whole lung and bronchoalveolar lavage (BAL) macrophage samples obtained from COPD patients, smokers, and non-smokers. Soluble ADAM15 protein levels were measured in BAL fluid (BALF) and plasma samples from COPD patients and controls. Cells expressing ADAM15 in the lungs were identified using immunostaining. Staining for ADAM15 in different cells in the lungs was related to forced expiratory volume in 1 s (FEV1), ratio of FEV1 to forced vital capacity (FEV1/FVC), and pack-years of smoking history.ResultsADAM15 gene expression and/or protein levels were increased in alveolar macrophages and whole lung samples from COPD patients versus smokers and non-smokers. Soluble ADAM15 protein levels were similar in BALF and plasma samples from COPD patients and controls. ADAM15 immunostaining was increased in macrophages, CD8+ T cells, epithelial cells, and airway α-smooth muscle (α-SMA)-positive cells in the lungs of COPD patients. ADAM15 immunostaining in macrophages, CD8+ T cells and bronchial (but not alveolar) epithelial cells was related inversely to FEV1 and FEV1/FVC, but not to pack-years of smoking history. ADAM15 staining levels in airway α-SMA-positive cells was directly related to FEV1/FVC. Over-expressing ADAM15 in THP-1 cells reduced their release of matrix metalloproteinases and CCL2.ConclusionsThese results link increased ADAM15 expression especially in lung leukocytes and bronchial epithelial cells to the pathogenesis of COPD.

Attenuation of Hyperoxic Lung Injury in Newborn Thioredoxin-1-Overexpressing Mice through the Suppression of Proinflammatory Cytokine mRNA Expression.

The role of thioredoxin-1 (TRX), a small redox-active protein with antioxidant effects, during hyperoxic lung injury in newborns remains undetermined. We investigated TRX impact on hyperoxic lung injury in newborn TRX transgenic (TRX-Tg) and wildtype (WT) mice exposed to 21% or 95% O2 for four days, after which some mice were allowed to recover in room air for up to 14 days. Lung morphology was assessed by hematoxylin/eosin and elastin staining, as well as immunostaining for macrophages. The gene expression levels of proinflammatory cytokines were evaluated using quantitative real-time polymerase chain reaction. During recovery from hyperoxia, TRX-Tg mice exhibited an improved mean linear intercept length and increased number of secondary septa in lungs compared with the WT mice. Neonatal hyperoxia enhanced the mRNA expression levels of proinflammatory cytokines in the lungs of both TRX-Tg and WT mice. However, interleukin-6, monocyte chemoattractant protein-1, and chemokine (C-X-C motif) ligand 2 mRNA expression levels were reduced in the lungs of TRX-Tg mice compared with the WT mice during recovery from hyperoxia. Furthermore, TRX-Tg mice exhibited reduced macrophage infiltration in lungs during recovery. These results suggest that in newborn mice TRX ameliorates hyperoxic lung injury during recovery likely through the suppression of proinflammatory cytokines.

Pharmacological Inhibition of Cathepsin S Suppresses Abdominal Aortic Aneurysm in Mice

Evidence suggests that cathepsin S (CTSS), a potent mammalian elastase, participates in abdominal aortic aneurysm (AAA) formation. This study examines the hypothesis that pharmacological inhibition of CTSS with an α-ketoamide based compound 6r might suppress AAA in mice. Experimental study of the CaCl2 induced AAA model in B6 mice and angiotensin II (AngII) infused AAA model in ApoE-/- mice. The effects of intraperitoneal administration of 6r (25mg/kg) and vehicle every three days since one day after AAA induction were evaluated at 28 days using CaCl2 induced (n=12 per group) and AngII infused (n=8 per group) models. Additionally, the effects of post-treatment with 6r and vehicle from seven days or 14 days after AAA induction were evaluated at 28 days using the CaCl2 induced model (n=6 per group). Aortic samples were harvested for histological and biochemical analyses, including cathepsin levels, Verhoeff Van Gieson staining, TUNEL assay, and immunostaining for macrophages. In the CaCl2 induced model, treatment with 6r suppressed aortic dilatation observed in vehicle treated controls (median: 0.58 vs. 0.92mm; p<.001), along with reduced CTSS and cathepsin K (CTSK) levels (both p<.001), preserved elastin integrity (p<.001), fewer medial apoptotic cells (p=.012) and less macrophage infiltration (p=.041). In the AngII infused model, the aortic diameter was smaller in 6r treated mice than in vehicle treated controls (median: 0.95 vs. 1.84mm; p=.047). The levels of CTSS (p<.001) and CTSK (p=.033) and the numbers of elastin breaks (p<.001), medial apoptotic cells (p<.001) and infiltrating macrophages (p=.030) were attenuated under 6r treatment. Finally, post-treatment with 6r from seven days (p=.046) or 14 days (p=.012) after AAA induction limited CaCl2 induced AAA. Pharmacological inhibition of CTSS by 6r suppresses AAA formation in mice. Also, post-treatment with 6r retards mouse AAA progression. These findings provide proof of concept validation for CTSS as a potential therapeutic target in AAA.

Allograft Inflammatory Factor 1 as an Immunohistochemical Marker for Macrophages in Multiple Tissues and Laboratory Animal Species.

Allograft inflammatory factor 1 (AIF1) is a commonly used marker for microglia in the brains of humans and some animal models but has had limited applications elsewhere. We sought to determine whether AIF1 can be used as a macrophage marker across common laboratory animal species and tissues. We studied tissues (that is, spleen, liver, and lung) with defined macrophage populations by using an AIF1 immunostaining technique previously validated in human tissue. Tissues were collected from various mouse strains (n = 20), rat strains (n = 15), pigs (n = 4), ferrets (n = 4), and humans (n = 4, lung only). All samples of liver had scattered immunostaining in interstitial cells, consistent with resident tissue macrophages (Kupffer cells). Spleen samples had cellular immunostaining of macrophages in both the red and white pulp compartments, but the red pulp had more immunostained cellular aggregates and, in some species, increased immunostaining intensity compared with white pulp. In lung, alveolar macrophages had weak to moderate staining, whereas interstitial and perivascular macrophages demonstrated moderate to robust staining. Incidental lesions and tissue changes were detected in some sections, including a tumor, inducible bronchus-associated lymphoid tissue, and inflammatory lesions that demonstrated AIF1 immunostaining of macrophages. Finally, we compared AIF1 immunostaining of alveolar macrophages between a hypertensive rat model (SHR strain) and a normotensive model (WKY strain). SHR lungs had altered intensity and distribution of immunostaining in activated macrophages compared with macrophages of WKY lungs. Overall, AIF1 immunostaining demonstrated reproducible macrophage staining across multiple species and tissue types. Given the increasing breadth of model species used to study human disease, the use of cross-species markers and techniques can reduce some of the inherent variability within translational research.

Tumor-resident macrophages modulate intestinal barrier function through sialylation of Mucin 1 in IBD and colitis-associated cancer.

Abstract Mucin 1 (MUC1) is a transmembrane glycoprotein overexpressed and hypoglycosylated in chronic inflammatory conditions and human adenocarcinoma compared to healthy tissues. We have reported that AOM/DSS-treated human MUC1.Tg mice showed higher tumor incidence and greater inflammation when compared to wild-type (WT) mice. Moreover, high expression levels of pro-inflammatory cytokines, including TNF-α and IL-6, were found in MUC1+ inflamed colon tissues and exogenous TNF-α promoted the transcriptional activity of MUC1 as well as over-expression of its hypoglycosylated form. As macrophages are the major source of TNF-α and IL-6, we analyzed infiltrating type 1 (M1) and type 2 (M2) macrophages into the inflamed and tumorigenic colon tissues. Immunofluorescence assay indicated increased presence of macrophages, and in particular CD206+ (M2), in inflamed colon tissues of MUC1.Tg mice compared to WT mice. In addition, immunostaining of macrophages in colon tissues from IBD patients showed expression of hypoglycosylated MUC1 and the presence of macrophages expressing CD163+, a human M2 marker. Interestingly, in vitro co-culture model system indicated that M1 and M2 polarized macrophages, induced different glycoforms of MUC1 in HT-29 colon cancer cells. In addition, macrophage-secreted factors modulated glycosyltrasferase enzymes, sush as ST6GalNAc1 and ST3GAL1, involved in O-glycan sialylation. We are currently dissecting the molecular basis by which macrophages induced hypoglycosylated and tumor forms of MUC1 during inflammation and colitis-associated cancer (CAC). Our findings will elucidate novel functions of tumor-associated macrophages in altering the intestinal barrier function in IBD and CAC.

Heparan sulfate 3-O-sulfotransferase 2 (HS3ST2) displays an unexpected subcellular localization in the plasma membrane

BackgroundHeparan sulfate (HS) 3-O-sulfation can be catalysed by seven 3-O-sulfotransferases (HS3STs) in humans, still it is the rarest modification in HS and its biological function is yet misunderstood. HS3ST2 and HS3ST3B exhibit the same activity in vitro. They are however differently expressed in macrophages depending on cell environment, which suggests that they may be involved in distinct cellular processes. Here, we hypothesized that both isozymes might also display distinct subcellular localizations. MethodsThe subcellular distribution of HS3ST2 and HS3ST3B was analysed by using overexpression systems in HeLa cells. The localization of endogenous HS3ST2 was confirmed by immunostaining in primary macrophages. ResultsWe found that HS3ST3B was only localized in the Golgi apparatus and no difference between full-length enzyme and truncated construct depleted of its catalytic domain was observed. In contrast, HS3ST2 was clearly visualized at the plasma membrane. Its truncated form remained in the Golgi apparatus, meaning that the catalytic domain might support correct addressing of HS3ST2 to cell surface. Moreover, we found a partial co-localization of HS3ST2 with syndecan-2 in HeLa cells and primary macrophages. Silencing the expression of this proteoglycan altered the localization of HS3ST2, which suggests that syndecan-2 is required to address the isozyme outside of the Golgi apparatus. ConclusionsWe demonstrated that HS3ST3B is a Golgi-resident isozyme, while HS3ST2 is addressed to the plasma membrane with syndecan-2. General significanceThe membrane localization of HS3ST2 suggests that this enzyme may participate in discrete processes that occur at the cell surface.

Guinea pig infection with the intracellular pathogen Rhodococcus equi

Rhodococcus equi is an opportunistic, intracellular pathogen that causes pyogranulomatous pneumonia in foals and immunocompromised people. Currently, there is no experimental model of R. equi pneumonia other than intra-bronchial experimental infection of foals with R. equi, which is labor-intensive and costly. This study’s objective was to develop a guinea pig (GP) model of R. equi pneumonia that would facilitate development of novel approaches for controlling and preventing this disease. Guinea pigs were infected with either 101, 102, 103, or 104 colony forming units (CFUs) of a virulent strain of R. equi using a Madison aerosol chamber, or 106 or 107 CFUs of this strain intratracheally. Animals were monitored daily for clinical signs of pneumonia, and were euthanized and necropsied on days 1, 3, 7, or 35 post-infection (PI). Lung homogenates were plated onto selective agar to determine bacterial load. No clinical signs of disease were observed regardless of the inoculum dose or infection method. No bacteria were recovered from GPs euthanized at 35 days PI. Histology and immunostaining of T-cells, B-cells, and macrophages in lungs showed that inflammatory responses in infected GPs were similarly unremarkable irrespective of dose or route of infection. Guinea pigs appear to be resistant to pulmonary infection with virulent R. equi even at doses that reliably produce clinical pneumonia in foals.

Abstract 28: Apolipoprotein A-I Vascular Gene Therapy Provides Durable Protection from Atherosclerosis in Hyperlipidemic Rabbits

Background: Gene therapy, delivered directly to the vascular wall, could potentially protect against atherosclerotic lesion growth or regress existing lesions. Previously, we demonstrated that infusion of HDAdApoAI [a helper-dependent adenoviral vector (HDAd) expressing apolipoprotein (apo) A-I] in carotid arteries of fat-fed rabbits reduced early carotid atherosclerosis development (4 wk after vector infusion). Here we tested whether the same HDAd could protect long-term against more severe atherosclerosis. Methods: 25 fat-fed rabbits underwent bilateral carotid gene transfer [HDAdApoAI on one side; control vector (HDAdNull) on the other, with sides randomized]. Postoperatively, diets were adjusted to maintain plasma cholesterols of 200-800mg/dl. This protocol yields large lipid- and macrophage-rich lesions. 6 mo later, carotids were harvested for analyses of DNA, RNA, protein, cholesterol, and histology (H&amp;E, oil red O), or immunostaining for macrophages, muscle actin, T-cells, ICAM-1 and VCAM-1. Results: Vector genomes persisted equally in HDAdApoAI and HDAdNull-infused arteries; however, apoA-I mRNA was detected only in HDAdApoAI-infused arteries. HDAdApoAI-infused arteries had ~60% less median intimal lesion volume than HDAdNull-infused arteries, with concomitant reductions (40-75%) in intimal lipid, macrophage, smooth muscle cell, VCAM-1 and ICAM-1. We used within-rabbit paired analyses to control for high correlations of lesion size and composition between left and right carotids of the same rabbit. 19 of 25 rabbits had smaller lesions in HDAdApoAI-infused vs HDAdNull-infused arteries; median decreased intimal area between paired carotids in the same rabbit was 30% ( P &lt;0.03). Within individual rabbits, intimal lipid, macrophage, smooth muscle cell, VCAM-1 and ICAM-1 were all less in HDAdApoAI arteries (median 23-36% less; P &lt;0.05 for all except ICAM-1). Conclusions: Vascular gene therapy with HDAdApoAI reduced lesion development and decreased intimal lipid, macrophage, and adhesion molecule expression 6 mo after treatment. Vector genomes and apo A-I mRNA remained high at 6 mo. A single application of vascular gene therapy durably retards development of atherosclerosis, even in a setting of severe hyperlipidemia.

Detection of Phenotypic Differences in Alveolar Macrophages Using Ionized Calcium‐Binding Adapter Molecule 1 Marker

Alveolar macrophages are resident immune cells in the distal lung and offer a first line of defense to inhaled pathogens and environmental particulates. In healthy states, these resident immune cells reside in the alveolar spaces of the lung as quiescent monitors, but upon stimulation, they play significant roles in host defense, removal of inhaled particulates and tissue remodeling. Upon activation, alveolar macrophages can exhibit morphological changes including increases in cell size, ruffled borders and altered expression of markers. In this study of 10 week old male rats, we had immunostained lung sections for the marker, ionized calcium‐binding adapter molecule 1 (IBA1). We evaluated whether quantitative or qualitative differences could be detected in alveolar macrophages between the normotensive WKY (Wistar Kyoto) strain and the hypertensive SHR (Spontaneous Hypertensive Strain) strains. For all analysis, statistical significance was set at P&lt;0.05. Immunocytochemistry of bronchoalveolar fluid showed similar immunostaining of macrophages in both groups with no overt group‐distinguishing features. However, examination of immunostained paraffin embedded tissue sections suggest that SHR lungs have subpopulations of macrophages with increased cell size. We evaluated macrophage size in these subpopulations using a semiquantitative scoring system and then by quantitative evaluation of macrophage diameter with both showing significant increases in SHR macrophages (P&lt;0.01 for both). To test the extent of IBA1 immunostaining in lung high resolution images were quantified. SHR lungs had more IBA1 immunostaining than WKY (P&lt;0.05). Interestingly, IBA1 immunostaining in small, quiescent macrophages was often homogenous throughout the cytoplasm whereas macrophages that were larger in size often had a central zone of cytoplasmic pallor with stronger immunostaining near the periphery/membrane of the cell. These findings suggest that quantitative and qualitative features of IBA1 immunostaining may be useful tools to detect differences in the activation state of alveolar macrophages.Support or Funding InformationThis work was supported in part by the NIH

Polarized M2 macrophages in dogs with visceral leishmaniasis

The objective of the present study was to analyze the skin (nasal surface and ear regions), lymph nodes (popliteal and pre-scapular), spleen and liver of dogs with visceral leishmaniasis (VL), in order to investigate the relationship between the parasite load measured as DNA copy number of Alpha gene of DNA polymerase of Leishmania infantum by quantitative PCR and the number of M2 macrophages by immunohistochemistry. A set of 29 naturally infected dogs from an endemic area for VL were sampled and another set of six dogs negative for VL and from a non-endemic area were analyzed as the control group (C). The spleen presented the highest number of Leishmania DNA copies, with significant differences between the groups G1 and G2 (with and without skin lesions, respectively). The M2 phenotype immunostaining predominated among the macrophages in granulomas and inflammatory infiltrates of samples from the skin, lymph nodes and spleens examined. The presence of M2 macrophages in dogs from infected group differed significantly from the control group, in all organs analyzed, excepted liver. The highest proportion of M2 macrophages coincided with the highest parasitism loads found in more susceptible organs of VL dogs, even in the skin, considered a more resistant organ, while the liver showed low parasitism load and low immunostaining for M2 macrophages with no significant differences between infected and negative groups. It was concluded that the predominance of M2 phenotype in VL dogs favored the multiplication of Leishmania infantum in organs of dogs that are more susceptible to Leishmania infection, as skin, lymph nodes and spleen.

Dipeptidyl Peptidase 4 Distribution in the Human Respiratory Tract: Implications for the Middle East Respiratory Syndrome

Dipeptidyl peptidase 4 (DPP4, CD26), a type II transmembrane ectopeptidase, is the receptor for the Middle Eastern respiratory syndrome coronavirus (MERS-CoV). MERS emerged in 2012 and has a high mortality associated with severe lung disease. A lack of autopsy studies from MERS fatalities has hindered understanding of MERS-CoV pathogenesis. We investigated the spatial and cellular localization of DPP4 to evaluate an association MERS clinical disease. DPP4 was rarely detected in the surface epithelium from nasal cavity to conducting airways with a slightly increased incidence in distal airways. DPP4 was also found in a subset of mononuclear leukocytes and in serous cells of submucosal glands. In the parenchyma, DPP4 was found principally in type I and II cells and alveolar macrophages and was also detected in vascular endothelium (eg, lymphatics) and pleural mesothelia. Patients with chronic lung disease, such as chronic obstructive pulmonary disease and cystic fibrosis, exhibited increased DPP4 immunostaining in alveolar epithelia (type I and II cells) and alveolar macrophages with similar trends in reactive mesothelia. This finding suggests that preexisting pulmonary disease could increase MERS-CoV receptor abundance and predispose individuals to MERS morbidity and mortality, which is consistent with current clinical observations. We speculate that the preferential spatial localization of DPP4 in alveolar regions may explain why MERS is characterized by lower respiratory tract disease.

Liquid fructose supplementation in LDL-R−/− mice fed a western-type diet enhances lipid burden and atherosclerosis despite identical calorie consumption

BackgroundStudies on humans have related the high consumption of fructose, especially in the form of sugar-sweetened beverages, to obesity, fatty liver, and hypercholesterolemia, all risk factors for atherosclerosis, and cardiovascular disease. We aimed to determine whether supplementation of liquid fructose (SLF), in either a normal, healthy chow or a Western-style chow, promoted the appearance of atherosclerosis in a rodent model. MethodsLDL receptor knockout mice were fed for twelve weeks with normal chow, normal chow plus ad libitum 15% fructose solution, Western chow, or Western chow plus ad libitum 15% fructose solution (W+F). Food and liquid intake and body weight were periodically monitored. At the end of the study, plasma and hepatic lipids, liver histology and expression of genes related to lipid handling were analyzed and histological and immunohistological analyses of atherosclerosis at the aortic origin was performed. ResultsTotal calorie intake was significantly increased in Western-fed vs normal chow-fed mice, but was not modified by SLF. SLF significantly increased body weight, visceral adiposity, plasma lipids and liver cholesterol content in Western-fed mice, probably due to an increase in de novo lipid synthesis. Aortic atherosclerotic total lesion area was significantly correlated to plasma lipids, being highest in W+F mice. Further, SLF induced higher immunostaining for macrophages and oxidized-LDL receptor, independently of lesion area and caloric burden. ConclusionsSLF, without changing total calorie intake, increases atherosclerosis, visceral adipose tissue and cholesterol burden in a background of overweight LDL receptor knockout mice consuming an unhealthy, Western-type solid rodent chow.