Visual interstitial fibrosis assessment as continuous variable in protocol renal transplant biopsies.

AIMS: In current renal transplant pathology practice, interstitial fibrosis is visually assessed in categories according to the Banff classification. As this has a moderate reproducibility, which is little ameliorated by morphometric analysis, we investigated whether visual renal fibrosis assessment is feasible on a continuous scale, i.e. as a percentage of affected area of the cortex. METHODS AND RESULTS: Protocol renal biopsies taken at transplantation (n = 125), three (n = 73) and 12 months (n = 88) after transplantation were visually scored in categories (Banff) and percentages for interstitial fibrosis (ci). Interobserver variation (ICC and weighted κ) was assessed, and morphometric analysis on Sirius red-stained sections was performed. Correlations between the different methods and their association with donor age and eGFR 1 and 5 years post-transplant were analysed using Pearson's or Spearman's rho. Interobserver agreement was equivalent for Banff and %ci (κ = 0.713 versus ICC = 0.792), and for Banff IF/TA and %IF/TA (κ = 0.615 versus ICC = 0.743). Both Banff and %ci were associated with Sirius red morphometry in 3 and 12 months. With all three methods, a significant correlation was found between donor age and fibrosis in the implantation biopsy and between fibrosis in the 12 months' biopsy and eGFR at 1 and 5 years (eGFR at 1 year: Sirius red ρ = 0.487, %ci ρ = 0.393, Banff ρ = 0.413, all P < 0.01, eGFR at 5 years: Sirius red ρ = 0.392, %ci ρ = 0.333, Banff ρ = 0.435, all P < 0.01). CONCLUSION: Interstitial fibrosis assessment on a continuous scale can be used next to scoring in categories according to the Banff classification in protocol renal transplant biopsies.

SOX2 expression in the pathogenesis of premalignant lesions of the uterine cervix: its histo-topographical distribution distinguishes between low- and high-grade CIN.

SOX2 expression in high-grade cervical intraepithelial neoplasia (CIN3) and cervical squamous cell carcinoma is increased compared to that in the normal cervical epithelium. However, data on the expression and histological distribution of SOX2 in squamous epithelium during progression of CIN are largely lacking. We studied SOX2 expression throughout the epithelium in 53 cases of CIN1, 2, and 3. In general, SOX2 expression increased and expanded from basal/parabasal to the intermediate/superficial compartment during early stages of progression of CIN. An unexpected, specific expression pattern was found in areas classified as CIN2 and CIN3. This pattern was characterized by the absence or low expression of SOX2 in the basal/parabasal compartment and variable levels in the intermediate and superficial compartments. It was significantly associated with CIN3 (p = 0.009), not found in CIN1 and only seen in part of the CIN2 lesions. When the different patterns were correlated with the genetic make-up and presence of HPV, the CIN3-related pattern contained HPV-positive cells in the basal/parabasal cell compartment that were disomic. This is in contrast to the areas exhibiting the CIN1 and CIN2 related patterns, which frequently exhibited aneusomic cells. Based on their SOX2 localisation pattern, CIN1 and CIN2 could be delineated from CIN3. These data shed new light on the pathogenesis and dynamics of progression in premalignant cervical lesions, as well as on the target cells in the epithelium for HPV infection.

A morphometric analysis of pathological alterations in hemorrhoidal disease versus normal controls: a controlled trial

Objective: Until today, the true pathophysiology of hemorrhoidal disease (HD) has not yet been unraveled. More and more evidence guides us towards the hypothesis that reduced connective tissue stability is associated with a higher incidence of hemorrhoids. The present study aimed to compare the quantity and quality of collagen, and vessel morphometrics, in patients with symptomatic HD compared with normal controls. Methods: Twenty-two samples of grade III and grade IV HD tissue from patients undergoing a hemorrhoidectomy between January 2004 and June 2015 were included in the study group. Samples of 15 individuals without symptomatic HD who donated their body to science and died a natural death served as controls. The quantity and quality of anal collagen, and anal vessel morphometrics were objectified. The quality of collagen was subdivided in young (immature) and old (mature) collagen. Results: Patients with HD had an increased percentage of total anal collagen (62.1 ± 13.8 versus 18.7 ± 14.5%; p = 0.0001), a decreased percentage of young collagen (0.00009 ± 0.00008 versus 0.0008 ± 0.0008%; p = 0.001), and a smaller surface area of the anal vessels (795.1 ± 1215.9 micrometre2 versus 1219.0 ± 1976.1; p = 0.003) compared with controls. The percentage of old collagen did not differ between the control and study groups (0.588 ± 0.286% versus 0.389 ± 0.242%; p = 0.06). Conclusion: The outcomes of the present study suggest that alterations in anal collagen composition may play a role in the formation of hemorrhoids. (AU)

Intestinal Goblet Cell Loss during Chorioamnionitis in Fetal Lambs: Mechanistic Insights and Postnatal Implications.

Chorioamnionitis, an important cause of preterm birth, is linked to necrotizing enterocolitis (NEC). NEC is characterized by a disrupted mucus barrier, goblet cell loss, and endoplasmic reticulum (ER) stress of the intestinal epithelium. These findings prompted us to investigate the mechanisms underlying goblet cell alterations over time in an ovine chorioamnionitis model. Fetal lambs were intra-amniotically (IA) exposed to lipopolysaccharides (LPS) for 5, 12, or 24 h, or 2, 4, 8, or 15 d before premature delivery at 125 d gestational age (GA). Gut inflammation, the number, distribution, and differentiation of goblet cells, ER stress, and apoptosis were measured. We found a biphasic reduction in goblet cell numbers 24 h-2 d after, and 15 d after IA LPS exposure. The second decrease of goblet cell numbers was preceded by intestinal inflammation, apoptosis, and crypt ER stress, and increased SAM-pointed domain-containing ETS transcription factor (SPDEF)-positive cell counts. Our combined findings indicated that ER stress drives apoptosis of maturating goblet cells during chorioamnionitis, ultimately reducing goblet cell numbers. As similar changes have been described in patients suffering from NEC, these findings are considered to be clinically important for understanding the predecessors of NEC, and targeting ER stress in this context is interesting for future therapeutics.

Sequential Exposure to Antenatal Microbial Triggers Attenuates Alveolar Growth and Pulmonary Vascular Development and Impacts Pulmonary Epithelial Stem/Progenitor Cells.

Perinatal inflammatory stress is strongly associated with adverse pulmonary outcomes after preterm birth. Antenatal infections are an essential perinatal stress factor and contribute to preterm delivery, induction of lung inflammation and injury, pre-disposing preterm infants to bronchopulmonary dysplasia. Considering the polymicrobial nature of antenatal infection, which was reported to result in diverse effects and outcomes in preterm lungs, the aim was to examine the consequences of sequential inflammatory stimuli on endogenous epithelial stem/progenitor cells and vascular maturation, which are crucial drivers of lung development. Therefore, a translational ovine model of antenatal infection/inflammation with consecutive exposures to chronic and acute stimuli was used. Ovine fetuses were exposed intra-amniotically to Ureaplasma parvum 42 days (chronic stimulus) and/or to lipopolysaccharide 2 or 7 days (acute stimulus) prior to preterm delivery at 125 days of gestation. Pulmonary inflammation, endogenous epithelial stem cell populations, vascular modulators and morphology were investigated in preterm lungs. Pre-exposure to UP attenuated neutrophil infiltration in 7d LPS-exposed lungs and prevented reduction of SOX-9 expression and increased SP-B expression, which could indicate protective responses induced by re-exposure. Sequential exposures did not markedly impact stem/progenitors of the proximal airways (P63+ basal cells) compared to single exposure to LPS. In contrast, the alveolar size was increased solely in the UP+7d LPS group. In line, the most pronounced reduction of AEC2 and proliferating cells (Ki67+) was detected in these sequentially UP + 7d LPS-exposed lambs. A similar sensitization effect of UP pre-exposure was reflected by the vessel density and expression of vascular markers VEGFR-2 and Ang-1 that were significantly reduced after UP exposure prior to 2d LPS, when compared to UP and LPS exposure alone. Strikingly, while morphological changes of alveoli and vessels were seen after sequential microbial exposure, improved lung function was observed in UP, 7d LPS, and UP+7d LPS-exposed lambs. In conclusion, although sequential exposures did not markedly further impact epithelial stem/progenitor cell populations, re-exposure to an inflammatory stimulus resulted in disturbed alveolarization and abnormal pulmonary vascular development. Whether these negative effects on lung development can be rescued by the potentially protective responses observed, should be examined at later time points.

Nerve Fibers in the Tumor Microenvironment Are Co-Localized with Lymphoid Aggregates in Pancreatic Cancer.

B cells and tertiary lymphoid structures (TLS) are reported to be important in survival in cancer. Pancreatic Cancer (PDAC) is one of the most lethal cancer types, and currently, it is the seventh leading cause of cancer-related death worldwide. A better understanding of tumor biology is pivotal to improve clinical outcome. The desmoplastic stroma is a complex system in which crosstalk takes place between cancer-associated fibroblasts, immune cells and cancer cells. Indirect and direct cellular interactions within the tumor microenvironment (TME) drive key processes such as tumor progression, metastasis formation and treatment resistance. In order to understand the aggressiveness of PDAC and its resistance to therapeutics, the TME needs to be further unraveled. There are some limited data about the influence of nerve fibers on cancer progression. Here we show that small nerve fibers are located at lymphoid aggregates in PDAC. This unravels future pathways and has potential to improve clinical outcome by a rational development of new therapeutic strategies.

Chorioamnionitis induces changes in ovine pulmonary endogenous epithelial stem/progenitor cells in utero.

BACKGROUND: Chorioamnionitis, an intrauterine infection of the placenta and fetal membranes, is a common risk factor for adverse pulmonary outcomes in premature infants including BPD, which is characterized by an arrest in alveolar development. As endogenous epithelial stem/progenitor cells are crucial for organogenesis and tissue repair, we examined whether intrauterine inflammation negatively affects these essential progenitor pools. METHODS: In an ovine chorioamnionitis model, fetuses were intra-amniotically exposed to LPS, 2d or 7d (acute inflammation) before preterm delivery at 125d of gestation, or to intra-amniotic Ureaplasma parvum for 42d (chronic inflammation). Lung function, pulmonary endogenous epithelial stem/progenitor pools, and downstream functional markers were studied. RESULTS: Lung function was improved in the 7d LPS and 42d Ureaplasma groups. However, intrauterine inflammation caused a loss of P63+ basal cells in proximal airways and reduced SOX-9 expression and TTF-1+ Club cells in distal airways. Attenuated type-2 cell numbers were associated with lower proliferation and reduced type-1 cell marker Aqp5 expression, indicative for impaired progenitor function. Chronic Ureaplasma infection only affected distal airways, whereas acute inflammation affected stem/progenitor populations throughout the lungs. CONCLUSIONS: Acute and chronic prenatal inflammation improve lung function at the expense of stem/progenitor alterations that potentially disrupt normal lung development, thereby predisposing to adverse postnatal outcomes. IMPACT: In this study, prenatal inflammation improved lung function at the expense of stem/progenitor alterations that potentially disrupt normal lung development, thereby predisposing to adverse postnatal outcomes. Importantly, we demonstrate that these essential alterations can already be initiated before birth. So far, stem/progenitor dysfunction has only been shown postnatally. This study indicates that clinical protocols to target the consequences of perinatal inflammatory stress for the immature lungs should be initiated as early as possible and ideally in utero. Within this context, our data suggest that interventions, which promote function or repair of endogenous stem cells in the lungs, hold great promise.

Comparability of histological outcomes in rats and humans in a hernia model.

BACKGROUND: Hernia repair is one of the most frequently performed operations. In search of the ideal mesh for hernia repair, animal research is required. Although rats are most often used in experimental mesh experiments, no correlation with clinical findings in humans has ever been shown. Therefore, the aim of our study was to investigate whether adhesion formation and foreign body reactions to meshes in rats are comparable with the reactions in humans. MATERIALS AND METHODS: A fixed type of mesh was implanted intraperitoneally in a group of 10 rats and 10 patients undergoing elective, temporary stoma formation. In case of the latter, meshes were placed around the stoma. After a follow-up period of 12 wk in rats and after a median follow-up of 6 mo in humans, samples of the mesh were collected. Adhesion assessments were performed, and (immuno-) histochemical evaluation was performed by a specialized experimental pathologist and an experienced clinical pathologist. RESULTS: After the follow-up period, adhesion formation did not differ significantly between rats and humans. Moreover, general inflammation scores were comparable, although granulocytes and giant cells were more present in rats, compared with humans. On the other hand, the presence of fibrosis was more evident in humans compared with rats. CONCLUSIONS: To our knowledge, this is the first study, which showed that a specific animal model, namely a rat model, correlates with adhesion formation and the foreign body reaction to meshes in humans. It can be recommended to use rats in future experimental mesh for incisional hernia research.

The influence of anesthetics on substantia nigra tyrosine hydroxylase expression and tau phosphorylation in the hypoxic-ischemic near-term lamb.

BackgroundGeneral anesthetics could protect key neurotransmitter systems, such as the dopaminergic system, from hypoxic-ischemic encephalopathy (HIE) by limiting excessive glutamatergic neurotransmission. However, anesthetics may adversely affect inflammation and tau phosphorylation.MethodsA near-term sheep model of HIE by umbilical cord occlusion (UCO) under anesthesia was used. The effect of propofol and isoflurane on the dopaminergic neurotransmitter phenotype in the substantia nigra (SN) was studied using tyrosine hydroxylase immunohistochemistry. The overall microglial response and tau phosphorylation were also measured in the SN, surrounding the midbrain gray matter structures and the hippocampal white matter.ResultsThe isoflurane-treated UCO group had fewer tyrosine hydroxylase-expressing neurons in the SN at 8 h after the insult than the propofol-treated UCO or sham-operated groups (P<0.05). The microglial response was unchanged in the SN region. In the thalamus and the hippocampal stratum moleculare layer, the propofol-treated UCO group had a lower microglial response than the corresponding sham-operated group. Both UCO and the use of anesthetics additively increased tau phosphorylation in the SN region, thalamus, and hippocampus.ConclusionThe choice of anesthetics is important for an emergency C-section. Propofol could potentially protect the dopaminergic neurotransmitter phenotype within the SN at the cost of a widespread increase in tau phosphorylation.

FXR agonism protects against liver injury in a rat model of intestinal failure-associated liver disease.

BACKGROUND: Intestinal failure-associated liver disease (IFALD) is a clinical challenge. The pathophysiol-ogy is multifactorial and remains poorly understood. Disturbed recirculation of bile salts, e.g. due to loss of bile via an enterocutaneous fistula, is considered a major contributing factor. We hypothesize that impaired signaling via the bile salt receptor FXR underlies the development of IFALD. The aim of this study was to investigate whether activation of FXR improves liver homeostasis during chronic loss of bile in rats. METHODS: To study consequences of chronic loss of bile, rats underwent external biliary drainage (EBD) or sham surgery for seven days, and the prophylactic potential of the FXR agonist INT-747 was assessed. RESULTS: EBD for 7 days resulted in liver test abnormalities and histological liver damage. Expression of the intestinal FXR target gene Fgf15 was undetectable after EBD, and this was accompanied by an anticipated increase in hepatic Cyp7a1 expression, indicating increased bile salt synthesis. Treatment with INT-747 improved serum biochemistry, reduced loss of bile fluid in drained rats and prevented development of drainage-associated histological liver injury. CONCLUSIONS: EBD results in extensive hepatobiliary injury and cholestasis. These data suggest that FXR activation might be a novel therapy in preventing liver dysfunction in patients with intestinal failure. RELEVANCE FOR PATIENTS: This study demonstrates that chronic loss of bile causes liver injury in rats. Abro-gated recycling of bile salts impairing of enterohepatic bile salt/FXR signaling underlies these pathological changes, as administration of FXR agonist INT747 prevents biliary drainage-induced liver damage. Phar-macological activation of FXR might be a therapeutic strategy to treat disorders accompanied by a per-turbed enterohepatic circulation such as intestinal failure-associated liver disease.

Intermittent pacing therapy favorably modulates infarct remodeling.

Despite early revascularization, remodeling and dysfunction of the left ventricle (LV) after acute myocardial infarction (AMI) remain important therapeutic targets. Intermittent pacing therapy (IPT) of the LV can limit infarct size, when applied during early reperfusion. However, the effects of IPT on post-AMI LV remodeling and infarct healing are unknown. We therefore investigated the effects of IPT on global LV remodeling and infarct geometry in swine with a 3-day old AMI. For this purpose, fifteen pigs underwent 2 h ligation of the left circumflex coronary artery followed by reperfusion. An epicardial pacing lead was implanted in the peri-infarct zone. After three days, global LV remodeling and infarct geometry were assessed using magnetic resonance imaging (MRI). Animals were stratified into MI control and IPT groups. Thirty-five days post-AMI, follow-up MRI was obtained and myofibroblast content, markers of extracellular matrix (ECM) turnover and Wnt/frizzled signaling in infarct and non-infarct control tissue were studied. Results showed that IPT had no significant effect on global LV remodeling, function or infarct mass, but modulated infarct healing. In MI control pigs, infarct mass reduction was principally due to a 26.2 ± 4.4% reduction in infarct thickness (P ≤ 0.05), whereas in IPT pigs it was mainly due to a 35.7 ± 4.5% decrease in the number of infarct segments (P ≤ 0.05), with no significant change in infarct thickness. Myofibroblast content of the infarct zone was higher in IPT (10.9 ± 2.1%) compared to MI control (5.4 ± 1.6%; P ≤ 0.05). Higher myofibroblast presence did not coincide with alterations in expression of genes involved in ECM turnover or Wnt/frizzled signaling at 5 weeks follow-up. Taken together, IPT limited infarct expansion and altered infarct composition, showing that IPT influences remodeling of the infarct zone, likely by increasing regional myofibroblast content.

Vessel wall and adventitial DCE-MRI parameters demonstrate similar correlations with carotid plaque microvasculature on histology.

PURPOSE: To assess parameter agreement of volume transfer coefficient (Ktrans ) between two vascular regions and to study the correlation with microvessel density on histology. The dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) parameter Ktrans is frequently used to study atherosclerotic plaque microvasculature. Ktrans has been reported using different descriptive statistics (mean, median, 75th percentile) either for the whole vessel wall or the adventitia in previous studies. MATERIALS AND METHODS: DCE-MRI parameter agreement was analyzed in 110 symptomatic patients with ≥2 mm carotid plaque that underwent a 3T carotid DCE-MRI examination. Ktrans was estimated in the entire vessel wall and adventitia. Twenty-three patients underwent carotid endarterectomy and were used for comparison with histological quantification of microvessel density of the plaque using CD31 immunohistochemistry. DCE-MRI parameters in the vessel wall regions were compared using Pearson's correlation coefficient, Bland-Altman analysis, and a two-sided paired samples t-test. Correlation of the DCE-MRI parameters with histology was studied using the Pearson's correlation coefficient. RESULTS: Median adventitial Ktrans was 5% higher (P = 0.003) than entire vessel wall Ktrans , with no differences for other descriptive statistics. Vessel wall and adventitial Ktrans showed similar moderately strong correlations with plaque microvessel density on histology (Pearson's ρ: 0.59-0.65 [P < 0.003] and 0.52-0.64 [P < 0.011], respectively). CONCLUSION: The similar moderately strong correlations for vessel wall and adventitial Ktrans with microvessel density on histology suggested that both regions reflected plaque microvessel density. Care should to be taken when comparing absolute values between studies. Future studies incorporating thresholds for risk stratification need to agree upon standardization of DCE-MRI parameters. LEVEL OF EVIDENCE: 2 Technical Efficacy: Stage 2 J. Magn. Reson. Imaging 2017;46:1053-1059.

Osseointegration and implant stability of extraoral implants in Göttingen minipigs after irradiation.

OBJECTIVES: The aim of this study was to compare the influence of implant surface treatment and irradiation dose on implant stability and osseointegration of 144 extraoral implants in irradiated frontal bone of minipigs. MATERIAL AND METHODS: 144 implants with 3 different surface treatments (machined, etched and HAVD-coated) were implanted in the frontal bone of 16 Göttingen minipigs. Three groups of four pigs received radiation with equivalent doses of 25, 50 and 70 Gy, and one group served as control. Resonance frequency analysis (RFA) was performed recording Implant Stability Quotients (ISQ) at implant placement and 3 months thereafter. Removal torque was measured whilst removing specific implants after 3 months. In addition, the bone-to-implant contact (BIC) was analyzed. RESULTS: Evaluation of ISQ, BIC-values showed no significant difference between the different surface treatments in irradiated and non-irradiated bone. Removal torque revealed statistically significant differences between machined and HAVD-coated implants in the irradiated bone. CONCLUSIONS: Implant stability and osseointegration, based on Removal Torque showed significant higher results for the HAVD-coated implants. No significant difference was observed between the irradiated and non-irradiated animals. This study shows that HAVD-coated extraoral implants can potentially be used for craniofacial rehabilitation in non-irradiated and irradiated bone.

Imaging Intraplaque Inflammation in Carotid Atherosclerosis With 18F-Fluorocholine Positron Emission Tomography-Computed Tomography: Prospective Study on Vulnerable Atheroma With Immunohistochemical Validation.

BACKGROUND: (18)F-fluorocholine ((18)F-FCH) uptake is associated with cell proliferation and activity in tumor patients. We hypothesized that (18)F-FCH could similarly be a valuable imaging tool to identify vulnerable plaques and associated intraplaque inflammation and atheroma cell proliferation. METHODS AND RESULTS: Ten consecutive stroke patients (90% men, median age 66.5 years, range, 59.4-69.7) with ipsilateral >70% carotid artery stenosis and who underwent carotid endarterectomy were included in the study. Before carotid endarterectomy, all patients underwent positron emission tomography to assess maximum (18)F-FCH uptake in ipsilateral symptomatic carotid plaques and contralateral asymptomatic carotid arteries, which was corrected for background activity, resulting in a maximum target-to-background ratio (TBRmax). Macrophage content was assessed in all carotid endarterectomy specimens as a percentage of CD68(+)-staining per whole plaque area (plaqueCD68(+)) and as a maximum CD68(+) percentage (maxCD68(+)) in the most inflamed section/plaque. Dynamic positron emission tomography imaging demonstrated that an interval of 10 minutes between (18)F-FCH injection and positron emission tomography acquisition is appropriate for carotid plaque imaging. TBRmax in ipsilateral symptomatic carotid plaques correlated significantly with plaqueCD68(+) (Spearman's ρ=0.648, P=0.043) and maxCD68(+) (ρ=0.721, P=0.019) in the 10 corresponding carotid endarterectomy specimens. TBRmax was significantly higher (P=0.047) in ipsilateral symptomatic carotid plaques (median: 2.0; interquartile range [Q1-Q3], 1.5-2.5) compared with the contralateral asymptomatic carotid arteries (median: 1.4; Q1-Q3, 1.3-1.6). TBRmax was not significantly correlated to carotid artery stenosis (ρ=0.506, P=0.135). CONCLUSIONS: In vivo uptake of (18)F-FCH in human carotid atherosclerotic plaques correlated strongly with degree of macrophage infiltration and recent symptoms, thus (18)F-FCH positron emission tomography is a promising tool for the evaluation of vulnerable plaques. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT01899014.

Cell kinetics during regeneration in the sponge Halisarca caerulea: how local is the response to tissue damage?

Sponges have a remarkable capacity to rapidly regenerate in response to wound infliction. In addition, sponges rapidly renew their filter systems (choanocytes) to maintain a healthy population of cells. This study describes the cell kinetics of choanocytes in the encrusting reef sponge Halisarca caerulea during early regeneration (0-8 h) following experimental wound infliction. Subsequently, we investigated the spatial relationship between regeneration and cell proliferation over a six-day period directly adjacent to the wound, 1 cm, and 3 cm from the wound. Cell proliferation was determined by the incorporation of 5-bromo-2'-deoxyuridine (BrdU). We demonstrate that during early regeneration, the growth fraction of the choanocytes (i.e., the percentage of proliferative cells) adjacent to the wound is reduced (7.0 ± 2.5%) compared to steady-state, undamaged tissue (46.6 ± 2.6%), while the length of the cell cycle remained short (5.6 ± 3.4 h). The percentage of proliferative choanocytes increased over time in all areas and after six days of regeneration choanocyte proliferation rates were comparable to steady-state tissue. Tissue areas farther from the wound had higher rates of choanocyte proliferation than areas closer to the wound, indicating that more resources are demanded from tissue in the immediate vicinity of the wound. There was no difference in the number of proliferative mesohyl cells in regenerative sponges compared to steady-state sponges. Our data suggest that the production of collagen-rich wound tissue is a key process in tissue regeneration for H. caerulea, and helps to rapidly occupy the bare substratum exposed by the wound. Regeneration and choanocyte renewal are competing and negatively correlated life-history traits, both essential to the survival of sponges. The efficient allocation of limited resources to these life-history traits has enabled the ecological success and diversification of sponges.

Cell turnover and detritus production in marine sponges from tropical and temperate benthic ecosystems.

This study describes in vivo cell turnover (the balance between cell proliferation and cell loss) in eight marine sponge species from tropical coral reef, mangrove and temperate Mediterranean reef ecosystems. Cell proliferation was determined through the incorporation of 5-bromo-2'-deoxyuridine (BrdU) and measuring the percentage of BrdU-positive cells after 6 h of continuous labeling (10 h for Chondrosia reniformis). Apoptosis was identified using an antibody against active caspase-3. Cell loss through shedding was studied quantitatively by collecting and weighing sponge-expelled detritus and qualitatively by light microscopy of sponge tissue and detritus. All species investigated displayed substantial cell proliferation, predominantly in the choanoderm, but also in the mesohyl. The majority of coral reef species (five) showed between 16.1±15.9% and 19.0±2.0% choanocyte proliferation (mean±SD) after 6 h and the Mediterranean species, C. reniformis, showed 16.6±3.2% after 10 h BrdU-labeling. Monanchora arbuscula showed lower choanocyte proliferation (8.1±3.7%), whereas the mangrove species Mycale microsigmatosa showed relatively higher levels of choanocyte proliferation (70.5±6.6%). Choanocyte proliferation in Haliclona vansoesti was variable (2.8-73.1%). Apoptosis was negligible and not the primary mechanism of cell loss involved in cell turnover. All species investigated produced significant amounts of detritus (2.5-18% detritus bodyweight(-1)·d(-1)) and cell shedding was observed in seven out of eight species. The amount of shed cells observed in histological sections may be related to differences in residence time of detritus within canals. Detritus production could not be directly linked to cell shedding due to the degraded nature of expelled cellular debris. We have demonstrated that under steady-state conditions, cell turnover through cell proliferation and cell shedding are common processes to maintain tissue homeostasis in a variety of sponge species from different ecosystems. Cell turnover is hypothesized to be the main underlying mechanism producing sponge-derived detritus, a major trophic resource transferred through sponges in benthic ecosystems, such as coral reefs.

Glyoxalase 1 overexpression does not affect atherosclerotic lesion size and severity in ApoE-/- mice with or without diabetes.

AIMS: Advanced glycation end-products (AGEs) and their precursors have been associated with the development of atherosclerosis. We recently discovered that glyoxalase 1 (GLO1), the major detoxifying enzyme for AGE precursors, is decreased in ruptured human plaques, and that levels of AGEs are higher in rupture-prone plaques. We here investigated whether overexpression of human GLO1 in ApoE(-/-) mice could reduce the development of atherosclerosis. METHODS AND RESULTS: We crossed C57BL/6 ApoE(-/-) mice with C57BL/6 GLO1 overexpressing mice (huGLO1(+/-)) to generate ApoE(-/-) (n = 16) and ApoE(-/-) huGLO1(+/-) (n = 20) mice. To induce diabetes, we injected a subset with streptozotocin (STZ) to generate diabetic ApoE(-/-) (n = 8) and ApoE(-/-) huGLO1(+/-) (n = 13) mice. All mice were fed chow and sacrificed at 25 weeks of age. The GLO1 activity was three-fold increased in huGLO1(+/-) aorta, but aortic root lesion size and phenotype did not differ between mice with and without huGLO1(+/-) overexpression. We detected no differences in gene expression in aortic arches, in AGE levels and cytokines, in circulating cells, and endothelial function between ApoE(-/-) mice with and without huGLO1(+/-) overexpression. Although diabetic mice showed decreased GLO1 expression (P < 0.05) and increased lesion size (P < 0.05) in comparison with non-diabetic mice, GLO1 overexpression also did not affect the aortic root lesion size or inflammation in diabetic mice. CONCLUSION: In ApoE(-/-) mice with or without diabetes, GLO1 overexpression did not lead to decreased atherosclerotic lesion size or systemic inflammation. Increasing GLO1 levels does not seem to be an effective strategy to reduce glycation in atherosclerotic lesions, likely due to increased AGE formation through GLO1-independent mechanisms.

Similar matrix alterations in alveolar and small airway walls of COPD patients.

BACKGROUND: Remodelling in COPD has at least two dimensions: small airway wall thickening and destruction of alveolar walls. Recent studies indicate that there is some similarity between alveolar and small airway wall matrix remodelling. The aim of this study was to characterise and assess similarities in alveolar and small airway wall matrix remodelling, and TGF-ß signalling in COPD patients of different GOLD stages. METHODS: Lung tissue sections of 14 smoking controls, 16 GOLD II and 19 GOLD IV patients were included and stained for elastin and collagens as well as hyaluronan, a glycosaminoglycan matrix component and pSMAD2. RESULTS: Elastin was significantly decreased in COPD patients not only in alveolar, but also in small airway walls. Interestingly, both collagen and hyaluronan were increased in alveolar as well as small airway walls. The matrix changes were highly comparable between GOLD stages, with collagen content in the alveolar wall increasing further in GOLD IV. A calculated remodelling index, defined as elastin divided over collagen and hyaluronan, was decreased significantly in GOLD II and further lowered in GOLD IV patients, suggesting that matrix component alterations are involved in progressive airflow limitation. Interestingly, there was a positive correlation present between the alveolar and small airway wall stainings of the matrix components, as well as for pSMAD2. No differences in pSMAD2 staining between controls and COPD patients were found. CONCLUSIONS: In conclusion, remodelling in the alveolar and small airway wall in COPD is markedly similar and already present in moderate COPD. Notably, alveolar collagen and a remodelling index relate to lung function.

Local delivery of polarized macrophages improves reperfusion recovery in a mouse hind limb ischemia model.

AIMS: Enhancement of collateral development in coronary or peripheral artery disease is a therapeutic target, but it has proven difficult to achieve. Macrophages are key players in collateral remodeling, yet the effect of different macrophage subsets on arteriogenesis has not been investigated. METHODS AND RESULTS: Murine macrophages were cultured from bone marrow and polarized into M1 (IFNγ), M2a (IL-4) or M2c (IL-10) subsets. C57BL/6 mice underwent femoral artery ligation followed by intramuscular injection of macrophage subsets. Using eGFP expressing macrophages, cells could be detected at least 6 days after ligation and were located in the perivascular space of collateral vessels. After 14 days, perfusion ratio was increased in animals treated with M1 as well as M2a and M2c macrophages compared to control. Depletion of circulating monocytes by clodronate liposome injections did not hamper reperfusion recovery, however, treatment with exogenous polarized macrophages improved perfusion ratio after 14 days again. We used IL10R(fl/fl)/LysMCre(+) mice to study the effect of inhibition of endogenous polarization towards specifically M2c macrophages on arteriogenesis. Deletion of the IL10-receptor (IL10R) in the myeloid lineage did not affect reperfusion recovery, yet the pro-arteriogenic effect of exogenously injected M2c macrophages was still present. CONCLUSIONS: Local injection of polarized macrophages promotes reperfusion recovery after femoral artery ligation and is not influenced by depletion of circulatory monocytes. Preventing endogenous M2c polarization did not affect reperfusion recovery suggesting that M2c's are not required for collateralization, but are sufficient to induce collateral formation upon exogenous administration. This is the first study using local injection of macrophage subsets showing the pro-arteriogenic effect of polarized macrophages.

Rapid insulin-mediated increase in microvascular glycocalyx accessibility in skeletal muscle may contribute to insulin-mediated glucose disposal in rats.

It has been demonstrated that insulin-mediated recruitment of microvascular blood volume is associated with insulin sensitivity. We hypothesize that insulin rapidly stimulates penetration of red blood cells (RBC) and plasma into the glycocalyx and thereby promotes insulin-mediated glucose uptake by increasing intracapillary blood volume. Experiments were performed in rats; the role of the glycocalyx was assessed by enzymatic degradation using a bolus of hyaluronidase. First, the effect of insulin on glycocalyx accessibility was assessed by measuring the depth of penetration of RBCs into the glycocalyx in microvessels of the gastrocnemius muscle with Sidestream Dark-field imaging. Secondly, peripheral insulin sensitivity was determined using intravenous insulin tolerance tests (IVITT). In addition, in a smaller set of experiments, intravital microscopy of capillary hemodynamics in cremaster muscle and histological analysis of the distribution of fluorescently labeled 40 kDa dextrans (D40) in hindlimb muscle was used to evaluate insulin-mediated increases in capillary blood volume. Insulin increased glycocalyx penetration of RBCs by 0.34±0.44 µm (P<0.05) within 10 minutes, and this effect of insulin was greatly impaired in hyaluronidase treated rats. Further, hyaluronidase treated rats showed a 35±25% reduction in whole-body insulin-mediated glucose disposal compared to control rats. Insulin-mediated increases in capillary blood volume were reflected by a rapid increase in capillary tube hematocrit from 21.1±10.1% to 29.0±9.8% (P<0.05), and an increase in D40 intensity in individual capillaries of 134±138% compared to baseline at the end of the IVITT. These effects of insulin were virtually abolished in hyaluronidase treated animals. In conclusion, insulin rapidly increases glycocalyx accessibility for circulating blood in muscle, and this is associated with an increased blood volume in individual capillaries. Hyaluronidase treatment of the glycocalyx abolishes the effects of insulin on capillary blood volume and impairs insulin-mediated glucose disposal.