Investigating paediatric airways by non-bronchoscopic lavage: Normal cellular data

Background: Bronchoscopic bronchoalveolar lavage in children to investigate bronchia disorders such as asthtna has both ethical and procedural difficulties.


Objective: The aim of this study was to establish a standardized non-bronchoscopic method to perform bronchoalveolar lavage in children attending for elective surgery to obtain normal cellular data.


Methods: Bronchoalveolar lavage was performed on normal children (n= 55) by infusing saline (20 mL) through an 8 FG suction catheter passed after endotracheal intubation. Oxygen saturation, heart and respiratory rate were monitored during the bronchoalveolar lavage procedure. Cellular analysis and total protein estimation of the lavage fluid were performed. Epithelial lining fluid volume was calculated (n = 15) using the urea dilution method.


Results: The procedure was well tolerated by all children. Total cell count and differential cell count for children (macrophages 70.8 ± 2.3%, lymphocytes 3.8 ± 0.6%, neutrophils 5,7 ± 1.0%, eosinophils 0.14 ± 0.03%. epithelial cells 19.6 ± 2.1%, mast cells 0.21 ± 0.02%) were similar to those reported for adults. Age and sex comparisons revealed no differences between groups. The mean total protein recovered in the cell free supernatant was 49.72 ± 4.29 mg/L and epithelial lining fluid volume was 0.82 ± 0.11% of return lavageate.


Conclusion This method allows bronchoalveolar lavage to be performed safely and quickly on children attending for routine elective surgery. Using this method and taking the ‘window of opportunity’ of elective surgery, the presence or absence of airway inflammation could be studied in children with various patterns of asthma during relatively asymptomatic periods.

Beta2 integrins target Rap GTPases to the plasma membrane by means of degranulation.

We report the novel observation that engagement of ß2 integrins on human neutrophils is accompanied by increased levels of the small GTPases Rap1 and Rap2 in a membrane-enriched fraction and a concomitant decrease of these proteins in a granule-enriched fraction. In parallel, we observed a similar time-dependent decrease of gelatinase B (a marker of specific and gelatinase B-containing granules) but not myeloperoxidase (a marker of azurophil granules) in the granule fraction, and release of lactoferrin (a marker of specific granules) in the extracellular medium. Furthermore, inhibition of Src tyrosine kinases, or phosphoinositide 3-kinase with PP1 or LY294002, respectively, blocked ß2 integrin-induced degranulation and the redistribution of Rap1 and Rap2 to a membrane-enriched fraction. Consequently, the ß2 integrin-dependent exocytosis of specific and gelatinase B-containing granules occurs via a Src tyrosine kinase/phosphoinositide 3-kinase signaling pathway and is responsible for the translocation of Rap1 and Rap2 to the plasma membrane in human neutrophils.

Fasciola hepatica: Histology of the testis in egg-producing adults of several laboratory-maintained isolates of flukes grown to maturity in cattle and sheep and in flukes from naturally infected hosts

A total of 8 calves approximately 6 months old and 22 lambs of similar age were infected with metacercariae of Fasciola hepatica of various laboratory-maintained isolates including: Cullompton (sensitive to triclabendazole) and Sligo, Oberon and Leon (reported as resistant to triclabendazole). Ten to 16 weeks after infection, flukes were harvested from these experimental animals and the histology of the testis tissue was examined in a representative sample of flukes from each population. Adult wild-type flukes were also collected from 5 chronically infected cattle and 7 chronically infected sheep identified at post-mortem inspection. The testis tissue of these flukes was compared with that of the various laboratory-maintained isolates. Whilst the testes of the wild-type, Oberon and Leon flukes displayed all the usual cell types associated with spermatogenesis in Fasciola hepatica (spermatogonia, spermatocytes, spermatids and mature sperm), the Cullompton flukes from both cattle and sheep showed arrested spermatogenesis, with no stages later than primary spermatocytes represented in the testis profiles. The presence of numerous eosinophilic apoptotic bodies and nuclear fragments suggested that meiotic division was anomalous and incomplete. In contrast to the wild-type flukes, no mature spermatozoa were present in the testes or amongst the shelled eggs in the uterus. A high proportion of the eggs collected from these flukes hatched to release normal-appearing miracidia after an appropriate incubation period, as indeed was the case with all isolates examined and the wild-type flukes. It is concluded that the eggs of Cullompton flukes are capable of development without fertilization, i.e. are parthenogenetic. The implications of this for rapid evolution of resistant clones following an anthelmintic selection event are discussed. Amongst the Sligo flukes examined, two subtypes were recognised, namely, those flukes with all stages of spermatogenesis and mature spermatozoa present in the testes (type 1), and those flukes with all stages of spermatogenesis up to spermatids present, but no maturing spermatozoa in the testes (type 2). Each sheep infected with the Sligo isolate had both type 1 (approximately 60%) and type 2 (approximately 40%) flukes present in the population. Spermatozoa were found amongst the eggs in the uterus in 64% of flukes and this did not necessarily reflect the occurrence of spermatozoa in the testis profiles of particular flukes, suggesting that cross-fertilization had occurred. The apparent disruption of meiosis in the spermatocytes of the Cullompton flukes is consistent with reports that Cullompton flukes are triploid (3n = 30), whereas the Sligo and wild-type flukes are diploid (2n = 20). In the Sligo flukes the populations are apparently genetically heterogenous, with a proportion of the flukes unable to produce fully formed spermatozoa perhaps because of a failure in spermiogenesis involving elongation of the nucleus during morphogenesis. (C) 2008 Elsevier B.V. All rights reserved.

Antiproteases as therapeutics to target inflammation in cystic fibrosis.

Cystic Fibrosis (CF) is the most common fatal inherited disease of Caucasians, affecting about 1 in 3000 births. Patients with CF have a recessive mutation in the gene encoding the CF transmembrane conductance regulator (CFTR). CFTR is expressed in the epithelium of many organs throughout the exocrine system, however, inflammation and damage of the airways as a result of persistent progressive endobronchial infection is a central feature of CF. The inflammatory response to infection brings about a sustained recruitment of neutrophils to the site of infection. These neutrophils release various pro-inflammatory compounds including proteases, which when expressed at aberrant levels can overcome the endogenous antiprotease defence mechanisms of the lung. Unregulated, these proteases can exacerbate inflammation and result in the degradation of structural proteins and tissue damage leading to bronchiectasis and loss of respiratory function. Other host-derived and bacterial proteases may also contribute to the inflammation and lung destruction observed in the CF lung. Antiprotease strategies to dampen the excessive inflammatory response and concomitant damage to the airways remains an attractive therapeutic option for CF patients.

Impaired Immune Tolerance to Porphyromonas gingivalis Lipopolysaccharide Promotes Neutrophil Migration and Decreased Apoptosis.

Periodontitis, a chronic inflammatory disease of the tissues supporting the teeth, is characterized by an exaggerated host immune and inflammatory response to periopathogenic bacteria. Toll-like receptor activation, cytokine network induction, and accumulation of neutrophils at the site of inflammation are important in the host defense against infection. At the same time, induction of immune tolerance and the clearance of neutrophils from the site of infection are essential in the control of the immune response, resolution of inflammation, and prevention of tissue destruction. Using a human monocytic cell line, we demonstrate that Porphyromonas gingivalis lipopolysaccharide (LPS), which is a major etiological factor in periodontal disease, induces only partial immune tolerance, with continued high production of interleukin-8 (IL-8) but diminished secretion of tumor necrosis factor alpha (TNF-) after repeated challenge. This cytokine response has functional consequences for other immune cells involved in the response to infection. Primary human neutrophils incubated with P. gingivalis LPS-treated naïve monocyte supernatant displayed a high migration index and increased apoptosis. In contrast, neutrophils treated with P. gingivalis LPS-tolerized monocyte supernatant showed a high migration index but significantly decreased apoptosis. Overall, these findings suggest that induction of an imbalanced immune tolerance in monocytes by P. gingivalis LPS, which favors continued secretion of IL-8 but decreased TNF- production, may be associated with enhanced migration of neutrophils to the site of infection but also with decreased apoptosis and may play a role in the chronic inflammatory state seen in periodontal disease.

Reduced Nitro-oxidative Stress and Neural Cell Death Suggests a Protective Role for Microglial Cells in TNFα−/− Mice in Ischemic Retinopathy

Purpose. Neovascularization occurs in response to tissue ischemia and growth factor stimulation. In ischemic retinopathies, however, new vessels fail to restore the hypoxic tissue; instead, they infiltrate the transparent vitreous. In a model of oxygen-induced retinopathy (OIR), TNFa and iNOS, upregulated in response to tissue ischemia, are cytotoxic and inhibit vascular repair. The aim of this study was to investigate the mechanism for this effect.

Methods. Wild-type C57/BL6 (WT) and TNFa-/- mice were subjected to OIR by exposure to 75% oxygen (postnatal days 7–12). The retinas were removed during the hypoxic phase of the model. Retinal cell death was determined by TUNEL staining, and the microglial cells were quantified after Z-series capture with a confocal microscope. In situ peroxynitrite and superoxide were measured by using the fluorescent dyes DCF and DHE. iNOS, nitrotyrosine, and arginase were analyzed by real-time PCR, Western blot analysis, and activity determined by radiolabeled arginine conversion. Astrocyte coverage was examined after GFAP immunostaining.

Results. The TNFa-/- animals displayed a significant reduction in TUNEL-positive apoptotic cells in the inner nuclear layer of the avascular retina compared with that in the WT control mice. The reduction coincided with enhanced astrocytic survival and an increase in microglial cells actively engaged in phagocytosing apoptotic debris that displayed low ROS, RNS, and NO production and high arginase activity.

Conclusions. Collectively, the results suggest that improved vascular recovery in the absence of TNFa is associated with enhanced astrocyte survival and that both phenomena are dependent on preservation of microglial cells that display an anti-inflammatory phenotype during the early ischemic phase of OIR.

Respiratory viral infection in lower airways of asymptomatic children.

Aim: The aim of this study was to determine if asthmatic children have viruses more commonly detected in lower airways during asymptomatic periods than normal children. Methods: Fifty-five asymptomatic children attending elective surgical procedures (14 with stable asthma, 41 normal controls) underwent non-bronchoscopic bronchoalveolar lavage. Differential cell count and PCR for 13 common viruses were performed. Results: Nineteen (35%) children were positive for at least one virus, with adenovirus being most common. No differences in the proportion of viruses detected were seen between asthmatic and normal ‘control’ children. Viruses other than adenovirus were associated with higher neutrophil counts, suggesting that they caused an inflammatory response in both asthmatics and controls (median BAL neutrophil count, 6.9% for virus detected vs. 1.5% for virus not detected, p = 0.03). Conclusions: Over one-third of asymptomatic children have a detectable virus (most commonly adenovirus) in the lower airway; however, this was not more common in asthmatics. Viruses other than adenovirus were associated with elevated neutrophils suggesting that viral infection can be present during relatively asymptomatic periods in asthmatic children.

Fasciola hepatica: Histological changes in the reproductive structures of triclabendazole (TCBZ)-sensitive and TCBZ-resistant flukes after treatment in vivo with TCBZ and the related benzimidazole derivative, Compound Alpha

Twenty-four shed-reared lambs were each infected orally with 250 metacercariae of Fasciola hepatica, using either the triclabendazole (TCBZ)-sensitive Cullompton isolate or the TCBZ-resistant Sligo isolate. Twelve weeks after infection the lambs were treated with TCBZ (10 mg/kg) or with the experimental fasciolicide, Compound Alpha (Cpd alpha), a benzimidazole derivative of TCBZ (15 mg/kg). The lambs were euthanised 48,72 and 96 h after TCBZ treatment, or 24, 48 and 72 h after Cpd a treatment, and flukes were collected from the liver and/or gall bladder of each animal. Untreated animals harbouring 12-week infections were euthanised 24 h after administration of anthelmintic to the treatment groups, and the untreated flukes provided control material. A semi-quantitative assessment of the degree of histological change induced by the two drugs after different times of exposure was achieved by scoring the intensity of three well-defined lesions that developed in the testes and uteri of a representative sample of flukes from each lamb. In general, it was found that in those tissues where active meiosis and/or mitosis occurred (testis, ovary, and vitelline follicles), there was progressive loss of cell content due to apparent failure of cell division to keep pace with expulsion of the mature or effete products. Further, actively dividing cell types tended to become individualised, rounded and condensed, characteristic of apoptotic cell death. Protein synthetic activity was apparently inhibited in the Mehlis' secretory cells. In the uterus, where successful formation of shelled eggs represents the culmination of a complex sequence of cytokinetic, cytological and synthetic activity involving the vitelline follicles, the ovary and the Mehlis' gland, histological evidence indicating failure of ovigenesis was evident from 24 h post-treatment onwards. The development of these lesions may be related to the known antitubulin activity of the benzimidazole class of anthelmintics, to the induction of apoptosis in cells where mitosis or meiosis has aborted due to failure of spindle formation, and to drug-induced inhibition of protein synthesis. The semi-quantitative findings indicated that Cpd a is slightly less efficacious than TCBZ itself in causing histological damage to the reproductive structures of TCBZ-sensitive flukes, and that, like TCBZ, it caused no histological damage in flukes of the TCBZ-resistant isolate. This study illustrates the potential utility of histological techniques for conveniently screening representative samples of flukes in field trials designed to validate instances of drug resistance or to test the efficacy of new products against known drug-resistant and drug-susceptible fluke isolates. It also provides reference criteria for drug-induced histopathological changes in fluke reproductive structures which may aid interpretation of TEM findings. (C) 2009 Elsevier B.V. All rights reserved.

Inhibition of platelet-derived growth factor promotes pericyte loss and angiogenesis in ischemic retinopathy

We investigated whether inhibition of platelet-derived growth factor (PDGF) receptor tyrosine kinase activity would affect pericyte viability, vascular endothelial growth factor (VEGF)/vascular endothelial growth factor receptor-2 (VEGFR-2) expression and angiogenesis in a model of retinopathy of prematurity (ROP). ROP was induced in Sprague Dawley rats by exposure to 80% oxygen from postnatal (P) days 0 to 11 (with 3 hours/day in room air), and then room air from P12-18 (angiogenesis period). Shams were neonatal rats in room air from P0-18. STI571, a potent inhibitor of PDGF receptor tyrosine kinase, was administered from P12-18 at 50 or 100 mg/kg/day intraperitoneal (i.p.). Electron microscopy revealed that pericytes in the inner retina of both sham and ROP rats appeared normal; however STI571 induced a selective pericyte and vascular smooth muscle degeneration. Immunolabeling for caspase-3 and a-smooth muscle cell actin in consecutive paraffin sections of retinas confirmed that these degenerating cells were apoptotic pericytes. In all groups, VEGF and VEGFR-2 gene expression was located in ganglion cells, the inner nuclear layer, and retinal pigment epithelium. ROP was associated with an increase in both VEGF and VEGFR-2 gene expression and blood vessel profiles in the inner retina compared to sham rats. STI571 at both doses increased VEGF and VEGFR-2 mRNA and exacerbated angiogenesis in ROP rats, and in sham rats at 100 mg/kg/day. In conclusion, PDGF is required for pericyte viability and the subsequent prevention of VEGF/VEGFR-2 overexpression and angiogenesis in ROP.

Histological and ultrastructural investigation of retinal microaneurysm development in diabetic patients

BACKGROUND: Although microaneurysms are a clinicopathological hallmark of diabetic retinopathy, there have been few ultrastructural studies of these important lesions. As a result, knowledge of the mechanisms involved in the pathogenesis of microaneurysms remains fragmentary. This study provides histological and ultrastructural evidence of various stages in microaneurysm formation within the retinal vasculature. METHODS: The eyes of three type II diabetic patients, obtained within 24 hours of death, were studied by the trypsin digest technique. Eyes from two further type II diabetics were fixed in 2.5% glutaraldehyde within 12 hours of death and processed for electron microscopy. RESULTS: In the trypsin digest preparations, small saccular and fusiform microaneurysms were observed in the peripheral retinal. In the central retina, the microaneurysms ranged in morphology from thin walled, cellular forms to dense, acellular, hyalinised forms. Ultrastructurally, four distinct groups of microaneurysm were observed. Type I showed an extensive accumulation of polymorphonuclear cells into the lumen. The endothelium remained intact, although pericytes were invariably absent. Type II microaneurysms were typified by large numbers of red blood cells (RBCs) in the lumen. Endothelial cells and pericytes were completely absent. The type III microaneurysm was also non-perfused and contained aggregates of irregularly shaped RBC profiles and RBC breakdown products. Recanalisation by new vessels into the occluded lumen was observed in one microaneurysm. Type IV microaneurysms were almost or completely sclerosed, with extensive fibrosis and lipid infiltration into the lumen and basement membrane wall. CONCLUSION: This investigation describes several distinctive stages in the formation of microaneurysms during diabetic retinopathy. With reference to the pathogenesis of retinal microaneurysms, the interaction of various cell types is discussed and the significance of vascular cell death and localised hypertensive events highlighted.

C5a-mediated neutrophil phagocytic dysfunction is RhoA-dependent and predicts nosocomial infection in critically ill patients.

Critically ill patients are at heightened risk for nosocomial infections. The anaphylatoxin C5a impairs phagocytosis by neutrophils. However, the mechanisms by which this occurs and the relevance for acquisition of nosocomial infection remain undetermined. We aimed to characterize mechanisms by which C5a inhibits phagocytosis in vitro and in critically ill patients, and to define the relationship between C5a-mediated dysfunction and acquisition of nosocomial infection. In healthy human neutrophils, C5a significantly inhibited RhoA activation, preventing actin polymerization and phagocytosis. RhoA inhibition was mediated by PI3Kd. The effects on RhoA, actin, and phagocytosis were fully reversed by GM-CSF. Parallel observations were made in neutrophils from critically ill patients, that is, impaired phagocytosis was associated with inhibition of RhoA and actin polymerization, and reversed by GM-CSF. Among a cohort of 60 critically ill patients, C5a-mediated neutrophil dysfunction (as determined by reduced CD88 expression) was a strong predictor for subsequent acquisition of nosocomial infection (relative risk, 5.8; 95% confidence interval, 1.5-22; P = .0007), and remained independent of time effects as assessed by survival analysis (hazard ratio, 5.0; 95% confidence interval, 1.3-8.3; P = .01). In conclusion, this study provides new insight into the mechanisms underlying immunocompromise in critical illness and suggests novel avenues for therapy and prevention of nosocomial infection.

The Carboxy-Terminal Modulator Protein (CTMP) Regulates Mitochondrial Dynamics

Background: Mitochondria are central to the metabolism of cells and participate in many regulatory and signaling events. They are looked upon as dynamic tubular networks. We showed recently that the Carboxy-Terminal Modulator Protein (CTMP) is a mitochondrial protein that may be released into the cytosol under apoptotic conditions.

Activated oncogenic pathways and therapeutic targets in extranodal nasal-type NK/T cell lymphoma revealed by gene expression profiling

We performed comprehensive genome-wide gene expression profiling (GEP) of extranodal nasal-type natural killer/T-cell lymphoma (NKTL) using formalin-fixed, paraffin-embedded tissue (n = 9) and NK cell lines (n = 5) in comparison with normal NK cells, with the objective of understanding the oncogenic pathways involved in the pathogenesis of NKTL and to identify potential therapeutic targets. Pathway and network analysis of genes differentially expressed between NKTL and normal NK cells revealed significant enrichment for cell cycle-related genes and pathways, such as PLK1, CDK1, and Aurora-A. Furthermore, our results demonstrated a pro-proliferative and anti-apoptotic phenotype in NKTL characterized by activation of Myc and nuclear factor kappa B (NF-kappa B), and deregulation of p53. In corroboration with GEP findings, a significant percentage of NKTLs (n = 33) overexpressed c-Myc (45.4%), p53 (87.9%), and NF-kappa B p50 (67.7%) on immunohistochemistry using a tissue microarray containing 33 NKTL samples. Notably, overexpression of survivin was observed in 97% of cases. Based on our findings, we propose a model of NKTL pathogenesis where deregulation of p53 together with activation of Myc and NF-kappa B, possibly driven by EBV LMP-1, results in the cumulative up-regulation of survivin. Down-regulation of survivin with Terameprocol (EM-1421, a survivin inhibitor) results in reduced cell viability and increased apoptosis in tumour cells, suggesting that targeting survivin may be a potential novel therapeutic strategy in NKTL. Copyright (C) 2011 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

T-box 2 represses NDRG1 through an EGR1-dependent mechanism to drive the proliferation of breast cancer cells

T-box 2 (TBX2) is a transcription factor involved in mammary development and is known to be overexpressed in a subset of aggressive breast cancers. TBX2 has previously been shown to repress growth control genes such as p14(ARF) and p21(WAF1/cip1). In this study we show that TBX2 drives proliferation in breast cancer cells and this is abrogated after TBX2 small interfering RNA (siRNA) knockdown or after the expression of a dominant-negative TBX2 protein. Using microarray analysis we identified a large cohort of novel TBX2-repressed target genes including the breast tumour suppressor NDRG1 (N-myc downregulated gene 1). We show that TBX2 targets NDRG1 through a previously undescribed mechanism involving the recruitment of early growth response 1 (EGR1). We show EGR1 is required for the ability of TBX2 to repress NDRG1 and drive cell proliferation. We show that TBX2 interacts with EGR1 and that TBX2 requires EGR1 to target the NDRG1 proximal promoter. Abrogation of either TBX2 or EGR1 expression is accompanied by the upregulation of cell senescence and apoptotic markers. NDRG1 can recapitulate these effects when transfected into TBX2-expressing cells. Together, these data identify a novel mechanism for TBX2-driven oncogenesis and highlight the importance of NDRG1 as a growth control gene in breast tissue. Oncogene (2010) 29, 3252-3262; doi: 10.1038/onc.2010.84; published online 29 March 2010

Obatoclax induces Atg7-dependent autophagy independent of beclin-1 and BAX/BAK

Direct pharmacological targeting of the anti-apoptotic B-cell lymphoma-2 (BCL-2) family is an attractive therapeutic strategy for treating cancer. Obatoclax is a pan-BCL-2 family inhibitor currently in clinical development. Here we show that, although obatoclax can induce mitochondrial apoptosis dependent on BCL-2 associated x protein/BCL-2 antagonist killer (BAX/BAK) consistent with its on-target pharmacodynamics, simultaneous silencing of both BAX and BAK did not abolish acute toxicity or loss of clonogenicity. This is despite complete inhibition of apoptosis. Obatoclax dramatically reduced viability without inducing loss of plasma membrane integrity. This was associated with rapid processing of light chain-3 (LC3) and reduction of S6 kinase phosphorylation, consistent with autophagy. Dramatic ultrastructural vacuolation, not typical of autophagy, was also induced. Silencing of beclin-1 failed to prevent LC3 processing, whereas knockout of autophagy-related (Atg) 7 abolished LC3 processing but failed to prevent obatoclax-induced loss of clonogenicity or ultrastructural changes. siRNA silencing of Atg7 in BAX/BAK knockout mouse embryonic fibroblasts did not prevent obatoclax-induced loss of viability. Cells selected for obatoclax resistance evaded apoptosis independent of changes in BCL-2 family expression and displayed reduced LC3 processing. In summary, obatoclax exhibits BAX- and BAK-dependent and -independent mechanisms of toxicity and activation of autophagy. Mechanisms other than autophagy and apoptosis are blocked in obatoclax resistant cells and contribute significantly to obatoclax's anticancer efficacy. Cell Death and Disease (2010) 1, e108; doi:10.1038/cddis.2010.86; published online 16 December 2010