Mast cells promote lung vascular remodelling in pulmonary hypertension.

Left heart disease (LHD) frequently causes lung vascular remodelling and pulmonary hypertension (PH). Yet pharmacological treatment for PH in LHD is lacking and its pathophysiological basis remains obscure. We aimed to identify candidate mechanisms of PH in LHD and to test their relevance and therapeutic potential. In rats, LHD was induced by supracoronary aortic banding. Whole genome microarray analyses were performed, candidate genes were confirmed by RT-PCR and Western blots and functional relevance was tested in vivo by genetic and pharmacological strategies. In lungs of LHD rats, mast cell activation was the most prominently upregulated gene ontology cluster. Mast cell gene upregulation was confirmed at RNA and protein levels and remodelled vessels showed perivascular mast cell accumulations. In LHD rats treated with the mast cell stabiliser ketotifen, or in mast cell deficient Ws/Ws rats, PH and vascular remodelling were largely attenuated. Both strategies also reduced PH and vascular remodelling in monocrotaline-induced pulmonary arterial hypertension, suggesting that the role of mast cells extends to non-cardiogenic PH. In PH of different aetiologies, mast cells accumulate around pulmonary blood vessels and contribute to vascular remodelling and PH. Mast cells and mast cell-derived mediators may present promising targets for the treatment of PH.

Prevalence of equine herpesvirus type 2 (EHV-2) DNA in ocular swabs and its cell tropism in equine conjunctiva.

Equine herpes virus 2 (EHV-2), a gamma(2)-herpesvirus, is common in horses of all ages. Its role as a primary pathogen is unclear but there is an association between EHV-2, respiratory disease and keratoconjunctivitis. The purpose of this study was to gain more information on the prevalence of EHV-2 DNA in conjunctival swabs from horses with and without ocular disease and to define the anatomical site and cell type harbouring viral genome or antigen. By polymerase chain reaction (PCR) 22 out of 77 (28.6%) ocular swabs of clinically healthy and only 4 out of 48 (8.3%) samples from diseased horses were positive. To define the main virus reservoir ocular tissue from 13 randomly selected horses without pathological evidence of ocular disease were analysed by nested PCR. In two horses optic nerve, lacrimal gland and conjunctiva, in further two cases lacrimal gland and conjunctiva and in four horses the conjunctiva only were EHV-2 PCR positive. For specifying the target cell we focused on conjunctivae and selected 3 out of 15 clinically healthy slaughterhouse horses positive for EHV-2 by PCR. In situ hybridisation on sections of these paraffin embedded conjunctivae localized viral genome in histiocyte-like cells of the submucosa. Immunohistochemical staining with an EHV-2 or S100 specific polyclonal antiserum demonstrated that Langerhans cells were co-localized in the same region of the sample section where virus positive cells were detected. Furthermore, we concluded that detection of viral antigen revealed a productive virus infection.

Tumor necrosis factor alpha sensitizes low epidermal growth factor receptor (EGFR)-expressing carcinomas for anti-EGFR therapy.

Analysis of 1,060 xenotransplants derived from cancer cell lines as wel as spontaneously occurring tumors from the larynx, pharynx, mammary gland, uterine cervix, and vulva revealed that tumor regression induced by treatment with monoclonal antibodies (EMD 55900 and EMD 72000 against the epidermal growth factor receptor (EGFR) could be enhanced by tumor necrosis factor alpha (TNF-alpha) treatment in vivo. Moreover, tumor that primarily do not respond to antibody treatment can be made suscep tible by additional TNF-alpha treatment. To investigate the in vivo effects of monoclonal antibodies, we treated tumors derived from cell lines (A431 and Detroit 562) as well as spontaneously occurring squamous cell carci nomas and adenocarcinomas (transplanted on NMRI-nu/nu mice) gener ally with EMD 55900 (40 microg/g mouse) and its humanized version EMD 72000 (40 microg/g mouse). When treated with EMD 55900 and EMD 72000 carcinomas with an EGFR concentration of > or = 70 fmol/mg protein showed significant reduction in tumor size compared with untreated controls. The degree of tumor regression correlated with the EGFR concentration of the tumor. In mice treated with TNF-alpha (0.5 microg/g mouse) and EMD 55900 72000 simultaneously, we observed enhanced antitumor effects up to complete tumor eradication. Carcinomas with an EGFR concentration <70 fmol/mg protein could be made susceptible to treatment with EMD 55900 and EMD 72000 by simultaneous treatment with TNF-alpha, resulting in a significant reduction in tumor size.

The fibronectin/α3ß1 integrin axis serves as molecular basis for keratinocyte invasion induced by ßHPV.

Organ-transplant-recipients exhibit cancerization of the skin from which multiple human papillomavirus (HPV)-positive squamous cell carcinomas (SCCs) arise. However, the molecular basis for HPV-induced invasion of skin keratinocytes is not known. We generated a transgenic mouse model expressing the E7 oncoprotein of HPV8 in the murine epidermis under the control of the keratin-14 promoter and showed that E7 is carcinogenic in mice. We further showed that both, the E7-expressing keratinocyte and mesenchymal components of the extracellular matrix as critical in eliciting the invasive behavior. E7 expression in basal keratinocytes, grown on fibronectin, led to epithelial-mesenchymal transition mediated by a cadherin switch. E7-positive keratinocytes displayed enhanced EDA-fibronectin expression and secretion and stimulated dermal fibroblasts to express EDA-fibronectin. Deposition of fibronectin was also detected in the peritumoral stroma of HPV8-positive skin SCC. When grown on fibronectin, E7-positive keratinocytes, in particular stem cell-like cells, exhibited increased cell surface levels of the α3-integrin chain. Functional blocking confirmed α3 as a critical molecule sufficient to induce E7-mediated invasion. This mechanistic link is further supported by expression of an E7-mutant, impaired in targeting α3 to the cell surface. These findings highlight the importance of epithelial-extracellular matrix interaction required for keratinocyte invasion and provide further mechanistic evidence for a role of HPV in skin carcinogenesis.

Substance P primes the formation of hydrogen peroxide and nitric oxide in human neutrophils.

Substance P (SP), a neurotransmitter of the central and peripheral nervous system, has been implicated as a mediator of the pulmonary inflammatory response through its stimulatory effects on neutrophils. We investigated the role of SP in priming the production of reactive oxygen species by human neutrophils with the cytochrome c reduction assay and by flow cytometry using the intracellular oxidizable probe dichlorofluorescein. We also investigated SP-induced formation of nitrite and nitrate as an index of nitric oxide (NO) production. Our results indicate that SP primes two distinct pathways with respect to the induction of reactive oxygen species in the human neutrophil: the production of superoxide anion and hydrogen peroxide by the calmodulin-dependent NADPH oxidase, and the generation of NO by a constitutive NO synthase. Preincubation of neutrophils with inhibitors of calmodulin and NO synthase diminished the oxidative response in an additive fashion. These results give insight into distinct signal transduction pathways in the SP-primed neutrophil with respect to the formation of superoxide anion, hydrogen peroxide, and NO.

"Ideal PEEP" is superior to high dose partial liquid ventilation with low PEEP in experimental acute lung injury.

OBJECTIVE: To determine the effects of a high dose partial liquid ventilation (PLV) approximating the amount of the functional residual capacity (FRC) with low levels of positive end-expiratory pressure (PEEP) compared to a lung-protective strategy with volume-controlled mechanical ventilation (vcMV) with a PEEP level above the lower inflection point (LIP) on pulmonary gas exchange, haemodynamics, respiratory mechanics and lung injury in an experimental model of acute lung injury (ALI). DESIGN: Prospective, randomised, controlled study. METHODS: Twenty-four anaesthetised, tracheotomised and mechanically ventilated (FIO(2) 1.0) pigs underwent induction of ALI by repeated saline wash-out of surfactant. Animals were randomly assigned to receive either PLV ( PLV, n=8) with 30 ml/kg of perfluorocarbons (PF 5080, 3 M, Germany) and a PEEP level of 5 cmH(2)O, to receive vcMV with a PEEP level of 1 cmH(2)O above the LIP ( (ideal) PEEP, n=8), or to receive vcMV with a PEEP level of 5 cmH(2)O ( Controls, n=8). MEASUREMENTS AND RESULTS: Measurements of pulmonary gas exchange, respiratory mechanics and haemodynamics were performed hourly for a 6 h period. In the (ideal) PEEP group, intra-pulmonary shunt (Qs/Qt) decreased from 55+/-5% after induction of ALI to 10+/-3% ( p<0.05 versus Controls and versus PLV) and PaO(2) increased from 52+/-4 to 566+/-19 mmHg after 6 h of treatment ( p<0.05 versus Controls and versus PLV). In the PLV group, Qs/Qt decreased from 50+/-5% after induction of ALI to 24+/-3% ( p<0.05 versus Controls) and PaO(2) increased from 59+/-5 to 306+/-35 mmHg after 6 h of treatment ( p<0.05 versus Controls). In the PLV group and in Controls, mean pulmonary artery pressure (MPAP) was significantly increased from 27+/-2 to 38+/-2 mmHg and from 29+/-1 to 40+/-1 mmHg, respectively, 6 h after induction of ALI ( p<0.05 versus (ideal) PEEP), while in the (ideal) PEEP group, MPAP was maintained between 26+/-1 and 31+/-2 mmHg for 6 h after ALI. Cardiac output (CO) decreased significantly in the (ideal) PEEP group compared to Controls ( p<0.05), while CO did not change in the PLV group and in Controls. The compliance of the respiratory system (C(RS)) increased in the (ideal) PEEP group after induction of ALI from 11+/-2 to 22+/-5 ml/mbar ( p<0.05 versus Controls and versus PLV) and in the PLV group from 10+/-2 to 13+/-3 ml/mbar after 6 h of treatment ( p<0.05 versus Controls). On histological examination, the highest total injury scores were found in animals of the PLV group ( p<0.05 versus Controls and versus (ideal) PEEP), while the lowest total lung injury score was found in the dependent lung regions of the (ideal) PEEP group ( p<0.05 versus Controls). CONCLUSION: In this porcine model of ALI, vcMV with a PEEP level of 1 cmH(2)O above the LIP was superior to high dose PLV with a PEEP of 5 cmH(2)O in improving gas exchange and lung mechanics. In terms of lung damage, the treatment in the (ideal) PEEP group resulted in the lowest total lung injury scores.

Renal dysplasia and benign ureteropelvic polyps associated with hydronephrosis in a foal.

A 4-month-old male Trakehner foal with a history of hematuria, poor growth, and abnormal hair was found to have unilateral hydronephrosis and hydroureter, as determined by ultrasonography and surgical exploration. Nephrectomy and ureterectomy were performed as treatment. Gross examination of the ureter and kidney revealed renal pelvic and ureteral polyps causing obstruction and subsequent hydronephrosis. The histologic features were consistent with renal dysplasia. The polyps and renal dysplasia were likely to be congenital, but the etiopathogenesis is not known. The finding that urinary outflow obstruction can disrupt nephrogenesis and lead to renal dysplasia supports the view that the polyps developed, blocked urine flow, and caused the hydronephrosis and renal dysplasia in the foal.

Range of blood lactate values in farm pigs prior to experimental surgery.

In biomedical research the pig is widely used as an animal model for experimental surgery. Feasible monitoring tools during anaesthesia are pivotal for successful and reliable research outcome. Blood lactate values are a monitoring tool and prognostic indicator during surgery both in humans and animals. Lactate levels in pigs might be influenced by various parameters including stressful handling, breed and weight differences. To determine blood lactate levels present prior to experimental surgery, values of 124 female farm pigs were measured in venous blood samples. Pigs presented with blood lactate concentrations ranging from 0.5 to 5.5 mmol/L (median, 1.2 mmol/L; interquartile range [IQR] 1.2). Considering genetic background, Rheinhybrid/Pietrain pigs (n = 51; median, 1.4 mmol/L; IQR, 1) had significantly higher blood lactate levels compared with Landrace/Pietrain crossbred animals (n = 73; median, 1.1 mmol/L; IQR, 1; P < 0.05). Body weight had no significant effect on blood lactate levels within the evaluated range. This report can benefit research projects monitoring blood lactate values in farm pigs during experimental surgery.

Selection of reference genes for quantitative real-time PCR analysis in canine mammary tumors using the GeNorm algorithm.

Eleven reference genes (18s ribosomal ribonucleic acid [RNA], 28s ribosomal RNA, ubiquitin, beta-actin, glycerine aldehyde dehydrogenase, ATP-synthase subunit 5B, hydroxymethyl-bilane synthase, hypoxanthine-phosphoribosyl transferase, ribosomal protein L32, tryptophan 5-monooxygenase activation protein (zeta polypeptide), and TATA-Box binding protein) were analyzed in use as references for gene expression profiling experiments using quantitative reverse transcription polymerase chain reaction (qRT-PCR) in canine mammary tumors. The transcription level of the candidates was measured in 22 histologically characterized excised tumor specimens from mammary gland tissue and 22 samples of non-neoplastic mammary tissue samples from the same individuals. Results were used to rank candidate reference genes using the GeNorm tool. It was determined that in samples of canine mammary gland tissue, a combination of hypoxanthine-phosphoribosyl transferase, ATP-synthase subunit 5B, ribosomal protein L32 and ubiquitin yields stable reference gene expression levels, whereas the use of glycerin aldehyde dehydrogenase or ribosomal RNA is unsuitable for normalization of qRT-PCR results in this tissue type.

Ozone-induced epithelial injury in the ferret is similar to nonhuman primates.

Extrapolation to humans from rodent ozone exposure is limited by the anatomic differences between the species. Ferrets have similar pulmonary structures to humans, with well developed respiratory bronchioles and submucosal glands. We exposed adult ferrets, monkeys, and rats to 1 ppm ozone (O(3)) or filtered air for 8 h followed by 1 h in filtered air. Bronchoalveolar lavage (BAL) analysis, histopathology, and confocal microscopy were used to evaluate ozone-induced epithelial injury and inflammation. BAL showed significantly increased numbers of neutrophils in ozone-exposed as compared with filtered air ferrets, monkeys, and rats. However, there were 3- to 4-fold more neutrophils in monkeys and ferrets per milliliter of BAL than in rats. Ozone-exposed lungs showed a severe, acute infiltration of neutrophils in regions with necrotic epithelial cells, especially in the centriacinar region that was more severe in ferrets and monkeys than rats. We conclude that acute ozone exposure in ferrets induce severe epithelial necrosis and inflammation, results in similar epithelial injury compared with monkeys, and represents a better model of humans than rodents.

Effects of the Pasteurella multocida toxin on osteoblastic cells in vitro.

Pasteurella multocida toxin induces localized osteolysis in the turbinate bones of swine. Osteolysis appears to be due to an increased level of osteoclastic bone resorption, although osteoblast activity may also be impaired. We studied the effects of purified toxin on the osteoblastic phenotype of the ROS 17/2.8 rat osteoblastic osteosarcoma cell line. Treatment of both embryonic bovine lung cells and a nonosteoblastic rat osteosarcoma cell line (ROS 25/1) with nanomolar doses of toxin produced marked cytotoxic actions. In the osteoblastic ROS 17/2.8 cells, this level of toxin reduced expression of an osteoblastic marker (alkaline phosphatase), was associated with matrix mineralization, but had no cytopathologic action. The osteoblastic cell population may be resistant to a direct cytotoxic effect but is nevertheless a target for toxin action.

Recombinant tumour necrosis factor-alpha and platelet-activating factor synergistically increase intercellular adhesion molecule-1 and E-selectin-dependent neutrophil adherence to endothelium in vitro.

Neutrophil adhesion to microvascular endothelium at sites of acute inflammation is regulated by both chemotactic peptides and lipid-derived mediators. Tumour necrosis factor-alpha (TNF-alpha) is a pro-inflammatory peptide that up-regulates endothelial expression of intercellular adhesion molecule-1 (ICAM-1) and endothelial leucocyte adhesion molecule-1 (E-selectin), while platelet-activating factor (PAF) is a potent lipid mediator that induces vascular changes via an unknown mechanism. Both have been shown to increase leucocyte-endothelial adhesion in various in vitro models of acute inflammation; however, the combined effects of recombinant TNF-alpha (rTNF-alpha) and PAF on neutrophil-endothelium adhesion have not been well described. In this study, we found rTNF-alpha at 0.5 ng/ml and PAF at 10 microM acted synergistically to increase neutrophil adherence to cultured umbilical vein endothelial cells 4 hr after stimulation. This increased neutrophil-endothelial adhesion was, in part, dependent on up-regulated expression of ICAM-1 and E-selectin since application of anti-ICAM1 and anti-E-selectin F(ab')2 fragments markedly diminished adhesion. Cultures stimulated with rTNF-alpha (0.5 ng/ml) or PAF (10 microM) alone did not show a significant increase in neutrophil adhesion, and neither ICAM-1 nor E-selectin expression was up-regulated as determined by flow cytometric analysis of endothelial cells. These results indicate that rTNF-alpha and PAF act synergistically to increase neutrophil-endothelial adhesion by stimulating endothelial expression of ICAM-1 and E-selectin and, thus, may play important roles in the onset and severity of acute inflammatory reactions.

Small dose of exogenous surfactant combined with partial liquid ventilation in experimental acute lung injury: effects on gas exchange, haemodynamics, lung mechanics, and lung pathology.

A combination of exogenous surfactant and partial liquid ventilation (PLV) with perfluorocarbons should enhance gas exchange, improve respiratory mechanics and reduce tissue damage of the lung in acute lung injury (ALI). We used a small dose of exogenous surfactant with and without PLV in an experimental model of ALI and studied the effects on gas exchange, haemodynamics, lung mechanics, and lung pathology. ALI was induced by repeated lavages (PaO2/FIO2 less than 13 kPa) in 24 anaesthesized, tracheotomized and mechanically ventilated (FIO2 1.0) juvenile pigs. They were treated randomly with either a single intratracheal dose of surfactant (50 mg kg(-1), Curosurf, Serono AG, München, Germany) (SURF-group, n=8), a single intratracheal dose of surfactant (50 mg kg(-1), Curosurf) followed by PLV with 30 ml kg(-1) of perfluorocarbon (PF 5080, 3M, Germany) (SURF-PLV-group, n=8) or no further intervention (controls, n=8). Pulmonary gas exchange, respiratory mechanics, and haemodynamics were measured hourly for a 6 h period. In the SURF-group, the intrapulmonary right-to-left shunt (QS/QT) decreased significantly from mean 51 (SEM 5)% after lavage to 12 (2)%, and PaO2 increased significantly from 8.1 (0.7) to 61.2 (4.7) kPa compared with controls and compared with the SURF-PLV-group (P<0.05). In the SURF-PLV-group, QS/QT decreased significantly from 54 (3)% after induction of ALI to 26 (3)% and PaO2 increased significantly from 7.2 (0.5) to 30.8 (5.0) kPa compared with controls (P<0.05). Static compliance of the respiratory system (C(RS)), significantly improved in the SURF-PLV-group compared with controls (P<0.05). Upon histological examination, the SURF-group revealed the lowest total injury score compared with controls and the SURF-PLV-group (P<0.05). We conclude that in this experimental model of ALI, treatment with a small dose of exogenous surfactant improves pulmonary gas exchange and reduces the lung injury more effectively than the combined treatment of a small dose of exogenous surfactant and PLV.

Neonatal capsaicin treatment increases the severity of ozone-induced lung injury.

To test the hypothesis that lung sensory C fibers protect the small distal airways and alveoli from oxidant injury, we compared the effects of inhalation of ozone (1 ppm) or filtered air for 8 h on lung injury and lung inflammation in four groups of rats: (1) normal rats exposed to filtered air; (2) normal rats exposed to ozone; (3) rats treated as neonates with capsaicin (50 mg/kg, intraperitoneally) and subsequently exposed to filtered air; and (4) rats treated as neonates with capsaicin and subsequently exposed to ozone. All rats were allowed to recover in filtered air for 0, 4, 16, and 40 h before necropsy. Rats exposed to filtered air (Groups 1 and 3) showed normal airway and parenchyma structure. Normal untreated rats exposed to ozone showed a random distribution of mild, interstitial inflammatory changes and epithelial necrosis of bronchi and bronchiolar epithelium. However, rats treated with capsaicin and subsequently exposed to ozone demonstrated severe acute interstitial inflammation and epithelial coagulate necrosis in all airways, especially in small, peripheral airways and parenchyma; all of these changes were statistically significant. These findings support our hypothesis that lung sensory C fibers protect the distal airways from oxidant injury during acute ozone inhalation.

Integrin alpha E beta 7 expression on BAL CD4+, CD8+, and gamma delta T-cells in bleomycin-induced lung fibrosis in mouse.

CD4, CD8, and gamma delta T-cells located in the epithelium express the integrin alpha E beta 7 that binds to E-cadherin on the epithelium. Gamma delta T-cells mediate specific cellular immune functions and can recognize damaged cells directly. It was, therefore, of interest to analyse the presence of gamma delta T-cells and the expression of alpha E beta 7 on gamma delta T-cells in the bleomycin (BLM) model of pulmonary fibrosis. Lung fibrosis was induced by a single intratracheal instillation of BLM (0.125 U.mouse-1), and bronchoalveolar lavage (BAL) T-cell subpopulations were examined at various time-points for the expression of the integrin alpha E beta 7 by flow cytometry. CD4+ T-cells accounted for about 40% of the lymphocytes, compared to about 10% of CD8+ T-cells and 10-14% gamma delta T-cells. Within the CD4+ T-cell population the proportion of alpha E beta 7+ cells decreased between Days 2 and 22 from 36 to 11%. The percentage of alpha E beta 7+ CD8+ T-cells increased at the same time from 4 to 68%. However, more than 80% of the gamma delta T-cells in BAL fluid expressed alpha E beta 7 at all time-points. The surface-expression of this integrin on gamma delta T-cells was 2-3 times higher than on CD4+ or CD8+ T-cells. This predominant expression of alpha E beta 7 on gamma delta T-cells suggests a role for these cells in the pathogenesis of bleomycin-induced lung fibrosis.