Exhaled breath profiling using broadband quantum cascade laser-based spectroscopy in healthy children and children with asthma and cystic fibrosis.

Exhaled breath analysis is a potential non-invasive tool for diagnosing and monitoring airway diseases. Gas chromatography-mass spectrometry and electrochemical sensor arrays are the main techniques to detect volatile organic compounds (VOC) in exhaled breath. We developed a broadband quantum cascade laser spectroscopy technique for VOC detection and identification. The objective of this study was to assess the repeatability of exhaled breath profiling with broadband quantum cascade laser-based spectroscopy and to explore the clinical applicability by comparing exhaled breath samples from healthy children with those from children with asthma or cystic fibrosis (CF). Healthy children and children with stable asthma or stable CF, aged 6-18 years, were included. Two to four exhaled breath samples were collected in Tedlar bags and analyzed by quantum cascade laser spectroscopy to detect VOCs with an absorption profile in the wavenumber region between 832 and 1262.55 cm(-1). We included 35 healthy children, 39 children with asthma and 15 with CF. Exhaled breath VOC profiles showed poor repeatability (Spearman's rho = 0.36 to 0.46) and agreement of the complete profiles. However, we were able to discriminate healthy children from children with stable asthma or stable CF and identified VOCs that were responsible for this discrimination. Broadband quantum cascade laser-based spectroscopy detected differences in VOC profiles in exhaled breath samples between healthy children and children with asthma or CF. The combination of a relatively easy and fast method and the possibility of molecule identification makes broadband quantum cascade laser-based spectroscopy attractive to investigate the diagnostic and prognostic potential of volatiles in exhaled breath.

Evaluation of the LigaSure(™) Vessel Sealing System for bowel transection and intestinal anastomosis-an experimental study in a porcine model.

BACKGROUND: The purpose of the present study is to assess the value of the LigaSure™ Vessel Sealing System (LVSS) as a means for bowel transection and intestinal anastomosis. METHODS: We compared the LVSS for (1) transecting bowel and (2) creation of an intestinal anastomosis with standard methods such as stapler (S) and hand-sewn (HS) in a porcine model. For each study arm, i.e., bowel transection and anastomosis creation, both the small bowel and colon were examined. In total, ten transections and ten anastomoses were performed for each. Burst and anastomotic leak pressures were compared. RESULTS: In the study arm 1, LVSS achieved lowest burst pressures in both small bowel (LVSS 39.8 ± 3.6 mmHg, S 81.9 ± 3.9, HS 111.9 ± 14.7 mmHg, p < 0.0001) and colon transections (LVSS 21.5 ± 2.6 mmHg, S 79.5 ± 4.9, HS 91.0 ± 5.2 mmHg, p < 0.0001). There was no difference in burst pressures between S and HS in both small bowel and colon transections. In the study arm 2, LVSS showed the lowest anastomotic leak pressures for small bowel (LVSS 26.4 ± 2.6 mmHg, S 52.1 ± 6.2, HS 87.4 ± 7.0 mmHg, p < 0.0001) and colonic anastomoses (LVSS 16.9 ± 1.3 mmHg, S 55.9 ± 4.3, HS 74.4 ± 4.4 mmHg, p < 0.0001). Furthermore, small bowel and colonic anastomoses using S demonstrated significantly lower leak pressures than HS anastomosis p < 0.001 and p = 0.004, respectively. CONCLUSIONS: The LVSS achieves significantly lower burst pressures and anastomotic leak pressures for bowel transection and intestinal anastomosis than S and HS techniques. However, due to the achieved pressure levels of 39.8 ± 3.6 mmHg, LVSS appears to be a sufficient stand-alone method for bowel transection. Whether it can be used to perform intestinal anastomosis warrants further research in a survival model.

Comparison of two different transection techniques in liver surgery-an experimental study in a porcine model.

AIMS: Postoperative morbidity and mortality after liver resection is closely related to the degree of intraoperative blood loss; the majority of which occurs during transection of the liver parenchyma. Many approaches and devices have therefore been developed to limit bleeding, but none has yet achieved perfect results up to now. The aim of this standardized chronic animal study was to compare the safety and efficacy of the LigaSure™ Vessel Sealing System (LVSS) with the stapler technique, which is one of the modern techniques for transecting the parenchyma in liver surgery. METHODS: Sixteen pigs underwent a left liver resection (LLR). Eight pigs received a LLR by means of an Endo GIA, whereas the other eight pigs underwent liver parenchymal transection followed by simultaneous sealing by the LVSS. The operating time, transection time, blood loss during transection, and time of hemostasis were measured on the day of LLR (postoperative day 0/POD 0). Animals were re-explored on postoperative day 7 (POD 7) and the transection surface of remnant liver was observed for fluid collection (hematoma, biloma, and abscess), necrosis, and other pathologies. A biopsy was taken from the area of transection for histopathological examination. RESULTS: All animals survived until POD 7. Operating time and transection time of the liver parenchyma on POD 0 was significantly shorter in the stapler group. There was no significant difference between the two groups in terms of blood loss during transection, time of hemostasis and number of sutures for hemostasis on POD 0, morbidity rate, as well as the histopathological examination on POD 7. Furthermore, the material costs were significantly higher in the stapler group than in the LVSS group. CONCLUSION: In this standardized chronic animal study concerning transection of the parenchyma in liver surgery, LVSS seems not only to be safe, but also comparable with the stapler technique in terms of morbidity and mortality. Additionally, LVSS significantly reduces material costs. However, the transection time is significantly longer for LVSS than for the stapler resection technique.

Diffusion-weighted imaging in rectal carcinoma patients without and after chemoradiotherapy: a comparative study with histology.

Diffusion-weighted imaging (DWI) can be used to quantitatively assess functional parameters in rectal carcinoma that are relevant for prognosis and treatment response assessment. However, there is no consensus on the histopathological background underlying the findings derived from DWI. The aim of this study was to perform a comparison of DWI and histologic parameters in two groups of rectal carcinoma patients without (n=12) and after (n=9) neoadjuvant chemoradiotherapy (CRT). The intravoxel incoherent motion (IVIM) model was used to calculate the diffusion coefficient D and the perfusion fraction f in rectal carcinoma, the adjacent rectum and fat in the two patient groups. Immunohistological analysis was performed to assess the cellularity, vascular area fraction and vessel diameter for comparison and correlation. Out of 36 correlations between parameters from DWI and histology, four were found to be significant. In rectal carcinoma of patients without CRT, the diffusion D and the perfusion f correlated with the vascular area fraction, respectively, which could not be found in the group of patients who received CRT. Further correlations were found for the rectum and fat. Histological evaluation revealed significant differences between the tissues on the microscopic level concerning the cellular and vascular environment that influence diffusion and perfusion. In conclusion, DWI produces valuable biomarkers for diffusion and perfusion in rectal carcinoma and adjacent tissues that are highly dependent of the underlying cellular microenvironment influenced by structural and functional changes as well as the administered treatment, and consequently can be beyond histological ascertainability.

Effectiveness of ozone against endodontopathogenic microorganisms in a root canal biofilm model.

AIM: To assess the antimicrobial efficacy of aqueous (1.25-20 microg mL(-1)) and gaseous ozone (1-53 g m(-3)) as an alternative antiseptic against endodontic pathogens in suspension and a biofilm model. METHODOLOGY: Enterococcus faecalis, Candida albicans, Peptostreptococcus micros and Pseudomonas aeruginosa were grown in planctonic culture or in mono-species biofilms in root canals for 3 weeks. Cultures were exposed to ozone, sodium hypochlorite (NaOCl; 5.25%, 2.25%), chlorhexidine digluconate (CHX; 2%), hydrogen peroxide (H(2)O(2); 3%) and phosphate buffered saline (control) for 1 min and the remaining colony forming units counted. Ozone gas was applied to the biofilms in two experimental settings, resembling canal areas either difficult (setting 1) or easy (setting 2) to reach. Time-course experiments up to 10 min were included. To compare the tested samples, data were analysed by one-way anova. RESULTS: Concentrations of gaseous ozone down to 1 g m(-3) almost and aqueous ozone down to 5 microg mL(-1) completely eliminated the suspended microorganisms as did NaOCl and CHX. Hydrogen peroxide and lower aqueous ozone concentrations were less effective. Aqueous and gaseous ozone were dose- and strain-dependently effective against the biofilm microorganisms. Total elimination was achieved by high-concentrated ozone gas (setting 2) and by NaOCl after 1 min or a lower gas concentration (4 g m(-3)) after at least 2.5 min. High-concentrated aqueous ozone (20 microg mL(-1)) and CHX almost completely eliminated the biofilm cells, whilst H(2)O(2) was less effective. CONCLUSION: High-concentrated gaseous and aqueous ozone was dose-, strain- and time-dependently effective against the tested microorganisms in suspension and the biofilm test model.

Invasion front-specific overexpression of tissue inhibitor of metalloproteinase-1 in liver metastases from colorectal cancer.

BACKGROUND: Matrix metalloproteinases (MMPs) and their inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), have been implicated in invasion and metastasis. The distribution of MMPs and TIMPs in the invasion front of liver metastases from colorectal cancer were investigated in order to understand their potential role in invasiveness. MATERIALS AND METHODS: Freshly frozen material of colorectal metastases of the liver was microdissected into four separate compartments, namely pure liver, liver invasion, tumour invasion and pure tumour. RNA was isolated and analyzed on Affymetrix microarrays. Expression of TIMP-1 was confirmed by quantitative polymerase chain reaction in 10 colorectal liver metastases. Cellular localisation of TIMP-1 was examined by immunohistochemistry. RESULTS: Affymetrix microarray data revealed that several MMP and TIMP genes including MMP-2, -3, -7, -9, -11, -12, -14, -15, -16, -19 and -24, and TIMP-1, -2 and -3 were generally up-regulated in both invasion front compartments. Among these genes, TIMP-1 showed the highest expression. The qPCR results indicated an average 15-fold upregulation of TIMP-1 in the liver invasive front and an average 13-fold up-regulation in the tumor invasive front, each compared to normal liver tissue. Immunohistochemistry revealed expression of TIMP-1 in tumour epithelia as well as in host tissue cells, including fibroblastic cells. CONCLUSION: Our data indicate that tumour invasion in colorectal liver metastasis is associated with increased TIMP-1 RNA and protein levels in both tumour and host cells.

Enhanced VDUP-1 gene expression by PPARgamma agonist induces apoptosis in human macrophage.

The fate and phenotype of lesion macrophages is regulated by cellular oxidative stress. Thioredoxin-1 (Trx-1) plays a major role in the regulation of cellular redox balance, with resultant effects on gene expression and cellular responses including cell growth and death. Trx-1 activity is inhibited by interaction with vitamin D-upregulated protein-1 (VDUP-1). Peroxisome proliferator-activated receptor gamma (PPARgamma) is expressed by human monocyte-derived macrophages (HMDM) and PPARgamma agonism has been reported to decrease expression of inflammatory genes and to promote apoptosis of these cells. To determine whether VDUP-1 may be involved in regulating the effects of PPARgamma agonists in macrophages, we investigated the effect of a synthetic PPARgamma agonist (GW929) on the expression of VDUP-1 in HMDM. GW929 concentration-dependently increased HMDM expression of VDUP-1 (mRNA and protein). Transfection of different fragments of the VDUP-1 promoter as well as gel shift analysis revealed the presence of functional PPARgamma response elements (PPRE) in the promoter. Under conditions in which PPAR agonism altered levels of VDUP-1, caspase-3 activity, and macrophage apoptosis were also elevated. The results suggest that PPARgamma activation stimulates apoptosis in human macrophages by altering the cellular redox balance via regulation of VDUP-1.

Effect of aqueous ozone on the NF-kappaB system.

Ozone has been proposed as an alternative oral antiseptic in dentistry, due to its antimicrobial power reported for gaseous and aqueous forms, the latter showing a high biocompatibility with mammalian cells. New therapeutic strategies for the treatment of periodontal disease and apical periodontitis should consider not only antibacterial effects, but also their influence on the host immune response. Therefore, our aim was to investigate the effect of aqueous ozone on the NF-kappaB system, a paradigm for inflammation-associated signaling/transcription. We showed that NF-kappaB activity in oral cells stimulated with TNF, and in periodontal ligament tissue from root surfaces of periodontally damaged teeth, was inhibited following incubation with ozonized medium. Under this treatment, IkappaBalpha proteolysis, cytokine expression, and kappaB-dependent transcription were prevented. Specific ozonized amino acids were shown to represent major inhibitory components of ozonized medium. In summary, our study establishes a condition under which aqueous ozone exerts inhibitory effects on the NF-kappaB system, suggesting that it has an anti-inflammatory capacity.

Generation and characterisation of monoclonal antibodies to oleanolic acid.

Monoclonal antibodies to oleanolic acid, a pentacyclic triterpene, were generated using the hybridoma fusion method described by Kohler and Milstein . Protein conjugates of the target molecule for immunisation were prepared either by directly linking the isoprenoid to different protein carriers via the carboxylic function or after introduction of a succinic spacer between the protein carrier and position 3 of the target molecule. Antibodies of three different cell lines were further analysed by competitive ELISA and were shown to be directed either against both rings A and B or to ring E of the pentacyclic system. Thus these antibodies can be used in the specific detection of oleanolic acid itself or of a broad range of oleanolic acid derivatives sharing a specific structural moiety. Therefore these antibodies may be interesting tools for the screening of putative terpenoid-containing plants.

Reduced beta3-endonexin levels are associated with enhanced urokinase-type plasminogen activator receptor expression in ApoE-/- mice.

Proteolysis of extracellular matrix components is required for cell migration occurring in atherosclerotic lesion formation. In the present study, gene expression of the urokinase plasmingen activator receptor (uPAR) and underlying mechanisms were analyzed during the development of atherosclerosis in the aorta of apolipoprotein E-deficient mice (apoE(-/-)). A significant increase of uPAR expression was detected in the atherosclerotic tissue with advancing plaque-dimension. As uPAR gene transcription involves the transcription factor nuclear factor-kappaB (NF-kappaB), we analyzed nuclear NF-kappaB activity in vascular tissue of apoE-deficient mice. Congruent to uPAR, we could detect an increase in NF-kappaB activity, which underlines the chronic inflammatory component of the disease. Recently we reported that beta(3)-endonexin, a protein that modulates beta(3)-integrins, regulates uPAR expression through direct interaction with subunits of the NF-kappaB-complex. Herein we could show that beta(3)-endonexin protein is expressed in aortic tissue of mice. Moreover, in contrast to uPAR or NF-kappaB, the expression of beta(3)-endonexin was reduced in extracts of advanced atherosclerotic aortic tissue. The cytoplasmic protein beta(3)-endonexin regulates function of beta(3)-integrins. We revealed that integrin stimulation of endothelial cells led to an enhanced NF-kappaB activity and secretion of the NF-kappaB dependent chemokine monocyte chemoattractant protein-1 (MCP-1). The beta(3)-integrin dependent increase in MCP-1 was notedly reduced in cells that overexpressed beta(3)-endonexin. These results provide strong evidence that beta(3)-endonexin acts as a regulating factor in the integrin-mediated signal transduction and the present findings imply a pathophysiological role of beta(3)-endonexin in atherogenesis.

Retrospective evidence that the MHC (B haplotype) of chickens influences genetic resistance to attenuated infectious bronchitis vaccine strains in chickens.

Infectious bronchitis is a respiratory disease of chickens that is caused by the coronavirus infectious bronchitis virus (IBV). Virtually all broiler and layer breeder flocks are routinely vaccinated against IBV. Two hatches of 1-day-old chicks from four lines were mistakenly vaccinated for infectious bronchitis using a moderately attenuated vaccine designed for chicks of an older age. The vaccination resulted in high mortality, and chicks from three of four lines died with signs typical of infectious bronchitis. The mortality that occurred using this less-attenuated vaccine was significantly influenced by the genetic line, and the MHC (B) haplotype in chickens of three B congenic lines. B congenic chickens possessing the B*15 haplotype were resistant in contrast to chickens possessing the B*13 or B*21 haplotypes. Chicks from two further hatches of the four lines were vaccinated appropriately with a more attenuated IBV vaccine, and only limited chick mortality was seen. These retrospective data from two repeated hatches confirm earlier data indicating chicken genes influence resistance to IBV, and indicate for the first time that genes tightly linked to the B haplotype are relevant in resistance to IBV. Due to extenuating circumstances it was not possible to verify results with chicks from F2 matings. Factors that may enhance definition of the role of the B haplotype in immune response to IBV, and the desirability for further analysis of a B haplotype-linked influence on immunity to IBV are discussed.

Increased IkappaB kinase activity is associated with activated NF-kappaB in acute myeloid blasts.

NF-kappaB/Rel transcription factors are modulators of immune and inflammatory processes and are also involved in malignancy. Phosphorylation of the IkappaB inhibitors by the IkappaB kinase (IKK) complex leads to their proteasomal degradation, resulting in activated NF-kappaB. Here, we investigated the activation status of NF-kappaB and the IKK complex in acute myeloid leukemia (AML). Gelshift assays revealed an increased level of activated nuclear NF-kappaB in myeloid blasts. Both bone marrow and peripheral blood blasts from AML patients showed enhanced IKK activity relative to controls, whereas the IKK protein concentrations were comparable. In addition, an increased level of IkappaB-alpha was detected in AML blast cells, although this appeared to be insufficient to block nuclear translocation of NF-kappaB, also confirmed by immunofluorescence. In subtype M4 and M5 AML cells a more extensive NF-kappaB activation and higher IKK activity was found than in M1/M2 specimens. Isolated AML blasts cultured ex vivo responded to external stimulation (TNF, LPS) by further IKK activation, IkappaB degradation and NF-kappaB activation. Preincubation with the proteasome inhibitor PSI inhibited the NF-kappaB system in isolated AML blasts. This study established for the first time a dysregulation of IKK signaling in AML leading to increased NF-kappaB activity suggesting potential therapeutic avenues.

Limitations to empirical extrapolation studies: the case of BMD ratios.

Extrapolation relationships are of keen interest to chemical risk assessment in which they play a prominent role in translating experimentally derived (usually in animals) toxicity estimates into estimates more relevant to human populations. A standard approach for characterizing each extrapolation relies on ratios of pre-existing toxicity estimates. Applications of this "ratio approach" have overlooked several sources of error. This article examines the case of ratios of benchmark doses, trying to better understand their informativeness. The approach involves mathematically modeling the process by which the ratios are generated in practice. Both closed form and simulation-based models of this "data-generating process" (DGP) are developed, paying special attention to the influence of experimental design. The results show the potential for significant limits to informativeness, and revealing dependencies. Future applications of the ratio approach should take imprecision and bias into account. Bootstrap techniques are recommended for gauging imprecision, but more complicated techniques will be required for gauging bias (and capturing dependencies). Strategies for mitigating the errors are suggested.

Predominant role of toll-like receptor 2 versus 4 in Chlamydia pneumoniae-induced activation of dendritic cells.

Chlamydia pneumoniae is an obligate intracellular human pathogen causing diseases such as pneumonia, bronchitis, and pharyngitis. Because of its intracellular replication, cell-mediated immune responses are needed to mediate successful defenses of the host. Because dendritic cells play a central role in linking innate immunity and Ag-specific cell-mediated immune responses we asked whether dendritic cells are activated upon contact with C. pneumoniae and whether known Toll like receptors (TLR) are involved in this process. Here we show that C. pneumoniae was taken up by bone marrow-derived murine dendritic cells. Ingested C. pneumoniae appeared to be unable to develop mature inclusion inside dendritic cells. Furthermore, upon contact with C. pneumoniae dendritic cells were potently stimulated because NF-kappaB was activated and translocated to the nucleus, cytokines like IL-12p40 and TNF-alpha were secreted, and expression of MHC class II molecules, CD40, CD80, and CD86 was up-regulated. Importantly, secretion of cytokines as well as translocation of NF-kappaB were dependent on the presence of TLR2 and independent from TLR4 with the exception of IL-12p40 secretion, which was attenuated in the absence of either a functional TLR2 or 4. In conclusion, we show here that recognition of the Gram-negative bacterium C. pneumoniae depends largely on TLR2 and only to a minor extent on TLR4.

Characterization of a novel human cell-cycle-regulated homologue of Drosophila dlg1.

Cell cycle defects have been associated with the process of carcinogenesis in many studies. Here we report the cloning and analysis of the novel gene KIAA0008 (GenBank acc. no. D13633). Chromosomal localization experiments assigned the gene to chromosome 14q22-q23. The mRNA transcript was found to be cell cycle regulated, expressed at S-phase, and maintained at both G2-and M-phases. In situ hybridization showed expression in proliferating colon and breast (tumor) tissues. Structurally, KIAA0008 shares homology with the Drosophila melanogaster discs large-1 (dlg1) tumor suppressor gene and membrane-associated guanylate kinase protein family members. The potential involvement of KIAA0008 in cell proliferation is discussed, along with its sequence identity and tissue distribution.

Regression of large tumors expressing a suicide gene.

Using the HSV-tk/GCV system, complete remissions of tumors up to a size of 5 mm in diameter have been reported in mice and rats. In order to examine whether larger tumors of clinically relevant size can also be successfully treated, we established hepatic and peritoneal tumors consisting of retrovirally pretransduced tk-positive CC531 colon adenocarcinoma cells in syngenic Wag/Ola rats. In this model, we evaluated whether large tumors respond to therapy under the ideal condition of 100% gene transfer. Within 10 days, GCV treatment led to a marked regression of the adenocarcinomas. Tumors up to a size of 4000 mm3 could be completely eradicated. Passing through several stages of degeneration and cytolytic necrosis, a complete replacement by fibrous tissue was seen. There was no histological evidence for apoptosis and cell mediated immunity. These results suggest that complete regression of tumors of clinically relevant size can be achieved by direct toxic effects of the HSV-tk/GCV system if 100% of the cells are transduced.

Effects of adenoviral wild-type p53 gene transfer in p53-mutated lymphoma cells.

The present study assessed the role of adenoviral vector-mediated wild-type p53 gene transfer in B lymphoma cells. Deficiency of p53-mediated cell death is common in human cancer contributing to both tumorigenesis and chemoresistance. Lymphoma cells are being considered as suitable targets for gene therapy protocols. Recently, we reported an adenoviral protocol leading to highly efficient gene transfer to B lymphoma cells. All lymphoma cell lines (n=5) tested here showed mutations in the p53 gene locus. The aim of this work was to transduce lymphoma cells with the wild-type p53 gene. Using this protocol, 88% of Raji, 75% of Daudi, and 45% of OCI-Ly8-LAM53 cells were transfected with the reporter gene green fluorescent protein at a multiplicity of infection of 200. The expression of green fluorescent protein in CA46 and BL41 cells was 27% and 42%, respectively. At this multiplicity of infection, growth characteristics of lymphoma cell lines were not changed significantly. In contrast, cells transduced with wild-type p53 gene showed an inhibition of proliferation as well as an increase in apoptosis. Cell loss by apoptosis after p53 gene transfer was up to 40% as compared to transduction with an irrelevant vector. In addition, we determined the effects of DNA damage produced by the DNA topoisomerase II inhibitor etoposide on wild-type p53 transfected lymphoma cells. In Ad-p53-transfected Raji cells, treatment with the drug resulted in a marked increase of cell loss in comparison to Ad-beta-Gal-transfected cells (45% vs. 77%). Interestingly, performing cytotoxicity studies, we could show an increased sensitivity of Raji and Daudi cells against immunological effector cells. In conclusion, transduction of wild-type p53 into lymphoma cells expressing mutated p53 was efficient and led to inhibition of proliferation and increase in apoptotic rate in some cell lines dependent on p53 mutation. This protocol should have an impact on the use of lymphoma cells in cancer gene therapy protocols.