Food and Indoor Air Isolated Bacillus Non-Protein Toxins: : Structures, Physico-Chemical Properties and Mechanisms of Effects on Eukaryotic Cells

We report here the structures and properties of heat-stable, non-protein, and mammalian cell-toxic compounds produced by spore-forming bacilli isolated from indoor air of buildings and from food. Little information is available on the effects and occurrence of heat-stable non-protein toxins produced by bacilli in moisture-damaged buildings. Bacilli emit spores that move in the air and can serve as the carriers of toxins, in a manner similar to that of the spores of toxic fungi found in contaminated indoor air. Bacillus spores in food cause problems because they tolerate the temperatures applied in food manufacture and the spores later initiate growth when food storage conditions are more favorable. Detection of the toxic compounds in Bacillus is based on using the change in mobility of boar spermatozoa as an indicator of toxic exposure. GC, LC, MS, and nuclear magnetic resonance NMR spectroscopy were used for purification, detection, quantitation, and analysis of the properties and structures of the compounds. Toxicity and the mechanisms of toxicity of the compounds were studied using boar spermatozoa, feline lung cells, human neural cells, and mitochondria isolated from rat liver. The ionophoric properties were studied using the BLM (black-lipid membrane) method. One novel toxin, forming ion channels permeant to K+ > Na+ > Ca2+, was found and named amylosin. It is produced by B. amyloliquefaciens isolated from indoor air of moisture-damaged buildings. Amylosin was purified with an RP-HPLC and a monoisotopic mass of 1197 Da was determined with ESI-IT-MS. Furthermore, acid hydrolysis of amylosin followed by analysis of the amino acids with the GS-MS showed that it was a peptide. The presence of a chromophoric polyene group was found using a NMR spectroscopy. The quantification method developed for amylosin based on RP-HPLC-UV, using the macrolactone polyene, amphotericin B (MW 924), as a reference compound. The B. licheniformis strains isolated from a food poisoning case produced a lipopeptide, lichenysin A, that ruptured mammalian cell membranes and was purified with a LC. Lichenysin A was identified by its protonated molecules and sodium- and potassium- cationized molecules with MALDI-TOF-MS. Its protonated forms were observed at m/z 1007, 1021 and 1035. The amino acids of lichenysin A were analyzed with ESI-TQ-MS/MS and, after acid hydrolysis, the stereoisomeric forms of the amino acids with RP-HPLC. The indoor air isolates of the strain of B. amyloliquefaciens produced not only amylosin but also lipopeptides: the cell membrane-damaging surfactin and the fungicidal fengycin. They were identified with ESI-IT-MS observing their protonated molecules, the sodium- and potassium-cationized molecules and analysing the MS/MS spectra. The protonated molecules of surfactin and fengycin showed m/z values of 1009, 1023, and 1037 and 1450, 1463, 1493, and 1506, respectively. Cereulide (MW 1152) was purified with RP-HPLC from a food poisoning strain of B. cereus. Cereulide was identified with ESI-TQ-MS according to the protonated molecule observed at m/z 1154 and the ammonium-, sodium- and potassium-cationized molecules observed at m/z 1171, 1176, and 1192, respectively. The fragment ions of the MS/MS spectrum obtained from the protonated molecule of cereulide at m/z 1154 were also interpreted. We developed a quantification method for cereulide, using RP-HPLC-UV and valinomycin (MW 1110, which structurally resembles cereulide) as the reference compound. Furthermore, we showed empirically, using the BLM method, that the emetic toxin cereulide is a specific and effective potassium ionophore of whose toxicity target is especially the mitochondria.

Adenoviral gene therapy for non-small cell lung cancer

Adenoviral gene therapy is an experimental approach to cancer refractory to standard cancer therapies. Adenoviruses can be utilized as vectors to deliver therapeutic transgenes into cancer cells, while gene therapy with oncolytic adenoviruses exploits the lytic potential of viruses to kill tumor cells. Although adenoviruses demonstrate several advantages over other vectors - such as the unparalleled transduction efficacy and natural tropism to a wide range of tissues - the gene transfer efficacy to cancer cells has been limited, consequently restricting the therapeutic effect. There are, however, several approaches to circumvent this problem. We utilized different modified adenoviruses to obtain information on adenovirus tropism towards non-small cell lung cancer (NSCLC) cells. To enhance therapeutic outcome, oncolytic adenoviruses were evaluated. Further, to enhance gene delivery to tumors, we used mesenchymal stem cells (MSCs) as carriers. To improve adenovirus specificity, we investigated whether widely used cyclooxygenase 2 (Cox-2) promoter is induced by adenovirus infection in nontarget cells and whether selectivity can be retained by the 3 untranslated region (UTR) AU-rich elements. In addition, we investigated whether switching adenovirus fiber can retain gene delivery in the presence of neutralizing antibodies. Our results show that adenoviruses, whose capsids were modified with arginine-glycine-aspartatic acid (RGD-4C), the serotype 3 knob, or polylysins displayed enhanced gene transfer into NSCLC cell lines and fresh clinical specimens from patients. The therapeutic efficacy was further improved by using respective oncolytic adenoviruses with isogenic 24bp deletion in the E1A gene. Cox-2 promoter was also shown to be induced in normal and tumor cells following adenovirus infection, but utilization of 3 UTR elements can increase the tumor specificity of the promoter. Further, the results suggested that use of MSCs could enhance the bioavailability and delivery of adenoviruses into human tumors, although cells had no tumor tropism per se. Finally, we demonstrated that changing adenovirus fiber can allow virus to escape from existing neutralizing antibodies when delivered systemically. In conclusion, these results reveal that adenovirus gene transfer and specificity can be increased by using modified adenoviruses and MSCs as carriers, and fiber modifications simultaneously decrease the effect of neutralizing antibodies. This promising data suggest that these approaches could translate into clinical testing in patients with NSCLC refractory to current modalities.

Microarrays in Lung Cancer Research: From Comparative Analyses to Verified Findings

Keuhkosyöpä on yleisimpiä syöpätauteja. Se jaetaan kahteen päätyyppiin: pienisoluiseen ja ei-pienisoluiseen keuhkosyöpään. Ei-pienisoluinen keuhkosyöpä jaetaan lisäksi alatyyppeihin, joista suurimmat ovat levyepiteeli-, adeno- ja suurisoluinen karsinooma. Keuhkosyövän tärkein riskitekijä on tupakointi, mutta muutkin työ- ja elinympäristön altisteet, kuten asbesti, voivat johtaa syöpään. Väitöstyössä tutkittiin kahdenlaisten keuhkosyöpäryhmien erityispiirteitä. Työssä kartoitettiin, onko löydettävissä muutoksia, jotka erottavat asbestikeuhkosyövät muista syövistä sekä luuytimeen varhaisessa vaiheessa leviävät keuhkosyövät leviämättömistä syövistä. Tutkimusten ensimmäisessä vaiheessa käytettiin mikrosirupohjaisia menetelmiä, jotka mahdollistavat jopa kaikkien geenien tarkastelun yhden kokeen avulla. Vertailevien mikrosirututkimusten avulla on mahdollista paikantaa geenejä tai kromosomialueita, joiden muutokset erottelevat ryhmät toisistaan. Asbestiin liittyvissä tutkimuksissa paikannettiin kuusi kromosomialuetta, joissa geenien kopiolukumäärän sekä ilmenemistason muutokset erottelivat potilaat altistushistorian mukaan. Riippumattomilla laboratoriomenetelmillä tehtyjen jatkoanalyysien avulla pystyttiin varmistamaan, että 19p-alueen häviämä oli yhteydessä asbestialtistukseen. Työssä osoitettiin myös, että 19p-alueen muutoksia voidaan indusoida altistamalla soluja asbestille in vitro. Tutkimuksessa saatiin lisäksi viitteitä asbestispesifisistä muutoksista signaalinvälitysreiteissä, sillä yhdessä toimivien geenien ilmentymisessä havaittiin eroja asbestille altistuneiden ja altistumattomien välillä. Vertailemalla luuytimeen syövän aikaisessa vaiheessa levinneiden ja leviämättömien keuhkoadenokarsinoomien muutosprofiileita toisiinsa, paikannettiin viisi aluetta, joilla geenien kopiolukumäärä- sekä ilmenemistason muutokset erottelivat ryhmät toisistaan. Jatkoanalyyseissä havaittiin, että 4q-alueen häviämää esiintyi adenokarsinoomien lisäksi levyepiteelikarsinoomiin, jotka olivat levinneet luuytimeen. Myös keuhkosyöpien aivometastaaseissa alue oli toistuvasti hävinnyt. Väitöstyön tutkimukset osoittavat, että vertailevien mikrosiruanalyysien avulla saadaan tietoa syöpäryhmien erityispiirteistä. Työssä saadut tulokset osoittavat, että 19p-alueen muutokset ovat tyypillisiä asbestikeuhkosyöville ja 4q-alueen muutokset luuytimeen aikaisessa vaiheessa leviäville keuhkosyöville.

Microbe-induced activation of inflammatory cytokine response in human cells

Human body is in continuous contact with microbes. Although many microbes are harmless or beneficial for humans, pathogenic microbes possess a threat to wellbeing. Antimicrobial protection is provided by the immune system, which can be functionally divided into two parts, namely innate and adaptive immunity. The key players of the innate immunity are phagocytic white blood cells such as neutrophils, monocytes, macrophages and dendritic cells (DCs), which constantly monitor the blood and peripheral tissues. These cells are armed for rapid activation upon microbial contact since they express a variety of microbe-recognizing receptors. Macrophages and DCs also act as antigen presenting cells (APCs) and play an important role in the development of adaptive immunity. The development of adaptive immunity requires intimate cooperation between APCs and T lymphocytes and results in microbe-specific immune responses. Moreover, adaptive immunity generates immunological memory, which rapidly and efficiently protects the host from reinfection. Properly functioning immune system requires efficient communication between cells. Cytokines are proteins, which mediate intercellular communication together with direct cell-cell contacts. Immune cells produce inflammatory cytokines rapidly following microbial contact. Inflammatory cytokines modulate the development of local immune response by binding to cell surface receptors, which results in the activation of intracellular signalling and modulates target cell gene expression. One class of inflammatory cytokines chemokines has a major role in regulating cellular traffic. Locally produced inflammatory chemokines guide the recruitment of effector cells to the site of inflammation during microbial infection. In this study two key questions were addressed. First, the ability of pathogenic and non-pathogenic Gram-positive bacteria to activate inflammatory cytokine and chemokine production in different human APCs was compared. In these studies macrophages and DCs were stimulated with pathogenic Steptococcus pyogenes or non-pathogenic Lactobacillus rhamnosus. The second aim of this thesis work was to analyze the role of pro-inflammatory cytokines in the regulation of microbe-induced chemokine production. In these studies bacteria-stimulated macrophages and influenza A virus-infected lung epithelial cells were used as model systems. The results of this study show that although macrophages and DCs share several common antimicrobial functions, these cells have significantly distinct responses against pathogenic and non-pathogenic Gram-positive bacteria. Macrophages were activated in a nearly similar fashion by pathogenic S. pyogenes and non-pathogenic L. rhamnosus. Both bacteria induced the production of similar core set of inflammatory chemokines consisting of several CC-class chemokines and CXCL8. These chemokines attract monocytes, neutrophils, dendritic cells and T cells. Thus, the results suggest that bacteria-activated macrophages efficiently recruit other effector cells to the site of inflammation. Moreover, macrophages seem to be activated by all bacteria irrespective of their pathogenicity. DCs, in contrast, were efficiently activated only by pathogenic S. pyogenes, which induced DC maturation and production of several inflammatory cytokines and chemokines. In contrast, L. rhamnosus-stimulated DCs matured only partially and, most importantly, these cells did not produce inflammatory cytokines or chemokines. L. rhamnosus-stimulated DCs had a phenotype of "semi-mature" DCs and this type of DCs have been suggested to enhance tolerogenic adaptive immune responses. Since DCs have an essential role in the development of adaptive immune response the results suggest that, in contrast to macrophages, DCs may be able to discriminate between pathogenic and non-pathogenic bacteria and thus mount appropriate inflammatory or tolerogenic adaptive immune response depending on the microbe in question. The results of this study also show that pro-inflammatory cytokines can contribute to microbe-induced chemokine production at multiple levels. S. pyogenes-induced type I interferon (IFN) was found to enhance the production of certain inflammatory chemokines in macrophages during bacterial stimulation. Thus, bacteria-induced chemokine production is regulated by direct (microbe-induced) and indirect (pro-inflammatory cytokine-induced) mechanisms during inflammation. In epithelial cells IFN- and tumor necrosis factor- (TNF-) were found to enhance the expression of PRRs and components of cellular signal transduction machinery. Pre-treatment of epithelial cells with these cytokines prior to virus infection resulted in markedly enhanced chemokine response compared to untreated cells. In conclusion, the results obtained from this study show that pro-inflammatory cytokines can enhance microbe-induced chemokine production during microbial infection by providing a positive feedback loop. In addition, pro-inflammatory cytokines can render normally low-responding cells to high chemokine producers via enhancement of microbial detection and signal transduction.

Noninvasive biomarkers for early smoking related lung disease

Chronic obstructive pulmonary disease (COPD) is a slowly progressive disease characterized by airway inflammation and largely irreversible airflow limitation. One major risk factor for COPD is cigarette smoking. Since the inflammatory process starts many years prior to the onset of clinical symptoms and still continues after smoking cessation, there is an urgent need to find simple non-invasive biomarkers that can be used in the early diagnosis of COPD and which could help in predicting the disease progression. The first aim of the present study was to evaluate the involvement of different oxidative/nitrosative stress markers, matrix metalloproteinases (MMPs) and their tissue inhibitor-1 (TIMP-1) in smokers and in COPD. Elevated numbers of inducible nitric oxide synthase (iNOS), nitrotyrosine, myeloperoxidase (MPO) and 4-hydroxy-2-nonenal (4-HNE) positive cells and increased levels of 8-isoprostane and lactoferrin were found in sputum of non-symptomatic smokers compared to non-smokers, and especially in subjects with stable mild to moderate COPD, and they correlated with the severity of airway obstruction. This suggests that an increased oxidant burden exists already in the airways of smokers with normal lung function values. However, none of these markers could differentiate healthy smokers from symptomatic smokers with normal lung function values i.e. those individuals who are at risk of developing COPD. In contrast what is known about asthma exhaled nitric oxide (FENO) was lower in smokers than in non-smokers, the reduced FENO value was significantly associated with neutrophilic inflammation and the elevated oxidant burden (positive cells for iNOS, nitrotyrosine and MPO). The levels of sputum MMP-8 and plasma MMP-12 appeared to differentiate subjects who have a risk for COPD development but these finding require further investigations. The levels of all studied MMPs correlated with the numbers of neutrophils, and MMP-8 and MMP-9 with markers of neutrophil activation (MPO, lactoferrin) suggesting that especially neutrophil derived oxidants may stimulate the tissue destructive MMPs already in lungs of smokers who are not yet experiencing any airflow limitation. When investigating the role of neutrophil proteases (neutrophil elastase, MMP-8, MMP-9) during COPD exacerbation and its recovery period, we found that levels of all these proteases were increased in sputum of patients with COPD exacerbation as compared to stable COPD and controls, and decreased during the one-month recovery period, giving evidence for a role of these enzymes in COPD exacerbations. In the last study, the effects of subject`s age and smoking habits were evaluated on the plasma levels of surfactant protein A (SP-A), SP-D, MMP-9 and TIMP-1. Long-term smoking increased the levels of all of these proteins. SP-A most clearly correlated with age, pack years and lung function decline (FEV1/FVC), and based on the receiver operating characteristic curve analysis, SP-A was the best marker for discriminating subjects with COPD from controls. In conclusion, these findings support the hypothesis that especially neutrophil derived oxidants may activate MMPs and induce an active remodeling process already in the lungs of smokers with normal lung function values. The marked increase of sputum levels of neutrophil proteases in smokers, stable COPD and/or during its exacerbations suggest that these enzymes play a role in the development and progression of COPD. Based on the comparison of various biomarkers, SP-A can be proposed to serve as sensitive biomarker in COPD development.