The Ca2+-dependent pathway contributes to changes in the subcellular localization and extracellular release of interleukin-33.

Interleukin-33 (IL-33) is a member of the IL-1 cytokine family and plays critical roles in facilitating type-2 immune responses. IL-33 is localized in the nucleus and released to the extracellular milieu during cell death, although the precise mechanisms underlying IL-33 mobilization remain unclear. Here, we found that nigericin, a toxin derived from Streptomyces hygroscopicus, promoted IL-33 translocation from the nucleus to the cytosol before extracellular release. This translocation was inhibited by chelating Ca2+ with EGTA or membrane protection by glycine treatment. Ca2+ ionophore A23187 stimulation caused IL-33 translocation to the cytoplasm but was not sufficient for extracellular release. However, IL-33 release was induced by detergent treatment, which indicates that membrane rupture is required for IL-33 release. The pore-forming pyroptosis executor gasdermin D was cleaved following nigericin stimulation, and overexpression of the cleaved gasdermin D-N-terminal fragment that forms the membrane pore sufficiently induced IL-33 release, which was blocked by EGTA and glycine. Together, these findings suggest that Ca2+-dependent signals and gasdermin D pore formation are required for robust IL-33 production.

Comparative transcriptome expression analysis in susceptible and resistant potato (Solanum tuberosum) cultivars to common scab (Streptomyces scabies) revealed immune priming responses in the incompatible interaction.

Common scab disease in potato has become a widespread issue in major potato production areas, leading to increasing economic losses. Varietal resistance is seen as a viable and long-term scab management strategy. However, the genes and mechanisms of varietal resistance are unknown. In the current study, a comparative RNA transcriptome sequencing and differential gene signaling and priming sensitization studies were conducted in two potato cultivars that differ by their response to common scab (Streptomyces scabies), for unraveling the genes and pathways potentially involved in resistance within this pathosystem. We report on a consistent and contrasted gene expression pattern from 1,064 annotated genes differentiating a resistant (Hindenburg) and a susceptible (Green Mountain) cultivars, and identified a set of 273 co-regulated differentially expressed genes in 34 pathways that more likely reflect the genetic differences of the cultivars and metabolic mechanisms involved in the scab pathogenesis and resistance. The data suggest that comparative transcriptomic phenotyping can be used to predict scab lesion phenotype in breeding lines using mature potato tuber. The study also showed that the resistant cultivar, Hindenburg, has developed and maintained a capacity to sense and prime itself for persistent response to scab disease over time, and suggests an immune priming reaction as a mechanism for induced-resistance in scab resistant potato cultivars. The set of genes identified, described, and discussed in the study paves the foundation for detailed characterizations towards tailoring and designing procedures for targeted gene knockout through gene editing and phenotypic evaluation.

Analysis of the Tunicamycin Biosynthetic Gene Cluster of Streptomyces chartreusis Reveals New Insights into Tunicamycin Production and Immunity.

The tunicamycin biosynthetic gene cluster of Streptomyces chartreusis consists of 14 genes (tunA to tunN) with a high degree of apparent translational coupling. Transcriptional analysis revealed that all of these genes are likely to be transcribed as a single operon from two promoters, tunp1 and tunp2. In-frame deletion analysis revealed that just six of these genes (tunABCDEH) are essential for tunicamycin production in the heterologous host Streptomyces coelicolor, while five (tunFGKLN) with likely counterparts in primary metabolism are not necessary, but presumably ensure efficient production of the antibiotic at the onset of tunicamycin biosynthesis. Three genes are implicated in immunity, namely, tunI and tunJ, which encode a two-component ABC transporter presumably required for export of the antibiotic, and tunM, which encodes a putative S-adenosylmethionine (SAM)-dependent methyltransferase. Expression of tunIJ or tunM in S. coelicolor conferred resistance to exogenous tunicamycin. The results presented here provide new insights into tunicamycin biosynthesis and immunity.

Carbon catabolite regulation in Streptomyces: new insights and lessons learned.

One of the most significant control mechanisms of the physiological processes in the genus Streptomyces is carbon catabolite repression (CCR). This mechanism controls the expression of genes involved in the uptake and utilization of alternative carbon sources in Streptomyces and is mostly independent of the phosphoenolpyruvate phosphotransferase system (PTS). CCR also affects morphological differentiation and the synthesis of secondary metabolites, although not all secondary metabolite genes are equally sensitive to the control by the carbon source. Even when the outcome effect of CCR in bacteria is the same, their essential mechanisms can be rather different. Although usually, glucose elicits this phenomenon, other rapidly metabolized carbon sources can also cause CCR. Multiple efforts have been put through to the understanding of the mechanism of CCR in this genus. However, a reasonable mechanism to explain the nature of this process in Streptomyces does not yet exist. Several examples of primary and secondary metabolites subject to CCR will be examined in this review. Additionally, recent advances in the metabolites and protein factors involved in the Streptomyces CCR, as well as their mechanisms will be described and discussed in this review.

Development of Series of Affinity Tags in Streptomyces.

Streptomyces are of great biological and industrial significance due to their complex morphological development and ability to produce numerous secondary metabolites. However, the intrinsic biochemical mechanisms underlying morphogenesis and secondary metabolism are rarely revealed, partially because of the limited availability of the biochemical tools in Streptomyces. Here we provided series of integrative vectors with various affinity tags, including single tags 3×FLAG, 3×HA, 3×Strep-tag II, 18×His, 13×Myc, and dual tags, all of which were driven from a strong constitutive promoter ermEp*. Using a sigma factor SigT from S. coelicolor as a model, we successfully expressed and immuno-detected SigT fused with all tags. Moreover, after SigT was N-terminally tagged with 3×FLAG and C-terminally tagged with 18×His, we isolated SigT-interactive proteins from the S. coelicolor lysate based on the tandem affinity purification (TAP). Particularly, among the proteins purified, the SigT cognate anti-sigma factor RstA ranked the top with the most total independent spectra. These data suggested the feasibility of these affinity tags in Streptomyces, which will be widely employed to explore the biochemical mechanisms to further understand the dynamic and elaborate regulation in this genus.

Atividade antimicrobiana de moléculas bioativas produzidas por Streptomyces parvulus DPUA 1573 frente a Staphylococcus spp. multirresistentes de mastite bubalina / Antimicrobial activity of bioactive molecules produced by Streptomyces parvulus DPUA 1573 against Staphylococcus spp. multi-drug resistant isolates from mastitis buffalo

A mastite é uma inflamação na glândula mamária que pode acarretar perdas na produção e na qualidade do leite, gerando prejuízos econômicos para a pecuária leiteira. O tratamento é baseado na utilização de antibióticos, sendo, em muitos casos, ineficazes devido à resistência bacteriana já conhecida para esta doença. O objetivo deste trabalho foi selecionar linhagens de Streptomyces spp. produtoras de biocompostos com atividade antimicrobiana frente a isolados do gênero Staphylococcus multirresistentes de búfalas com mastite. Bem como, determinar os melhores parâmetros de produção, e avaliar a produção simultânea de ácido clavulânico. A seleção de Streptomyces spp. com capacidade de produzir compostos com atividade antimicrobiana foi realizada através da técnica bloco de gelose. Dentre as 30 espécies de Streptomyces spp. testadas, o micro-organismo Streptomyces parvulus DPUA 1573 apresentou melhores resultados, sendo capaz de inibir o crescimento de 7 isolados Staphylococcus spp. multirresistentes. Posteriormente, a espécie selecionada Streptomyces parvulus DPUA 1573 foi cultivada em diferentes condições pré-determinadas pelo planejamento fatorial 24, onde as variáveis independentes foram: concentração de soja (0,5; 1,0; 1,5%), glicose (0; 0,5; 1g/L), agitação (150; 200; 250rpm) e temperatura (28; 32; 37°C) e todos os ensaios do planejamento foram monitorados até 120 horas de cultivo. Todas as variáveis independentes influenciaram positivamente no crescimento celular, enquanto que para atividade antimicrobiana apenas as variáveis temperatura e agitação apresentaram efeitos significativos positivos. O líquido metabólito produzido por Streptomyces parvulus DPUA 1573 foi capaz de inibir o crescimento de sete Staphylococcus spp. multirresistentes. As melhores condições de cultivo para a produção de moléculas bioativas por este micro-organismo foi a 37?C, com 250rpm de agitação por período de 72 horas. Nos ensaios que apresentaram atividade antimicrobiana, foi avaliada a produção de ácido clavulânico ao longo do cultivo. A maior concentração de ácido clavulânico foi de 269,84g/L obtidas nas condições de 1,5% de farinha de soja em ausência de glicose no tempo de 96 horas. A linhagem Streptomyces parvulus DPUA 1573 foi eficiente contra Staphylococcus spp. multirresistentes isolados de mastite em búfalas, ainda apresentando concomitantemente produção de ácido clavulânico com o potencial uso farmacêutico.(AU)

Mastitis is an inflammation in one or more mammary glands which can lead to reduction in production and quality of milk causing economic losses to dairy farming. The use of antibiotics is the key for the treatment of this disease, but in many cases ineffective due to bacterial resistance already known for this condition. The aim of this study was to select strains of Streptomyces spp. producing biomolecules with antimicrobial activity against multidrug-resistant Staphylococcus isolated from buffaloes with mastitis, as well as to determine the best production parameters to the evaluation of simultaneous production of clavulanic acid. Thirty species of Streptomyces spp. were used to selecting the greatest producer spectrum of antimicrobial activity (agar block technique), with selection of Streptomyces parvulus DPUA 1573, and 7 multidrug-resistant Staphylococcus spp. sensitive to its biocompounds. The selected strain of Streptomyces parvulus DPUA 1573 was cultured in different conditions predetermined by the factorial design 24, where the independent variables were: soybean concentration (0.5, 1.0, 1.5%), glucose (0, 0.5, 1g/L), agitation (150, 200, 250rpm) and temperature (28, 32, 37°C); all the tests were monitored up to 120 hours of cultivation. All independent variables influenced positively the cell growth, while for antimicrobial activity only the variables temperature and agitation showed positive effects. The antimicrobial bio compounds showed activity against seven multidrug-resistant Staphylococcus spp under the conditions: temperature 37°C, agitation 250rpm, with 72 hours of production process. In the tests which showed antimicrobial activity, was also assessed the production of clavulanic acid along with the cultivation. The highest concentration of clavulanic acid was 269.84g/L obtained under the conditions of 1.5% of soybean flour and absence of glucose in 96 hours. The strain Streptomyces parvulus DPUA 1573 was effective against multidrug-resistant strains of Staphylococcus spp. of mastitis from buffaloes, still showing concomitantly production of clavulanic acid for pharmaceutical use.(AU)

[Hypersensitivity pneumonitis related to Penicillium chrysogenum and mesophilic Streptomyces: the usefulness of the Medical Indoor Environment Councelor (MIEC)]. / Pneumopathie d'hypersensibilité à Penicillium chrysogenum et Streptomyces mésophile: intérêt de l'enquête environnementale à domicile.

Hypersensitivity pneumonitis (HP) occurred after organic antigens inhalation at home is rare and the diagnosis is very often difficult. We report the case of a 55-year male patient with allergic asthma since childhood, well controlled with inhaled corticosteroids, twice hospitalized for respiratory distresses. The patient presented fever (39°C), dry cough, rapidly progressive dyspnea, chest pain and crackles. Blood gas analysis found a hypoxemia of 52 mmHg, and CT-scan showed ground glass images in the upper lobes. Respiratory function tests showed severe obstructive syndrome and a decrease of diffusion test. HP was suspected because the symptoms were triggered by domestic environmental. The Medical Indoor Environment Councelor (MIEC) visited the patient's house and camper and performed air and dust samples. Moldy walnuts were found in the camper. The identification of microorganisms present in the air and on the surfaces in the camper was used for serum precipitins research by double diffusion (DD) and electrosyneresis (E) methods. From the 14 antigens tested, serological tests were considered significant for mesophilic Streptomyces (five arcs DD, six arcs E) and Penicillium chrysogenum (one arc DD, four arcs E). After removal from the camper of the objects suspected to be contaminated, the patient's symptoms regressed. This is a typical case of domestic HP to mesophilic Streptomyces and P. chrysogenum. The MIEC's intervention was useful in both diagnosis and treatment.

Modulation of LPS induced inflammatory response by Lawsonyl monocyclic terpene from the marine derived Streptomyces sp.

In continuing research for compounds with immunosuppressive activity, Lawsonone (1), a novel Lawsonyl derivative isolated from marine-derived bacteria Streptomyces sp. was evaluated for its potent immunosuppressive activity on immune system. The effect of Lawsonone (1) was elucidated on the immune cells (splenocytes and macrophages) collected from BALB/c mice. Study was carried out to find the effect of Lawsonone (1) on Con-A and LPS stimulated splenocyte proliferation, LPS-induced NO, IL-1ß, IL-6 and TNF-α production in macrophages. Furthermore, the effect of Lawsonone (1) on T-cell subsets (CD4 and CD8) and total B-cell (CD19) population was analyzed by flow cytometry. The results obtained in the present study showed that Lawsonone (1) inhibited the proliferation of both T and B splenocytes. It inhibited the nitric oxide (NO) and pro-inflammatory cytokine (IL-1ß, IL-6 and TNF-α) production in LPS-stimulated macrophages in a dose-dependent manner. Moreover, flow cytometric analysis indicated the prominent inhibition of CD4, CD8 and CD19 cell populations in the spleen of mice treated with the variable doses of Lawsonone (1), with the maximum inhibition at the lowest dose (0.1µM). Taken together, the present results suggest that Lawsonone (1) may act as a potent molecule for immunosuppression and anti-inflammation, supporting its immunopharmacologic application to modify the immune system.

[cDNA library constructing and specific antigen expression of Streptomyces thermohydroscopicus].

OBJECTIVE: To construct a cDNA library from Streptomyces thermohydroscopicus and screen genes with virulence, obtain the recombinant fusion virulence proteins by prokaryotic expression system. METHODS: The Streptomyces thermohydroscopicus cDNA library was constructed by switching mechanism at 5'end of RNA transcript approach. A total of 1020 clones randomly selected from the cDNA library were sequenced and these expressed sequence tags (EST) were further analyzed for the screen of antigen-specific genes. The two candidate genes were subcloned into expression vector pET-28a. The recombinants were transformed into BL2 and proteins were expressed by the induction of isopropyl-ß-D-1-thiogalactopyranoside (IPTG). RESULTS: A high-quality cDNA library from Streptomyces thermohydroscopicus was constructed and a set of 978 valid sequences were obtained. Clustering and assembly of these cDNA sequences resulted in 347 unique genes, among which 2 potential antigen-specific genes were highly allied with outer membrane lipoprotein (51%) and transferring-binding protein B (42%) from Actinobacillus pleuropneumoniae serotype (APP). The open reading frame (ORF) of the two candidate genes are 1554 bp and 726 bp, which coded two peptides with 517 and 241 amino acids, respectively. The molecular weights of the recombinant fusion proteins were 63 000 and 30 000. CONCLUSION: The cDNA library of Streptomyces thermohydroscopicus reached the quality requirement of gene library. EST database in the library would greatly facilitate further screening of virulence genes.

Expression of a versatile DC-targeting fusion protein using an Adenovirus expression system.

The importance of viral and tumour vaccines in eliciting elicit strong CD8+ T-cell responses has been widely acknowledged. Strategies exploring ways to enhance CD8+ T-cell responses have been developed, including targeting of vaccine antigens to dendritic cell (DC) receptors to access to the cross presentation pathway. Many DC endocytic receptors could potentially lead to augmented CD8+ T-cell responses if antigens were targeted directly to them, however only a few receptors have been explored because current targeting reagents are limited in the number of receptors that they are able to target. Consequently, this study describes the production and purification of a streptavidin-fusion protein that provides a versatile and efficient means to target antigen to more than one DC receptor. A model antigen gene, CMV pp65, and a streptavidin core gene, were spliced together using an overlap-extension PCR technique. The resulting fusion gene was cloned into a vector allowing expression in an Adenovirus-based expression system. Expression was verified and optimised before Ni-NTA affinity chromatography purification. Evaluation of pp65-streptavidin immunogenicity revealed that it elicits similar levels of CD8+ T-cell proliferative responses as pp65 and is able to effectively target specific DC receptors when used in addition to biotinylated receptor-specific antibodies. Additionally, enhancement of CD8+ T-cell responses was shown after directing pp65-strep to selected DC receptors in preliminary in vitro experiments. Collectively, this highlights the ease of production of a streptavidin-fusion protein, and demonstrates its use as a promising strategy to evaluate numerous DC receptors as potential targets in vaccine strategies.

Phosphatidic acid is a leukocyte chemoattractant that acts through S6 kinase signaling.

Phosphatidic acid (PA) is a pleiotropic lipid second messenger in mammalian cells. We report here that extracellular PA acts as a leukocyte chemoattractant, as membrane-soluble dioleoyl-PA (DOPA) elicits actin polymerization and chemotaxis of human neutrophils and differentiated proleukemic HL-60 cells. We show that the mechanism for this involves the S6 kinase (S6K) signaling enzyme. Chemotaxis was inhibited >90% by the S6K inhibitors rapamycin and bisindolylmaleimide and by S6K1 silencing using double-stranded RNA. However, it was only moderately ( approximately 30%) inhibited by mTOR siRNA, indicating the presence of an mTOR-independent mechanism for S6K. Exogenous PA led to robust time- and dose-dependent increases in S6K enzymatic activity and Thr(421)/Ser(424) phosphorylation, further supporting a PA/S6K connection. We also investigated whether intracellular PA production affects cell migration. Overexpression of phospholipase D2 (PLD2) and, to a lesser extent, PLD1, resulted in elevation of both S6K activity and chemokinesis, whereas PLD silencing was inhibitory. Because the lipase-inactive PLD2 mutants K444R and K758R neither activated S6K nor induced chemotaxis, intracellular PA is needed for this form of cell migration. Lastly, we demonstrated a connection between extracellular and intracellular PA. Using an enhanced green fluorescent protein-derived PA sensor (pEGFP-Spo20PABD), we showed that exogenous PA or PA generated in situ by bacterial (Streptomyces chromofuscus) PLD enters the cell and accumulates in vesicle-like cytoplasmic structures. In summary, we report the discovery of PA as a leukocyte chemoattractant via cell entry and activation of S6K to mediate the cytoskeletal actin polymerization and leukocyte chemotaxis required for the immune function of these cells.

Co-cultivated damp building related microbes Streptomyces californicus and Stachybotrys chartarum induce immunotoxic and genotoxic responses via oxidative stress.

Oxidative stress has been proposed to be one mechanism behind the adverse health outcomes associated with living in a damp indoor environment. In the present study, the capability of damp building-related microbes Streptomyces californicus and Stachybotrys chartarum to induce oxidative stress was evaluated in vitro. In addition, the role of oxidative stress in provoking the detected cytotoxic, genotoxic, and inflammatory responses was studied by inhibiting the production of reactive oxygen species (ROS) using N-acetyl-l-cysteine (NAC). RAW264.7 macrophages were exposed in a dose- and time-dependent manner to the spores of co-cultivated S. californicus and S. chartarum, to their separately cultivated spore-mixture, or to the spores of these microbes alone. The intracellular peroxide production and cytotoxicity were measured by flow cytometric analysis, nitric oxide production was analyzed by the Griess method, DNA damage was determined by the comet assay, and cytokine production was measured by an immunochemical ELISA (enzyme-linked immunosorbent assay). All the studied microbial exposures triggered oxidative stress and subsequent cellular damage in RAW264.7 macrophages. The ROS scavenger, NAC, prevented growth arrest, apoptosis, DNA damage, and cytokine production induced by the co-culture since it reduced the intracellular level of ROS within macrophages. In contrast, the DNA damage and cell cycle arrest induced by the spores of S. californicus alone could not be prevented by NAC. Bioaerosol-induced oxidative stress in macrophages may be an important mechanism behind the frequent respiratory symptoms and diseases suffered by residents of moisture damaged buildings. Furthermore, microbial interactions during co-cultivation stimulate the production of highly toxic compound(s) which may significantly increase oxidative damage.

[Immunological activity of Streptomyces polysaccharide in normal and immunosuppressed mice].

OBJECTIVE: To investigate the immunological activity of Streptomyces polysaccharide (SMP) on normal and immunosuppressed mice. METHODS: The effect of SMP on the proliferating activity of normal mouse splenocytes was tested in the mixed lymphocyte culture, and the changes of peripheral blood T lymphocytes were evaluated with acid a-naphthyl acetate esterase (ANAE) method. The ratio of Lyt2+ and L3T4+ T cell subsets was measured by flow cytometry. RESULTS: SMP stimulated obvious proliferation of mixed lymphocytes, showed protective effects on T lymphocyte and increased the ratio of Lyt2+ and L3T4+ cell subsets to nearly normal level in immunosuppressed mice. CONCLUSIONS: SMP can regulate the immune function in mice.

Influence of a Streptomyces lividans SecG functional analogue on protein secretion

The membrane protein complex translocase mediates the translocation of bacterial proteins. In this complex, the SecY, SecE, and SecG proteins constitute an integral membrane domain. Sequence comparison revealed a potential secG-like gene in the gram-positive soil bacterium Streptomyces lividans. Chromosomal deletion of this gene resulted in a sporulation defect and an overall deficiency in secretion. The SecG-depleted strain was able to overproduce and secrete alpha-amylase, but the appearance of the oversynthesized protein outside the cell was delayed compared to the protein produced by the wildtype strain. SecG deficiency was found to result in more pronounced effects in S. lividans than in Bacillus subtilis or Escherichia coli (AU)

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Endophytic actinobacteria induce defense pathways in Arabidopsis thaliana.

Endophytic actinobacteria, isolated from healthy wheat tissue, which are capable of suppressing a number wheat fungal pathogens both in vitro and in planta, were investigated for the ability to activate key genes in the systemic acquired resistance (SAR) or the jasmonate/ethylene (JA/ET) pathways in Arabidopsis thaliana. Inoculation of A. thaliana (Col-0) with selected endophytic strains induced a low level of SAR and JA/ET gene expression, measured using quantitative polymerase chain reaction. Upon pathogen challenge, endophyte-treated plants demonstrated a higher abundance of defense gene expression compared with the non-endophyte-treated controls. Resistance to the bacterial pathogen Erwinia carotovora subsp. carotovora required the JA/ET pathway. On the other hand, resistance to the fungal pathogen Fusarium oxysporum involved primarily the SAR pathway. The endophytic actinobacteria appear to be able to "prime" both the SAR and JA/ET pathways, upregulating genes in either pathway depending on the infecting pathogen. Culture filtrates of the endophytic actinobacteria were investigated for the ability to also activate defense pathways. The culture filtrate of Micromonospora sp. strain EN43 grown in a minimal medium resulted in the induction of the SAR pathway; however, when grown in a complex medium, the JA/ET pathway was activated. Further analysis using Streptomyces sp. strain EN27 and defense-compromised mutants of A. thaliana indicated that resistance to E. carotovora subsp. carotovora occurred via an NPR1-independent pathway and required salicylic acid whereas the JA/ET signaling molecules were not essential. In contrast, resistance to F. oxysporum mediated by Streptomyces sp. strain EN27 occurred via an NPR1-dependent pathway but also required salicylic acid and was JA/ET independent.

Invasive Streptomyces infections: six cases and literature review.

Streptomyces are saprophytic soil organisms rarely known to cause invasive infections other than mycetoma. We report 6 cases of invasive Streptomyces infections and review 13 previously reported cases. Our series included 2 cases of lung abscess or pneumonitis, 3 cases of central venous catheter-related bloodstream infection, and I case of possible hypersensitivity pneumonitis. Most previous cases also included lung infections and bloodstream infections. Preexisting conditions, such as cancer, AIDS or HIV infection, presence of a central venous catheter, and prosthetic heart valve, were present in all cases since 1985. Diverse Streptomyces species were involved, consistent with the highly opportunistic nature of the infections. Clinical management depended on the clinical situation of individual cases without consensus. Available susceptibility data showed that Streptomyces organisms were consistently susceptible to amikacin; frequently susceptible to imipenem, clarithromycin or erythromycin, minocycline, and trimethoprim-sulfamethoxazole; and infrequently susceptible to ciprofloxacin and ampicillin. The diagnosis of Streptomyces infection required microbiologic and pathologic correlation to rule out contamination.

Mold-specific immunoglobulin G antibodies in a child population.

The determination of immunoglobulin G (IgG) antibodies to molds has been used as an objective evidence of significant mold exposure. Until present, no data have been published on antibody responses to molds in healthy children living in normal housing conditions. The microbe-specific IgG antibody concentrations of 21 molds and 3 actinobacteria were determined by enzyme-linked immunosorbent assay (ELISA) in 103 1- to 6-year-old children (12.4% of the population of that age), and in 111 7- to 14-year-old school children (12.1%). The international standard sera were available, and the IgG concentrations of the test sera could be expressed in mg/l. On average, IgG concentrations increased in relation to age until the age of 6-7 years. At school age the increase still continued but more slowly. Actinobacteria were the only exceptions; all three tested strains Sreptomyces albus, S. griseus and S. halstedii resulted in rather high concentrations until 3 years of age. If the children lived in a farm, mold-specific IgG concentrations increased at an earlier age than in other children. The results between farmers' children and other children differed significantly before school age for 20 of the 24 microbes tested, the four exceptions being the 3 actinobacteria and the mold Aspergillus versicolor. The reference values must be age related, and separate references are needed for farmers' children before school age.

A cross-reactive polyol-responsive monoclonal antibody useful for isolation of core RNA polymerase from many bacterial species.

The use of antibodies for protein purification is a powerful technique but the release of the target protein in its active form is often difficult. So called "polyol-responsive" monoclonal antibodies (PR-MAbs) have a feature that allows elution of the antigen under very gentle conditions, so that even multi-subunit proteins can be released in their active form. In this work a PR-MAb, 8RB13, was isolated that can purify RNA polymerase (RNAP) from many different bacterial species. High specificity towards RNAP with a broad species cross-reactivity was achieved by immunization with RNAP from Escherichia coli and screening with Bacillus subtilis RNA polymerase. The isolated MAb could detect the beta-subunit of RNA polymerase from 10 out of 12 species tested on a Western blot indicating its potential for purification of core RNAP from these organisms. Representatively, four of these species E. coli, B. subtilis, Pseudomonas aeruginosa, and Streptomyces coelicolor were subjected to immunoaffinity purification yielding RNA polymerases that were active in in vitro transcription and seemed to be primarily core polymerase, lacking sigma-subunits.

Immunoglobulin G antibodies against indoor dampness-related microbes and adult-onset asthma: a population-based incident case-control study.

Immunoglobulin G (IgG) antibodies against microbes related to indoor dampness problems have been used as potential biomarkers of fungal exposure in clinical investigations. There is limited information on their relation to asthma. We conducted a population-based incident case-control study to assess the risk of asthma in relation to specific IgG antibodies to eight dampness-related microbes: Aspergillus fumigatus, A. versicolor, Cladosporium cladosporioides, Fusarium oxysporum, Sporobolomyces salmonicolor, Stachybotrys chartarum, Streptomyces albus and Trichoderma citrinoviride. We recruited systematically all new cases of asthma during a 2.5-year study period and randomly selected controls from a source population of adults 21-63 years of age living in the Pirkanmaa Hospital District, South Finland. The clinically diagnosed case series consisted of 521 adults with newly diagnosed asthma and the control series of 932 controls selected randomly from the source population. IgG antibodies were analysed with ELISA. An increased risk of developing asthma in adulthood was significantly related to IgG antibodies to T. citrinoviride, but not to the other moulds. There was no evidence of a dose-response relation between the IgG antibody level and the risk of asthma. T. citrinoviride may play a role in the aetiology of adult-onset asthma or serve as an indicator of other causal factors.