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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 Ca 2+ with EGTA or membrane protection by glycine treatment. Ca 2+ 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 Ca 2+ -dependent signals and gasdermin D pore formation are required for robust IL-33 production., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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., Competing Interests: The authors have declared that no competing interests exist.

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, tun p1 and tun p2. 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., (Copyright © 2018 Widdick et al.)

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.

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.

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., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

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-1β, 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-1β, 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., (Copyright © 2013. Published by Elsevier B.V.)

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., (Copyright © 2012 Elsevier Inc. All rights reserved.)

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-β-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.

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.

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.

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.

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.

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.

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.

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 (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.

An immunoreactive role of lactic acid bacteria established in animals has seldom been investigated in humans. In a large-scale clinical study, children from day-care centers received either yoghurt (Y), milk fermented by yoghurt symbiosis and Lactobacillus casei (DN 114 001) (YC), or gelified milk (GM) as diet supplements during two 30-day supplementation periods separated by one 30-day period without supplementation. Feces samples were collected before, during, and after the 2nd supplementation period. Proteins were extracted in a buffer containing enzymatic inhibitors. IgA levels were assessed and adjusted to the weight of feces samples. Specific IgA to lactic acid bacteria strains (Streptococcus thermophilus 8901A, 8902A; Lactobacillus bulgaricus; Lactobacillus casei) present in Y and YC were assayed in ELISA and adjusted to individual IgA levels. Mean levels of fecal IgA were within reported ranges for pediatric populations of similar age. IgA levels decreased significantly but transiently in children receiving Y, and increased significantly in children receiving GM, but did not vary in the group of children who were given YC. Specific IgA to the 4 strains tested increased significantly during the supplementation period only in the group of children receiving GM, while it was transient and not significant in children receiving YC. No variation was noted in children given Y Specific IgA to lactic acid bacteria can be assayed in feces. Supplementation with fermented milks might induce a mucosal tolerance to environmental flora.

The immune responses in actinomycetoma lesions caused by Streptomyces somaliensis in Sudan were characterized by immunohistochemistry during 1997-1998. In sections stained with haematoxylin and eosin, the inflammatory reaction around the grain was of 2 types. In type I there were 3 zones: a neutrophil zone immediately around the grain, an intermediate zone containing mainly macrophages, and a peripheral zone consisting of lymphocytes and plasma cells. Zone 1 stained positively for CD15 (neutrophils), zone 2 for CD68 (macrophages) and CD3 (T lymphocytes), and zone 3 for CD20 (B lymphocytes). In the type II reaction, there was no neutrophil zone, the grains being surrounded only by macrophages and giant cells. This was confirmed by immunohistochemistry, which demonstrated the presence of CD3 positive cells. Immunoglobulins G and M and complement were demonstrated on the surface of the grain and on filaments inside the grain. Neutrophils and macrophages were recruited into the lesion by complement and were involved in the fragmentation of the grain. The cytokine profile in the lesion and regional lymph nodes was of a dominant Th2 pattern (interleukins-10 and 4).

Bleomycin-producing Streptomyces verticillus ATCC 15003 has two bleomycin resistance genes, designated blmA and blmB. Bleomycin N-acetyltransferase, encoded by blmB, was overproduced in Escherichia coli as a protein fused to the maltose-binding protein. The protein (fBAT), purified to homogeneity after digestion of the fusion product with blood coagulation factor Xa protease, had an additional 6 N-terminal amino acid residues, but retained its bleomycin-acetylating activity, as did the entire fusion protein. The K(m) and Vmax values of purified fBAT for the substrate bleomycin were 13.0 microM and 3.4 nmol [corrected] min-1 ml-1, respectively. The optimal pH for the acetylating activity was 6.0 in 10 mM phosphate buffer. The molecular mass and pI value of fBAT were estimated by polyacrylamide gel electrophoresis to be about 34500 and 6.13, respectively. An anti-fBAT monoclonal antibody was generated and used to show that bleomycin N-acetyltransferase is expressed simultaneously with bleomycin production in S. verticillus.

Serum samples obtained from 25 patients of mycetoma caused by Madurella mycetomi in 18, Streptomyces somaliensis in 5, Streptomyces madurae and streptomyces palletierii from 1 each and also from 25 healthy controls were evaluated for immunoglobulins IgG, IgM and IgA by radial immunodiffusion method using tripartigen plates. All the three classes of immunoglobulins showed increased concentration in cases of mycetoma by M. mycetomi and S. pelletierii. The raised levels of IgG and IgM in cases of mycetoma caused by S. madurae and S. somaliensis where bones were involved, were not statistically significant. IgA was consistently high in all cases of mycetoma irrespective of causative agent and tissue involved. These alterations may possibly reflect relatively poor IgG and IgM response in individuals with extension of mycetoma lesion to bones.

Changes in the levels of free intracellular calcium ([Ca2+]i) and the production of reactive oxygen metabolites (ROM) induced by opsonized indoor air fungi and bacteria in human polymorphonuclear leukocytes (PMNL) were measured. Moreover, modification of a chemotactic peptide (fMLP)-and a tumor promoter (PMA)-induced production of ROM by opsonized fungi and bacteria were studied. The cells were exposed to graded doses of opsonized Candida sp., Aspergillus sp., Cladosporium sp., Stachybotrys sp., Penicillium sp., Paecilomyces sp., or A4 or A91 Streptomyces sp. alone, or together with fMLP or PMA. All the organisms were isolated from air samples of mold-problem buildings. None of the fungi or bacteria induced changes in [Ca2+]i or the production of ROM without opsonization with human serum. Of all opsonized fungi and bacteria, only Candida sp. elevated [Ca2+]i. All fungi and bacteria, except Paecilomyces sp. and Stachybotrys sp., markedly increased the production of ROM in PMNL. Furthermore, A91 Streptomyces sp. and Aspergillus sp. amplified fMLP-induced production of ROM. Only Candida sp. increased PMA-induced phenomen that normally occurs in the lung, was required for biological activity of the fungi and bacteria. Amplification by opsonization of fungi- or bacteria-induced leukocyte activation revealed remarkable changes between these biologically active particles. The present results suggest that many indoor air fungi and bacteria may activate leukocytes to produce oxidative stress, perhaps associated with harmful effects in exposed individuals.

The crystal structure of a novel esterase from Streptomyces scabies, a causal agent of the potato scab disease, was solved at 2.1 A resolution. The tertiary fold of the enzyme is substantially different from that of the alpha/beta hydrolase family and unique among all known hydrolases. The active site contains a dyad of Ser 14 and His 283, closely resembling two of the three components of typical Ser-His-Asp(Glu) triads from other serine hydrolases. Proper orientation of the active site imidazol is maintained by a hydrogen bond between the N delta-H group and a main chain oxygen. Thus, the enzyme constitutes the first known natural variation of the chymotrypsin-like triad in which a carboxylic acid is replaced by a neutral hydrogen-bond acceptor.

Monoclonal antibodies were produced to Streptomyces lividans spore surface antigens. One particular hybridoma cell line, 43H6, produced a monoclonal antibody that reacted exclusively with Streptomyces cluster group 21 in an enzyme-linked immunosorbent assay (ELISA). Antibody 43H6 was found to be of subclass IgG1, kappa light chain. Western blot (immunoblot) analysis revealed that 43H6 recognized a major outer spore polypeptide of about 37,000 Da. The epitope was stably maintained in S. lividans spores over at least seven sporulation cycles on laboratory medium and for at least 14 weeks in sterile soil systems. The species group specificity of antibody 43H6 was exploited in the development of an immunocapture technique for the isolation of streptomycetes from soil. Magnetic beads coated with antibody 43H6 were mixed with soil samples seeded with S. lividans spores. Spore-bead complexes were recovered using magnets. Treatment of beads with blocking agents and the inclusion of detergents in the recovery system lessened non-specific binding of spores to beads and improved recovery. In buffer solutions decreasing the spore concentration increased the recovery values for a fixed bead concentration. At a spore concentration of 5 x 10(7) ml-1 the recovery was 4.3% whilst at 5 x 10(2) ml-1 it was 76% for a fixed bead concentration of 0.6 mg ml-1. Using a bead concentration of 2 mg per 10 g soil, approximately 30% of the target spore population of 10(6) c.f.u. was recovered from sterile soil and 4% from non-sterile soil. This method offers a rapid means of selectively recovering and concentrating Streptomyces spores from soil samples.

We attempted to screen a series of Streptomyces subtilisin inhibitor-like (SIL) proteins among several Streptomyces strains by using a highly sensitive assay system established by us. Of six randomly tested strains, four were found to produce SIL inhibitors as their major secreted proteins, suggesting that they might be distributed in a high frequency among this genus. Three inhibitors exhibited inhibition of both subtilisin BPN' and trypsin. Comparison of the amino terminal sequences of these isolated proteins with those of other reported SIL inhibitors revealed that the beta 1- and beta 2-sheets in SSI were highly conserved.

Two endo-beta-N-acetylglucosaminidases (PI and PII) have been isolated from the culture fluid of Pseudomonas sp. The substrate specificity of the PI enzyme was very similar to that of Endo-H from Streptomyces plicatus. On the contrary, the PII enzyme had a novel substrate specificity that degraded both high-mannose type and hybrid type oligosaccharides derived from ovalbumin, and the core structure of complex type oligosaccharides derived from human transferrin and porcine pancreatic lipase.

Rabbit antisera and monoclonal antibodies were raised against factor C, a regulatory protein of Streptomyces griseus. ELISA and immunoblotting techniques suitable to determine and characterize factor C antigen was detected in all the 23 Streptomyces strains and variants examined thus far and in one Bacillus subtilis too. Depending on the strain analysed it has a molecular mass of 34,000 or 70,000 in mycelial homogenates. Most of factor C was found excreted into the cultivation medium. The quantity of factor C antigen in different Streptomyces strains showed great variation. Amy+ strains were usually good producers of factor C while Amy- were not. This was consistent with our assumption that factor C was an inducer of reproductive phase in Streptomyces.

Antigenic extracts were prepared from culture filtrates of the principal agents known to cause actinomycetoma, namely Actinomadura madurae, Actinomadura pelletieri, Nocardia asteroides, Nocardia brasiliensis, Nocardia otitidis-caviarum, and Streptomyces somaliensis. These antigenic preparations were compared by immunodiffusion (ID), counterimmunoelectrophoresis (CIE), line immunoelectrophoresis (LIE) and rocket line immunoelectrophoresis (RLIE), with rabbit antisera prepared against each of the extracts. Cross-reactivity between antigenic extracts from the different actinomycetes, measured by determining the number of precipitin lines in homologous and heterologous systems, was common. Reactions were always stronger and more precipitin lines were present when antigenic extracts were tested against homologous antisera. Similarities between A. madurae and A. pelletieri antigens were of a low order and cross-reactivity did not exceed 33%. A. pelletieri resembled N. asteroides more closely than A. madurae, with 44% of detectable antigenic components cross-reacting. The three species of Nocardia had common antigenic epitopes, but the overall degree of similarity was of a low order (between 12 and 27% by LIE and RLIE). Antigenic extracts of S. somaliensis had few components in common with the other species tested and only one of the 34 lines present in the RLIE system for N. asteroides showed any reaction of identity with an antigenic component in the S. somaliensis extract. Single cross-reacting lines were also present in the CIE and LIE systems.

The immunological properties of ribonucleases from Act. rimosus were studied in comparison with RNA-ase from the cattle pancreas. The activity of anti-RNA-ase immune sera were determined by the method of specific neutralization of the effect of the above exzymes. The results of the study showed that with the use of homological RNA-ases as the antigens, the maximum level of enzymatic activity neutralization by the immune sera ranged within 70 to 90% and the capacity for induction of specific antibody production expressed in the serum titer was somewhat lower in the actinomycete RNA-ase than in the pancreatic one. When heterological antigens were used, neutralization of the RNA-ase effect by the immune sera was either not detectable or very low which is indicative of the antigenic differences in the actinomycete and pancreatic RNA-ases.

In autumn 1978 an epidemic of respiratory disease resembling allergic alveolitis occurred in a small Finnish community. The disease was caused by repeated exposures to tap water aerosol. The raw water of the community and the sand filters of the purification system were heavily contaminated with mesophilic actinomycetes. Fourteen different strains of actinomycetes were isolated. Exposed persons with and without symptoms as well as unexposed control persons were tested for antibodies against five of these actinomycetes and against Enterobacter agglomerans. Both the exposed and the control persons had antibodies to actinomycetes but the exposed persons had antibodies against more actinomycete strains than the control persons. Precipitating antibodies against E. agglomerans were also found in control persons as well as in patients. There was a significant difference between the patients and the exposed healthy persons in bacterial agglutination tests with flagellar antigen of one E. agglomerans strain. However, the role of mesophilic actinomycetes and E. agglomerans in the aetiology of the disease could not be firmly established.

Streptomyces thermohygroscopicus is a thermoactinomycetes which has been found recently to be related to farmer's lung in China. In the present study, a model of acute farmer's lung was successfully developed with extracellular proteinases of streptomyces thermohygroscopicus (EPST) in the rabbit, and the precipitating antibodies against EPST were detected in the sera from the patients with farmer's lung or bagassosis. Lack of cross reactivity was also showed between EPST, T. vulgaris antigen, and M. faeni antigen.

Mycelia from several strains of Streptomyces were potent B-lymphocyte mitogens for spleen cells from 3 inbred mouse strains (BALB/c, C3H/HeJ, C3H/nu/nu), inducing lymphocyte proliferation and differentiation into immunoglobulin-secreting cells. Streptomyces mycelia were also mitogenic towards human peripheral blood lymphocytes, where maximal stimulation was found on days 5 to 7. On the other hand, human lymphocytes incubated with the mycelia for 3 days, but not the supernatants of these cultures, strongly inhibited mixed leukocyte reactions. Irradiation experiments suggest the induction of both radioresistant and radiolabile suppressor-cell populations by the components.

Sera from 160 individuals resident in the Gezira, and 40 living in Khartoum, Sudan, were screened for antibodies to Streptomyces somaliensis and Actinomadura madurae using enzyme linked immunosorbent assay (ELISA). Antibodies were detected in 11.2% of those from the Gezira (6.8% to S. somaliensis, 4.3% to A. madurae, and 0.6% to both) compared to 2.5% of those resident in Khartoum. The ratio of men with antibodies compared to women was about 2:1 in those from Gezira. The results encouraged further epidemiological surveys are mycetoma in Sudan using ELISA.

A hemagglutinin has been purified 4000-fold from the culture filtrate of a strain of Streptomyces by affinity chromatography. The purified preparation was judged to be homogeneous by gel electrophoresis and its molecular weight was estimated to be about 70 000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. It may exhibit its full hemagglutinating activity in the monomer form. This hemagglutinin strongly agglutinated human blood group O erythrocytes and was inhibited by L-fucose. It was, however, distinct from the known L-fucose-specific hemagglutinins; first, the hemagglutinating activity of the purified preparation was more than 100-times stronger than that of others; second, D-mannose was a potent inhibitor of this hemagglutinin besides L-fucose but not or scarcely inhibitory to others; and third, p-nitrophenyl-beta-L-fucoside was more inhibitory to this hemagglutinin than p-nitrophenyl-alpha-L-fucoside as opposed to the case of others.

L-Fucose, D-mannose-specific lectin (SFL 100-2) particles produced by Streptomyces no. 100-2 were labeled with N-succinimidyl-[2,3-3H]propionate to investigate quantitatively their binding properties to human erythrocytes. The labeling did not influence the physical properties or the hemagglutinating activity of the lectin particles. The binding studies suggested that two kinds of receptor sites were present on the erythrocytes. Association constants (Ka's) of the lectin particles to the receptor sites and the numbers of the receptor sites (n) on human O erythrocytes were calculated to be 4.60 X 10(8) M-1 and 3.17 X 10(4)/cell for high-affinity receptor sites, and 7.5 X 10(7) M-1 and 1.33 X 10(5)/cell for low-affinity ones. The inhibition constants (Ki's) for L-fucose, p-nitrophenyl (PNP)-beta-L-fucoside, D-mannose, and PNP-alpha-D-mannoside were calculated to be 1.20 X 10(3), 1.82 X 10(3), 1.82 X 10(2), and 2.40 X 10(2) M-1, respectively. The numbers of carbohydrate-binding sites (m) on the lectin particles were estimated to be 2.82, 2.18, 2.19, and 2.21 for L-fucose, PNP-beta-L-fucoside, D-mannose, and PNP-alpha-D-mannoside, respectively, suggesting that SFL 100-2 has two carbohydrate-binding sites per particle.

From crude extracts of a Streptomyces strain exhibiting immunopotentiating effects, a tetrapeptide was isolated and its structure established as L Ala leads to D isoGlu leads to L, L Dap comes from Gly. This peptide was devoid of biological activity but its chemical coupling with lauric acid gave a substance endowed with adjuvant and immunostimulating properties. This substance and the corresponding synthetic lauroyltetrapeptide were as active in this respect as the muramyl-dipeptide, thus far considered as the minimal adjuvant-active structure of bacterial cell walls: the presence of a sugar moiety is therefore not a prerequisite for immunopotentiating activities.

Counterimmunoelectrophoresis used for the diagnosis of mycetoma was found superior to immunodiffusion (ID) especially when using neat serum and 1:2 dilution (P=less than 0.0001 with neat serum; and less than 0.01 with 1:2 dilution). It is recommended for routine use in mycology laboratories for following up patients on treatment and for confirming the diagnosis of sera that are weakly positive by ID.

Immunoprecipitation of polyribosomes from Streptomyces hydrogenans synthesizing 20beta-hydroxysteroid dehydrogenase was performed by immunoelectrophoresis. Only polyribosomes from cells induced by 11beta, 21-dihydroxy-4, 11(20)-pregnadien-3-one in vivo were precipitated by immunoglobulins specific for 20beta-hydroxysteroid dehydrogenase. No unspecific precipitations with polysomes from control cells cultivated in the absence of steroids were observed. Therefore, 11beta, 21-dihydroxy-4, 17(20)-pregnadien-3-one increases the number of biological active polyribosomes involved in 20beta-hydroxysteroid dehydrogenase synthesis, presumably by specific stimulation of mRNA synthesis in Streptomyces hydrogenans.

A monoclonal antibody (McAb) to Dermatophilus congolensis was produced from murine hybridoma cultures and purified by affinity chromatography. Species specificity was demonstrated using indirect immunofluorescent staining; the McAb was shown to react with 10 D congolensis isolates but not with 10 Nocardia species isolates, a Rhodococcus and a Streptomyces species isolate. The McAb was used to demonstrate D congolensis in clinical material from confirmed bovine and ovine cases and presumptive equine cases of dermatophilosis by indirect immunofluorescent staining.

Hypersensitivity pneumonitis is an immunologic lung disease known to be induced by the inhalation of either organic or inorganic antigens, and it may thus be associated with a variety of occupations and microorganisms. To identify an inciting allergen, environmental, bacteriologic, immunologic, and bronchial challenge studies must be employed. These techniques were used to demonstrate that a bacterium uniformly present in processed dirt, Streptomyces albus, was responsible for a biopsy-proven case of hypersensitivity pneumonitis.

The release of 3H 5-HT from murine mast cells is shown to be a simple reproducible method for studying the activation of such cells by various agents. 3H-serotonin was taken up by peritoneal cell suspensions in vitro and was released by antigen, Concanavalin A, Forssman antiserum, anti-mouse immunoglobulin, or a polypeptide antibiotic, Cinnamycin.

Streptomyces thermohygroscopicus (H9-4) is a new pathogenic strain of farmer's lung disease (FLD) which is wide spread in Hubei province. Experimental "farmer's lung" in mice C87 was induced with the metabolites of this strain. Rabbits also suffered from the same FLD with characteristic pathogenic changes after aspiration of dried powder of this organism. For analysis of antigen fractions, with diagnostic value from this crude antigen of H9-4 SDS-PAGE and immunoblotting technic was employed and sera of 82 patients suffering from FLD from several districts were tested with this technic and the results revealed IgG antibodies could recognize the antigen-peptides from Mw 73 Kd and 68 Kd which were main antigen-peptides or pathogenic antigen to the disease. Sera of patients with present illness could recognize the peptide antigen between 11-90 Kd. In addition, all serum samples from healthy controls and from patients suffering from other respiratory diseases gave negative results. Therefore we conclude that SDS-PAGE and immunoblotting technic can be used in the diagnosis of FLD.