Flexible enzymatic activation of artificial polyketide extender units by Streptomyces cinnamonensis into the monensin biosynthetic pathway.

Streptomyces cinnamonensis A495 is a variant of the monensin producer which instead of the native polyether antibiotic gives rise to antibiotic and anti-tumour shunt-product premonensin. Through the supplementation of the fermentation medium with suitable precursors, premonensin can be derivatized via the incorporation of new-to-nature extender units into the biosynthetic machinery. Polyketide extender units require activation, typically in form of coenzyme A-thioesters. These are membrane impermeable and thus in the past an artificial mimic was employed. Here, we show the use and preliminary characterization of a highly substrate promiscuous new enzyme for the endogenous thioester formation in a Streptomyces strain. These intracellularly activated alternative extender units are significantly better incorporated into premonensin than the synthetically activated counterparts. SIGNIFICANCE AND IMPACT OF THE STUDY: Polyketide natural products are of enormous relevance in medicine. The hit-rate in finding active compounds for the potential treatment of various diseases among this substance family of microbial origin is high. However, most polyketides require derivatization to render them suitable for the application. Of relevance in this field is the incorporation of artificial substances into the biogenesis of polyketides, hampered by both the microbial metabolism and the complexity of the enzymes involved. This manuscript describes the straightforward and selective biosynthetic incorporation of synthetic substances into a reduced polyketide and showcases a promising new enzyme to aid this purpose.

Quantification of cell surface proteins with bispecific antibodies.

Flow cytometry is an established method for fast and accurate quantitation of cellular protein levels and requires fluorescently labeled antibodies as well as calibration standards. A critical step for quantitation remains the production of suitable detection antibodies with a precisely defined ratio of antigen-binding sites to fluorophores. Problems often arise as a consequence of inefficient and unspecific labeling which can influence antibody properties. In addition, the number of incorporated fluorophores necessitates a special normalization step for quantitation. To address these problems, we constructed different mono- and bivalent bispecific antibodies with binding site(s) for the cell surface antigens, cMET, EGFR1/HER1, ErbB2/HER2 or ErbB3/HER3 and with an additional digoxigenin-binding single-chain Fv fusion. The fluorophore Cy5 was covalently coupled to digoxigenin and quantitatively bound by the bispecific antibody. A panel of tumor cell lines was assessed under different culture conditions for absolute receptor expression levels of the indicated antigens and the data were set in relation to mRNA, gene count and immunoblot data. We could reproducibly quantify these receptors, omit the otherwise required normalization step and demonstrate the superiority of a 1 + 1 bispecific antibody. The same antibodies were also used to quantify the number of proteins in intracellular vesicles in confocal microscopy. The antibodies can be stored like regular antibodies and can be coupled with different digoxigenin-labeled fluorophores which makes them excellent tools for FACS and imaging-based experiments.

Antibodies induced with recombinant VP1 from human rhinovirus exhibit cross-neutralisation.

Human rhinoviruses (HRVs) are the major cause of the common cold and account for 30-50% of all acute respiratory illnesses. Although HRV infections are usually harmless and invade only the upper respiratory tract, several studies demonstrate that HRV is involved in the exacerbation of asthma. VP1 is one of the surface-exposed proteins of the viral capsid that is important for the binding of rhinoviruses to the corresponding receptors on human cells. Here we investigated its potential usefulness for vaccination against the common cold. We expressed VP1 proteins from two distantly related HRV strains, HRV89 and HRV14, in Escherichia coli. Mice and rabbits were immunised with the purified recombinant proteins. The induced antibodies reacted with natural VP1 and with whole virus particles as shown by immunoblotting and immunogold electron microscopy. They exhibited strong cross-neutralising activity for different HRV strains. Therefore, recombinant VP1 may be considered a candidate HRV vaccine to prevent HRV-induced asthma exacerbations.

Characterization of Der p 21, a new important allergen derived from the gut of house dust mites.

BACKGROUND: The house dust mite (HDM) Dermatophagoides pteronyssinus is a major allergen source eliciting allergic asthma. The aim of the study was to identify new important HDM allergens associated with allergic asthma. METHODS: A cDNA coding for a new mite allergen, designated Der p 21, was isolated using immunoglobulin E (IgE) antibodies from patients with allergic asthma out of a D. pteronyssinus expression cDNA library and expressed in Escherichia coli. RESULTS: Circular dichroism analysis of the purified allergen showed that rDer p 21 (14 726 Da) is one of the few mite allergens with an alpha-helical secondary structure. The protein exhibited high thermal stability and refolding capacity, and, as determined by small angle X-ray scattering, formed a dimer consisting of two flat triangles. rDer p 21 bound high levels of patients' IgE antibodies and showed high allergenic activity in basophil activation experiments. Rabbit anti-Der p 21 IgG antibodies inhibited mite-allergic patients' IgE binding and allowed the ultrastructural localization of the allergen in the midgut (epithelium, lumen and faeces) of D. pteronyssinus by immunogold electron microscopy. Der p 21 revealed sequence homology with group 5 mite allergens, but IgE and IgG reactivity data and cross-inhibition studies identified it as a new mite allergen. CONCLUSIONS: Der p 21 is a new important mite allergen which is liberated into the environment via faecal particles and hence may be associated with allergic asthma.

Relative predispositional effects of HLA class II DRB1-DQB1 haplotypes and genotypes on type 1 diabetes: a meta-analysis.

The direct involvement of the human leukocyte antigen class II DR-DQ genes in type 1 diabetes (T1D) is well established, and these genes display a complex hierarchy of risk effects at the genotype and haplotype levels. We investigated, using data from 38 studies, whether the DR-DQ haplotypes and genotypes show the same relative predispositional effects across populations and ethnic groups. Significant differences in risk within a population were considered, as well as comparisons across populations using the patient/control (P/C) ratio. Within a population, the ratio of the P/C ratios for two different genotypes or haplotypes is a function only of the absolute penetrance values, allowing ranking of risk effects. Categories of consistent predisposing, intermediate ('neutral'), and protective haplotypes were identified and found to correlate with disease prevalence and the marked ethnic differences in DRB1-DQB1 frequencies. Specific effects were identified, for example for predisposing haplotypes, there was a statistically significant and consistent hierarchy for DR4 DQB1*0302s: DRB1*0405 =*0401 =*0402 > *0404 > *0403, with DRB1*0301 DQB1*0200 (DR3) being significantly less predisposing than DRB1*0402 and more than DRB1*0404. The predisposing DRB1*0401 DQB1*0302 haplotype was relatively increased compared with the protective haplotype DRB1*0401 DQB1*0301 in heterozygotes with DR3 compared with heterozygotes with DRB1*0101 DQB1*0501 (DR1). Our results show that meta-analyses and use of the P/C ratio and rankings thereof can be valuable in determining T1D risk factors at the haplotype and amino acid residue levels.

High oral prevalence of Candida krusei in leprosy patients in northern Thailand

Although Candida albicans is the most common human yeast pathogen, other Candida species such as C. krusei are now recognized as emerging agents, especially in patients with human immunodeficiency virus (HIV) disease. C. krusei is inherently resistant to the widely used triazole antifungal fluconazole and poses therapeutic problems, especially in systemic candidiasis. In a surveillance study of leprosy patients (with arrested or burnt-out disease) in a leprosarium in northern Thailand, we found a rate of oral carriage of C. krusei (36 per cent) significantly (P smaller 0.05) higher than that for a healthy control group (10 per cent). Among the Candida-positive patients, 16 of 35 (46 per cent) carried C. krusei, while C. albicans was the second most common isolate (12 of 35 patients; 34 per cent). The corresponding figures for the control group were 2 of 13 (15 per cent) and 6 of 13 (46 per cent), respectively. Studies of the antifungal resistance of the C. krusei isolates from patients indicated that all except one of the isolates were resistant to fluconazole, two isolates were resistant to ketoconazole, and all isolates were sensitive to amphotericin B. Evaluation of their genetic profiles by randomly amplified polymorphic DNA analysis with three different primers and subsequent analysis of the gel profiles by computerized cluster-derived dendrograms revealed that the C. krusei isolates from patients belonged to 10 disparate clusters, despite the origin from a single locale. These nascent findings indicate an alarmingly high prevalence of a Candida species resistant to a widely used antifungal in a part of the world where HIV disease is endemic.

High oral prevalence of Candida krusei in leprosy patients in northern Thailand.

Although Candida albicans is the most common human yeast pathogen, other Candida species such as C. krusei are now recognized as emerging agents, especially in patients with human immunodeficiency virus (HIV) disease. C. krusei is inherently resistant to the widely used triazole antifungal fluconazole and poses therapeutic problems, especially in systemic candidiasis. In a surveillance study of leprosy patients (with arrested or burnt-out disease) in a leprosarium in northern Thailand, we found a rate of oral carriage of C. krusei (36%) significantly (P < 0.05) higher than that for a healthy control group (10%). Among the Candida-positive patients, 16 of 35 (46%) carried C. krusei, while C. albicans was the second most common isolate (12 of 35 patients; 34%). The corresponding figures for the control group were 2 of 13 (15%) and 6 of 13 (46%), respectively. Studies of the antifungal resistance of the C. krusei isolates from patients indicated that all except one of the isolates were resistant to fluconazole, two isolates were resistant to ketoconazole, and all isolates were sensitive to amphotericin B. Evaluation of their genetic profiles by randomly amplified polymorphic DNA analysis with three different primers and subsequent analysis of the gel profiles by computerized cluster-derived dendrograms revealed that the C. krusei isolates from patients belonged to 10 disparate clusters, despite the origin from a single locale. These nascent findings indicate an alarmingly high prevalence of a Candida species resistant to a widely used antifungal in a part of the world where HIV disease is endemic.

Oral manifestations in 45 HIV-positive children from Northern Thailand.

Forty-five Northern Thai children with HIV infection or AIDS were examined for oral manifestations. Of these children, 51.1% (n=23) were asymptomatic (category N), 48.9% were mildly, moderately or severely symptomatic (category A, B, C) and 48.9% (n=22) revealed oral lesions. Eleven patients (24.4%) showed one oral lesion, eight (17.8%) had two and three (6.6%) had three oral lesions. Erythematous candidiasis was the most common lesion (17.8%). Oral hairy leukoplakia was seen in 6.7% (n=3). Geographic tongue, not usually considered to be associated with HIV infection, was seen in 6.7% (n=3). Only 15 patients (33.3%) received antiretroviral therapy (ART). Comparison of patients with or without ART did not show differences in the prevalence of oral lesions. More studies in Thai HIV-infected children are needed to reveal the prevalence of oral manifestations, as well as for the predictive value of the most common or specific oral manifestations.

Release of allergen-bearing cytoplasm from hydrated pollen: a mechanism common to a variety of grass (Poaceae) species revealed by electron microscopy.

BACKGROUND: The release of submicronic particles from grass pollen after rainfall was suggested to be responsible for outbreaks of grass pollen asthma. Recently, we provided evidence for the release of respirable allergen-bearing particles from hydrated ryegrass (Lolium perenne ) pollen as a possible explanation for this phenomenon. OBJECTIVE: We investigated whether water-induced release of respirable allergen-bearing particles could be a mechanism common to several members of the sweet grass family Poaceae (Gramineae). METHODS: Pollens from 6 different Poaceae species were hydrated in water and examined by means of scanning electron microscopy for release of cytoplasmic materials. Rabbit antisera raised against purified recombinant group 1 and 5 allergens were used for immunogold labeling of expelled materials by means of field emission scanning electron microscopy. In addition, group 1 and 5 allergens were immunogold-localized on ultrathin sections. RESULTS: Fresh Poaceae pollens expelled cytoplasmic materials containing group 1 and 5 allergens on hydration in water. Expulsion of submicronic particles strongly decreased after 1 month of storage. CONCLUSIONS: Our results suggest expulsion of cytoplasm after hydration as a mechanism common to pollens of important allergenic grasses. The water-induced release of respirable allergen-bearing particles from grass pollens might explain asthma attacks observed after rainfall during the grass pollen season.

Oral manifestations in HIV-positive adults from Northern Thailand.

Eighty-seven HIV-infected patients in a provincial hospital in Northern Thailand were examined for oral manifestations of HIV disease and AIDS. The median age was 31.3 years. Seventy-four of the patients were women, 13 were men. 96.6% had a history of heterosexual transmission. Sixty-one patients were CDC-category A, 20 were category B and 6 were category C (AIDS). Thirty-eight percent of the patients revealed oral lesions; 23% had one oral lesion and 13.8% had two oral lesions. Common lesions were oral candidiasis (10.3% pseudomembranous candidiasis, 6.9% erythematous candidiasis and 3.4% both forms), oral hairy leukoplakia (11.5%) and exfoliative cheilitis (6.9%). Gingival linear erythema was seen in 8% of the patients; periodontal lesions and necrotising ulcerative gingivitis were not observed. Men were more commonly affected by oral manifestations than women (P < 0.004). The spectrum of oral lesions is comparable to other studies from the region, although most of these reported more men than women. Also, the degree of immunosuppression was more marked (AIDS).

Impact of PAF antagonist BN 52021 (Ginkolide B) on post-ischemic graft function in clinical lung transplantation.

BACKGROUND: Platelet activating factor (PAF) is associated with ischemia/reperfusion injury (I/R) after lung transplantation. Following promising experimental results, this prospective trial investigated the potential effect of PAF antagonist BN 52021 (ginkolide B) on clinical Euro-Collins (EC)-based lung preservation. METHODS: We analyzed 8 double-lung transplant patients in each of 3 groups. In the low-dose group (LDG), donor lungs were perfused with EC containing 2 mg/kg BN 52021, whereas we used 10 mg/kg in the high-dose group (HDG) and placebo in the control group (CG). Before reperfusing the first lung, we administered intravenously 120 mg BN 52021 (LDG), 600 mg BN 52021 (HDG), or placebo (CG). Hemodynamics in terms of pulmonary arterial pressure, pulmonary vascular resistance and serial determinations of the alveolo-arterial oxygen difference (AaDO(2)) were recorded. We measured blood levels of PAF pre-operatively and post-operatively, after 10 minutes and after 3, 8, 24, 48, and 144 hours. RESULTS: Within 32 hours, we noted a tendency toward better AaDO(2) in the LDG and the HDG compared with the CG (p > 0.05). We observed a significant improvement of AaDO(2) after 3 hours (HDG, p = 0.033) and 8 hours (LDG, p = 0.024), with poorest values in the CG. The PAF concentrations were lowest in the HDG, with significant deterioration 10 minutes after reperfusion. In contrast, placebo led to higher PAF levels. We measured significantly lower PAF concentrations (HDG vs CG) at 10 minutes and at 6 days post-operatively. CONCLUSIONS: Use of high-dose PAF antagonist BN 52021 can easily be combined with clinical preservation methods and may help optimize pulmonary function with reduced PAF levels, in the early post-ischemic period.

Expulsion of allergen-containing materials from hydrated rye grass (Lolium perenne) pollen revealed by using immunogold field emission scanning and transmission electron microscopy.

BACKGROUND: Several studies demonstrated episodes of grass pollen-induced allergic asthma after heavy rainfalls. It has been hypothesized that these asthma attacks might be due to the release of respirable allergen-bearing particles from pollen cytoplasm. OBJECTIVE: In this study we investigated the release mechanism of the most potent and frequently recognized grass pollen allergens, group 1 and group 5, from freshly harvested and subsequently hydrated rye grass pollen at the ultrastructural level. METHODS: Rabbit antisera against purified recombinant group 1 and group 5 allergens were used to investigate, by using field emission scanning and transmission immunogold electron microscopy, the allergen release from rye grass pollen grains into isotonic aqueous solutions or water. RESULTS: Pollen grains exposed to isotonic aqueous solutions remained intact and released allergens by means of diffusion. However, pollen grains hydrated in distilled water or rainwater expelled starch grains and cytoplasmic debris of respirable size. Group 1 and group 5 allergens were observed on and within these materials. CONCLUSIONS: Exposure of rye grass pollen to water leads to an expulsion of subcellular allergen-containing pollen components of respirable size. Our ultrastructural data thus support the idea that this release of allergen-containing respirable pollen materials may be a cause of asthma attacks after heavy rainfalls.

Purification, biochemical, and immunological characterisation of a major food allergen: different immunoglobulin E recognition of the apo- and calcium-bound forms of carp parvalbumin.

BACKGROUND: Almost 4% of the population suffer from food allergy which is an adverse reaction to food with an underlying immunological mechanism. AIMS: To characterise one of the most frequent IgE defined food allergens, fish parvalbumin. METHODS: Tissue and subcellular distribution of carp parvalbumin was analysed by immunogold electron microscopy and cell fractionation. Parvalbumin was purified to homogeneity, analysed by mass spectrometry and circular dichroism (CD) spectroscopy, and its allergenic activity was analysed by IgE binding and basophil histamine release tests. RESULTS: The isoelectric point (pI) 4.7 form of carp parvalbumin, a three EF-hand calcium-binding protein, was purified to homogeneity. CD analysis revealed a remarkable stability and refolding capacity of calcium-bound parvalbumin. This may explain why parvalbumin, despite cooking and exposure to the gastrointestinal tract, can sensitise patients. Purified parvalbumin reacted with IgE of more than 95% of individuals allergic to fish, induced dose-dependent basophil histamine release and contained, on average, 83% of the IgE epitopes present in other fish species. Calcium depletion reduced the IgE binding capacity of parvalbumin which, according to CD analysis, may be due to conformation-dependent IgE recognition. CONCLUSIONS: Purified carp parvalbumin represents an important cross reactive food allergen. It can be used for in vitro and in vivo diagnosis of fish-induced food allergy. Our finding that the apo-form of parvalbumin had a greatly reduced IgE binding capacity indicates that this form may be a candidate for safe immunotherapy of fish-related food allergy.

In situ localization of latex allergens in 3 different brands of latex gloves by means of immunogold field emission scanning and transmission electron microscopy.

BACKGROUND: Latex proteins represent relevant allergens, particularly for those persons who are frequently exposed to latex products (eg, health care workers and patients with chronic disorders). Although several latex allergens have been characterized by biochemical and molecular biologic techniques, little information is available concerning the in situ localization of allergenic proteins in latex products. OBJECTIVE: The objective of the present study was the in situ localization of latex allergens. METHODS: Serum IgE from patients with latex allergy reacting with a broad range (5-200 kd) of latex allergens was used for the in situ localization of latex allergens. One surgical and 2 examination latex glove brands were investigated by using immunogold field emission scanning and transmission electron microscopy. RESULTS: Allergens were detected on the inner and outer surface of the gloves, particularly near the edges, crests, or folds of the bleb-like structures visible on the surface of the latex material at high magnifications. In ultrathin cross-sections, latex allergens were found throughout the sections. CONCLUSIONS: Latex allergens were localized on the outer and inner surface but also in the interior of latex gloves. The occurrence of latex allergens on the surface of latex products may be related to their potential to induce local reactions and, perhaps, to sensitize individuals by means of contact.

Comparison of degradation state and stability of different humic acids by means of chemolysis with tetramethylammonium hydroxide.

In the chemolysis products of extracted humic acids (HAs) and tetramethylammonium hydroxide, different compounds can be identified which allow a description of the sources of soil organic matter (SOM). It is possible to draw conclusions concerning stability, degree of intramolecular cross-linking and degradation of aliphatic and lignin components from the distinctive product ratios. The ratio of lignin derived acidic phenolic derivatives and their analogous aldehydes and the ratio of phenylpropenoic acids and analogous benzoic acids provide comparably good parameters for the characterization of the state of degradation of lignin compounds to relatively stable HA building blocks. Regarding the aliphatic chemolysis products, alpha,omega-dicarboxylic acid and methoxy fatty acid contents indicate an intramolecular cross-linking state within the HA molecules and the degree of stable aliphatic constituent parts. High contents of unsaturated fatty acids can be considered as an indicator of an easy degradable humic skeleton because with their double bonds they represent active sites for further transformations. The suitability of the carbon preference index as an indicator of a biogenic carbon source can be confirmed. It is remarkable how the results obtained from extracted humic acids are in accordance with the expectations for the different SOMs derived from land use.

Molecular, immunological, and structural characterization of Phl p 6, a major allergen and P-particle-associated protein from Timothy grass (Phleum pratense) pollen.

Due to the wide distribution and heavy pollen production of grasses, approximately 50% of allergic patients are sensitized against grass pollen allergens. cDNAs coding for two isoforms and four fragments of a major timothy grass (Phleum pratense) pollen allergen, Phl p 6, were isolated by IgE immunoscreening from a pollen expression cDNA library. Recombinant Phl p 6 (rPhl p 6), an acidic protein of 11.8 kDa, was purified to homogeneity as assessed by mass spectrometry and exhibited almost exclusive alpha-helical secondary structure as determined by circular dichroism spectroscopy. Phl p 6 reacted with serum IgE from 75% of grass pollen-allergic patients (n = 171). IgE binding experiments with rPhl p 6 fragments indicated that the N terminus of the allergen is required for IgE recognition. Purified rPhl p 6 elicited dose-dependent basophil histamine release and immediate type skin reactions in patients allergic to grass pollen. A rabbit antiserum raised against purified rPhl p 6 identified it as a pollen-specific protein that, by immunogold electron microscopy, was localized on the polysaccharide-containing wall-precursor bodies (P-particles). The association of Phl p 6 with P-particles may facilitate its intrusion into the deeper airways and thus be responsible for the high prevalence of IgE recognition of Phl p 6. Recombinant native-like Phl p 6 can be used for in vitro as well as in vivo diagnoses of grass pollen allergy, whereas N-terminal deletion mutants with reduced IgE binding capacity may represent candidates for immunotherapy of grass pollen allergy with a low risk of anaphylactic side effects.

pET-prof, a plasmid for high-level expression of recombinant peptides fused to a birch profilin-derived hexadecapeptide tag: a system for the detection and presentation of recombinant antigens.

We have previously identified a birch pollen profilin hexadecapeptide (Bp36/51), which was recognized by a monoclonal antibody (moAb 4A6) with high affinity. Here, we report the construction of a T7 RNA polymerase-driven high-level plasmid expression system, pET-prof, capable of producing proteins and peptides containing the Bp36/51 birch profilin-derived peptide fused to their N-terminus. As examples, the cDNAs coding for two major timothy grass (Phleum pratense) pollen allergens, Phl p 2 and Phl p 6, as well as for an alder (Alnus glutinosa) pollen allergen, Aln g 4, were overexpressed in Escherichia coli as BP36/51-tagged proteins. All three recombinant allergens were readily detected in nitrocellulose-blotted E. coli extracts by the Bp36/51-specific moAb 4A6. We demonstrate comparable IgE recognition of Bp36/51-tagged and untagged recombinant allergens by immunoblotting. A sandwich ELISA was developed using plate-bound moAb 4A6 to immobilize and present Bp36/51-tagged recombinant allergens to IgE antibodies of allergic patients. Using immunoelectronmicroscopy, we demonstrate that even under harsh fixation conditions, tagged allergens can be localized simultaneously in situ by moAb 4A6 and allergen-specific antisera. We suggest the use of the pET-prof system for the high-level expression of Bp36/51-tagged polypeptides that can be rapidly detected in total protein extracts, immunolocalized in situ, immobilized and presented to other antigen-specific antibodies (e.g. IgE), even when they occur in minute concentrations.