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Respiratory infections caused by multidrug-resistant Acinetobacter baumannii are difficult to treat and associated with high mortality among critically ill hospitalized patients. Bacteriophages (phages) eliminate pathogens with high host specificity and efficacy. However, the lack of appropriate preclinical experimental models hampers the progress of clinical development of phages as therapeutic agents. Therefore, we tested the efficacy of a purified lytic phage, vB_AbaM_Acibel004, against multidrug-resistant A. baumannii clinical isolate RUH 2037 infection in immunocompetent mice and a human lung tissue model. Sham- and A. baumannii-infected mice received a single-dose of phage or buffer via intratracheal aerosolization. Group-specific differences in bacterial burden, immune and clinical responses were compared. Phage-treated mice not only recovered faster from infection-associated hypothermia but also had lower pulmonary bacterial burden, lower lung permeability, and cytokine release. Histopathological examination revealed less inflammation with unaffected inflammatory cellular recruitment. No phage-specific adverse events were noted. Additionally, the bactericidal effect of the purified phage on A. baumannii was confirmed after single-dose treatment in an ex vivo human lung infection model. Taken together, our data suggest that the investigated phage has significant potential to treat multidrug-resistant A. baumannii infections and further support the development of appropriate methods for preclinical evaluation of antibacterial efficacy of phages.

Introduction: In 2014, the WHO proclaimed the beginning of a post-antibiotic era [1]. As alternatives to antibiotics, bacteriophages are increasingly coming into focus to combat the increasing number of resistant bacteria. In order to reach infected sites in the lungs and reduce unwanted systemic effects, nebulization of drugs is a favorable way of application. The infectivity of phages with different tail lengths after nebulization was tested under various conditions. This project is part of Phage4Cure, a BMBF-funded study examining the use of phages against lung infections. Methods: Long-tailed (Siphoviridae), short-tailed (Podoviridae) and medium long tailed (Myoviridae) phages from the order Caudovirales were analyzed. Four different phages per morphotype were nebulized with a JET nebulizer. Additionally, three phages (two Myoviridae, one Podovirus) were purified according to laboratory standards. By using the double-agar overlay method, titers after nebulization were compared to the stock solution. Results: Phages of the type Siphoviridae showed the highest titer losses during nebulization and Podoviridae the lowest. Among the Myoviridae variable and phage-dependent titer loss was observed. In the mesh nebulization system, all three phages showed a loss of titer after nebulization independent of the purification degree. Only one of the purified phages showed a higher titer loss after nebulization compared to lysate. Conclusion: For successful phage inhalation therapy, the effects of nebulizer technology, the size of the phage tail and purity must be considered. [1] www.who.int/mediacentre/news/releases/2014/amr-report/en/

Background Efficacy testing of immunotherapy in field studies is often hampered by variation of airborne allergens. Standardized allergen exposure in challenge chamber settings might be an alternative. Therefore, we developed a universal technique to create an atmosphere loaded with allergen particles of adjustable size from aqueous solutions of licensed allergen extracts. Objective The aim of this study was to apply this technique and test the safety and efficacy of challenges with house dust mite (HDM) allergen in the Fraunhofer allergen challenge chamber. Methods Aerosol particles carrying HDM allergen were produced by spray-drying of an aqueous solution containing HDM allergen and lactose. In a monocenter, placebo-controlled, single-blind, dose-escalation pilot study, 18 subjects with perennial allergic rhinitis and sensitization to HDM were exposed to HDM allergen for 4 h at either 250, 500, 1000 SQE/m3 or lactose alone (0 SQE/m3 ) 7 days apart. The dose of 500 SQE/m³ was repeated to investigate reproducibility. Total nasal symptom score (TNSS) was the primary endpoint. Results Exposure to HDM increased TNSS (mean ± SD) to 3.4 ± 1.8, 3.3 ± 2.1, and 3.6 ± 2.0 at 250, 500 and 1000 SQE/m3 , respectively, while lactose alone did not change TNSS (0.7 ± 0.6). The results were reproducible at 500 SQE/m3 . Pulmonary function and adverse event frequency did not change with escalation of allergen dose. Conclusion This HDM allergen particle generation is safe, specific and reproducible and can therefore be used for efficacy testing of immunotherapy and for basic clinical research.

Introduction: Rhinovirus (RV) infection is the most important trigger for asthma exacerbation. However, no GMP grade RV preparation and established protocols are available for human RV challenge studies. Aim: To establish safety and tolerability of a new GMP grade U-BIOPRED human RV16 (RV16UB) and to identify an appropriate dose to study biomarkers in asthmatics following RV challenge. Methods: Study 1: Single doses of 10, 100 and 1000 TCID50 of RV16UB were administered intranasally in healthy subjects (n=18) and 10 and 100 TCID50 in mild-moderate asthmatics on ICS control therapy (n=18), taking LABA and not taking LABA. Study 2: Single doses of 100 TCID50 were given to asthmatics on ICS not taking LABA (n=23) and changes from baseline in lung function, Asthma Control Diary (ACD), Common Cold Symptoms Score (CSS) and FeNO were determined (time weighted average days 1-7; TWA1-7). Results: Study 1: All doses of RV16UB were well tolerated in asthma and controls. A dose of 100 TCID50 induced cold symptoms and detectable viral RNA titers in most asthmatics. Study 2: Following challenge with 100 TCID50 morning and evening TWA1-7 FEV1 were significantly reduced by -1.7% and – 2.1% respectively. Max. FEV1 drop post challenge was 6.8%. TWA1-7 ACD and CSS were significantly increased after challenge (2.7 and 4.0 respectively). No significant changes in FeNO were found. Conclusion: A dose of 100 TCID50 of RV16UB was safe in asthmatics and induced a small, but significant reduction in FEV1 (-2%). Significant increases in ACD and CSS were found. Changes in biomarkers are under investigation.

Background: Global spread of multidrug resistant (MDR) bacteria is one of the biggest threats in modern medicine. Acinetobacter baumannii is a feared cause of lung infections particularly in critically ill patients lacking antibiotic options. Therefore, rediscovery of phage therapy has gained attention as treatment option due to its specificity and effectivity. Aims: This preclinical study evaluated the efficacy and safety of a novel phage preparation in a mouse model of A. baumannii lung infection. Methods: Mice were infected with a MDR A. baumannii strain [1]. Phage Acibel004 [2] was produced as suspension including efficient depletion of endotoxins using a newly established protocol. Solvent or phages were sprayed intratracheally 12 hours after infection. Clinical parameters, bacterial load, cell influx, lung permeability and cytokine release were quantified and histopathological examination was performed. Results: Phage-treated mice recovered faster from infection-associated hypothermia. Phage treatment reduced bacterial loads in lungs and BAL 48 hours after infection. Moreover, lung permeability and cytokine release were reduced. Histopathological examination of lungs revealed lesser extent of inflammation in phage-treated mice with unaffected cellular recruitment. No adverse effects were observed. Conclusion: For the first time, a highly purified phage against A. baumannii was successfully used to treat lung infection in vivo. The current preclinical data support the concept of phage-based therapy against pulmonary A. baumannii infections. [1] Knapp, S. et al. Am J Respir Crit Care Med 2006; 1;173(1):122-9 [2] Merabishvili, M. et al. PLoS One 2014; 11;9(8):e104853

Introduction: Chronic lung diseases are frequently complicated by lung and airway infections. The rise of new resistance mechanisms is alarming, and MDR bacteria, including Acinetobacter baumannii, are spreading globally. Rediscovery of phage-therapy could be a solution to overcome this problem. High specificity of bacteriophages and their effectivity in lysis make phages attractive as antibiotics are failing more and more. However, phages need to be properly chosen and characterized according to quality standards, and preparations must be highly purified.Objectives:This preclinical study aimed at determining the efficacy, safety and tolerability of a novel phage preparation in anticipation of a future clinical trial applying aerosolized lytic phages against A. baumannii. Methods: The specific phage Acibel004 [1], a myovirus, was produced as high-titer suspension subjected to final processing including highly efficient depletion of endotoxins by chromatography. Mice were transnasally infected with a MDR A. baumannii strain [2]. 12h p.i. mice were treated intratracheally with phage or solvent. At defined time points clinical parameters were measured, bacterial burdens in different organs determined and cell-influx analyzed. Lung permeability and cytokine release were measured and histological analyses performed. Furthermore, presence of vital phages was determined. Results: Phage application in uninfected mice did not cause measurable immune responses. In A. baumannii infected mice, phage therapy enhanced recovery from infection-associated hypothermia and improved clinical outcome. Bacterial loads in lungs and BALF of the phage-treated group were significantly reduced by 48h p.i.. Cellular recruitment to the lung and blood was unaffected by phage treatment, while lung permeability and cytokine production were significantly reduced. Hematoxylin and eosin stained lung slides of phage treated mice showed less spreading of bacteria to periphery. Phages were detected in BALF, lungs and plasma of treated mice. No adverse effects of phage therapy were observed. Conclusion: For the first time a highly purified phage preparation against A. baumannii was used successfully in vivo. The current preclinical data further support the concept of developing a phage-based therapy against pulmonary A. baumannii infections in patients.

Shiga toxin (Stx, synonymous to verotoxin, VT) binds with high and low affinity to the globo-series neutral glycosphingolipids (GSLs), globotriaosylceramide (Gb3Cer or Galα4Galβ4Glcβ1Cer, also known as CD77) and globotetraosylceramide (Gb4Cer or GalNAcβ3Galα4Galβ4Glcβ1Cer), respectively, which represent the targets of Stxs on many different cell types. B-cell-derived Raji cells and THP-1 cells of monocytic origin are widely used for the investigation of Stx-mediated cellular response, because Stx is known to cause cell death in both cell lines. Despite their functional importance, the Stx receptors of Raji and THP-1 cells have so far not been investigated. This prompted us to explore the structures of their GSL receptors in detail by means of nanoelectrospray ionization quadrupole time-of-flight mass spectrometry (nanoESI-QTOF-MS) with collision-induced dissociation (CID) in conjunction with Stx1 as well as anti-Gb3Cer and anti-Gb4Cer antibodies. Using the combination of a thin-layer chromatography (TLC) overlay assay and MS1 and MS2 analysis we identified Gb3Cer (d18:1, C24:1/C24:0) as the prevalent Stx1-receptor accompanied by less abundant Gb3Cer (d18:1, C16:0) in the neutral GSL fraction of Raji cells. The same Gb3Cer species but with almost equal proportions of the C24:1/C24:0 and C16:0 variants were found in THP-1 cells. In addition, unusual hydroxylated Gb3Cer (d18:1, C24:1/C24:0) and Gb3Cer (d18:1, C26:1) could be identified in trace quantities in both cell lines. As the most obvious difference between Raji and THP-1 cells we observed the expression of Gb4Cer in THP-1 cells, whereas Raji cells failed to express this elongation product of Gb3Cer. Both short- and long-chain fatty acid carrying Gb4Cer (d18:1, C16:0) and Gb4Cer (d18:1, C24:1/C24:0), respectively, were the prevalent Gb4Cer variants. This first report on the differential expression of Gb3Cer and Gb4Cer and their structural diversity in lymphoid and myeloid cell lines supports the hypothesis that such heterogeneities might play a functional role in the molecular assembly of GSLs in membrane organization and cellular signaling of Stx-susceptible cells. Copyright © 2010 John Wiley & Sons, Ltd.

Background Recombinant cell lines developed for therapeutic antibody production often suffer instability or lose recombinant protein expression during long-term culture. Heterogeneous gene expression among cell line subclones may result from epigenetic modifications of DNA or histones, the protein component of chromatin. We thus investigated in such cell lines, DNA methylation and the chromatin environment along the human eukaryotic translation elongation factor 1 alpha 1 (EEF1A1) promoter in an antibody protein-expression vector which was integrated into the Chinese hamster ovary (CHO) cell line genome. Results We analyzed four PT1-CHO cell lines which exhibited losses of protein expression at advanced passage number (>P35) growing in adherent conditions and in culture medium with 10 % FCS. These cell lines exhibited different integration sites and transgene copy numbers as determined by fluorescence in situ hybridization (FISH) and quantitative PCR (qPCR), respectively. By qRT-PCR, we analyzed the recombinant mRNA expression and correlated it with DNA methylation and with results from various approaches interrogating the chromatin landscape along the EEF1A1 promoter region. Each PT1-CHO cell line displayed specific epigenetic signatures or chromatin marks correlating with recombinant mRNA expression. The cell line with the lowest recombinant mRNA expression (PT1-1) was characterized by the highest nucleosome occupancy and displayed the lowest enrichment for histone marks associated with active transcription. In contrast, the cell line with the highest recombinant mRNA expression (PT1-55) exhibited the highest numbers of formaldehyde-assisted isolation of regulatory elements (FAIRE)-enriched regions, and was marked by enrichment for histone modifications H3K9ac and H3K9me3. Another cell line with the second highest recombinant mRNA transcription and the most stable protein expression (PT1-7) had the highest enrichments of the histone variants H3.3 and H2A.Z, and the histone modification H3K9ac. A further cell line (PT1-30) scored the highest enrichments for the bivalent marks H3K4me3 and H3K27me3. Finally, DNA methylation made a contribution, but only in the culture medium with reduced FCS or in a different expression vector. Conclusions Our results suggest that the chromatin state along the EEF1A1 promoter region can help predict recombinant mRNA expression, and thus may assist in selecting desirable clones during cell line development for protein production. Electronic supplementary material The online version of this article (doi:10.1186/s12896-016-0238-0) contains supplementary material, which is available to authorized users.

A preparative anion-exchange high-performance liquid chromatographic method for the separation of the closely allied monosialogangliosides GM3(Neu5Ac) and GM3(Neu5Gc) has been developed. Hybridoma cells, readily available material derived from industrial monoclonal antibody production, were used as ganglioside source and led to fractions with pure GM3(Neu5Ac) and GM3(Neu5Gc) in high milligram quantities. The crude ganglioside extract was loaded onto columns filled with the strong anion-exchanger trimethylaminoethyl (TMAE)-Fractogel. Gangliosides were eluted from the stationary phase with a gradient system of ammonium acetate in methanol. The scaled-up approach ranged over more than one order of magnitude from 20 to 500 mg batches of GM3 gangliosides. Thus, the high-resolution power of the strong anion-exchanger TMAE-Fractogel allowed the preparative isolation by one-step column chromatography of two GM3 specimens which only differ in one hydroxyl group at position 5 of the neuraminic acid (N-acetyl-versus N-glycolylneuraminic acid).

Increasing economic pressure is the main driving force to enhance the efficiency of existing processes. We developed a perfusion strategy for a seed train reactor to generate a higher inoculum density for a subsequent fed batch production culture. A higher inoculum density can reduce culture duration without compromising product titers. Hence, a better capacity utilization can be achieved. The perfusion strategy was planned to be implemented in an existing large scale antibody production process. Therefore, facility and process constraints had to be considered. This article describes the initial development steps. Using a proprietary medium and a Chinese hamster ovary cell line expressing an IgG antibody, four different cell retention devices were compared in regard to retention efficiency and reliability. Two devices were selected for further process refinement, a centrifuge and an inclined gravitational settler. A concentrated feed medium was developed to meet facility constraints regarding maximum accumulated perfundate volume. A 2-day batch phase followed by 5 days of perfusion resulted in cell densities of 1.6 × 10(10) cells L(-1) , a 3.5 fold increase compared to batch cultivations. Two reactor volumes of concentrated feed medium were needed to achieve this goal. Eleven cultivations were carried out in bench and 50 L reactors showing acceptable reproducibility and ease of scale up. In addition, it was shown that at least three perfusion phases can be combined within a repeated perfusion strategy.

The focus of this work was the investigation of correlations between cultivation parameters and cell surface physicochemistry (hydrophobicity, electrophoretic mobility, isoelectric point) of four strains (Pseudomonas aeruginosa DSM 1253, Escherichia coli 72, Arthrobacter oxydans DSM 420 and Corynebacterium glutamicum DSM 20300).

RATIONALE: There is an increasing demand for studies on indoor allergens, particularly on house dust mite (HDM) allergens, in order to perform basic clinical research and to test anti-allergic drugs under well-controlled conditions. At Fraunhofer ITEM, a novel technique to universally create an atmosphere loaded with allergen particles was recently developed. In brief, aerosol particles of defined size are produced by spray-drying of a solution that contains a licenced allergen extract and lactose as a carrier. The particle size distribution is dependent on the initial droplet size and the solute lactose concentration aiming to have a particle size above 10µm. The aim of this study was to test the safety and efficacy with dose-range finding and reproducibility of this novel HDM challenge in patients with allergic rhinitis in the Fraunhofer Environmental Challenge Chamber (ECC). METHODS: In a monocenter, placebo-controlled, single-blind, pilot study 18 subjects with allergic rhinitis and sensitization to HDM were exposed in the Fraunhofer ECC for 4 hours to house dust mite allergen (ALK lyophilisiert SQ503 Der p, ALK-Abello, Wedel, Germany) at either 250 SQE/m3, 500 SQE/m3, 1000 SQE/m3, or lactose alone (0 SQE/m3) seven days apart. The dose of 500 SQE/m3 was repeated to investigate reproducibility. Total nasal symptom score (TNSS), anterior rhinomanometry, nasal secretions, exhaled NO, FEV1 and adverse events were assessed prior to and during the exposures. RESULTS: At a lactose concentration of 10% (w/v) the mass median aerodynamic diameter of allergen particles was 13.5 µm and only 11% of particles were

Biological processes are becoming increasingly popular in dealing with waste air problems. Compared to chemical and physical treatment methods, they offer several advantages, especially in handling of waste air contaminated with low concentrations of biodegradable organic and, occasionally, inorganic substances. Two types of commercial systems are currently in use: biofilters and bioscrubbers. The aim of our studies was to develop an innovative concept of reactor design and engineering called “biocatalyst” (Biokatalysator). Our biocatalytic three-phase system lies in between the conventional biofilter and bioscrubber, using biomass immobilized on macroporous, tubiform supports installed in parallel to the air flow. In laboratory-scale investigations, a bioreactor was continuously fed with solvent-laden air, which produced a more cost-effective mode of operation than the conventional bioscrubber systems.

Shiga toxin (Stx, synonymous to verotoxin, VT) binds with high and low affinity to the globo-series neutral glycosphingolipids (GSLs), globotriaosylceramide (Gb3Cer or Galalpha4Galbeta4Glcbeta1Cer, also known as CD77) and globotetraosylceramide (Gb4Cer or GalNAcbeta3Galalpha4Galbeta4Glcbeta1Cer), respectively, which represent the targets of Stxs on many different cell types. B-cell-derived Raji cells and THP-1 cells of monocytic origin are widely used for the investigation of Stx-mediated cellular response, because Stx is known to cause cell death in both cell lines. Despite their functional importance, the Stx receptors of Raji and THP-1 cells have so far not been investigated. This prompted us to explore the structures of their GSL receptors in detail by means of nanoelectrospray ionization quadrupole time-of-flight mass spectrometry (nanoESI-QTOF-MS) with collision-induced dissociation (CID) in conjunction with Stx1 as well as anti-Gb3Cer and anti-Gb4Cer antibodies. Using the combination of a thin-layer chromatography (TLC) overlay assay and MS(1) and MS(2) analysis we identified Gb3Cer (d18:1, C24:1/C24:0) as the prevalent Stx1-receptor accompanied by less abundant Gb3Cer (d18:1, C16:0) in the neutral GSL fraction of Raji cells. The same Gb3Cer species but with almost equal proportions of the C24:1/C24:0 and C16:0 variants were found in THP-1 cells. In addition, unusual hydroxylated Gb3Cer (d18:1, C24:1/C24:0) and Gb3Cer (d18:1, C26:1) could be identified in trace quantities in both cell lines. As the most obvious difference between Raji and THP-1 cells we observed the expression of Gb4Cer in THP-1 cells, whereas Raji cells failed to express this elongation product of Gb3Cer. Both short- and long-chain fatty acid carrying Gb4Cer (d18:1, C16:0) and Gb4Cer (d18:1, C24:1/C24:0), respectively, were the prevalent Gb4Cer variants. This first report on the differential expression of Gb3Cer and Gb4Cer and their structural diversity in lymphoid and myeloid cell lines supports the hypothesis that such heterogeneities might play a functional role in the molecular assembly of GSLs in membrane organization and cellular signaling of Stx-susceptible cells.

The mouse monoclonal antibody 2D4, which recognizes the terminal GalNAcβ1-4Gal-disaccharide of GgOse3Cer and GgOse5Cer, was used for the detection of ganglio-series gangliosides. The method involves separation of gangliosides on thin layer chromatography plates, followed by silica gel fixation, Arthrobacter ureafaciens neuraminidase treatment and final immunostaining of desialylated gangliosides with the monoclonal antibody 2D4. Both neuraminidase and the hybridoma 2D4 producing the specific monoclonal antibody are commercially available and therefore accessible to all researchers working in this field. Gangliosides from mouse T lymphocytes and the mouse T cell lymphoma YAC-1 have been used as examples. This technique may be used for fast screening of gangliosides with the GgOse5Cer core structure which have been described as T cell markers, antigens in human neuronal disease and receptors for certain pathogenic bacteria.

Background Efficacy testing of immunotherapy in field studies is often hampered by variation of airborne allergens. Standardized allergen exposure in challenge chamber settings might overcome aforementioned limitations. However, airborne allergens such as house dust mite (HDM) or cat allergen are not available as standardized source material with distinct particle size. Therefore, we developed a method for universal allergen particle generation and conducted a pilot study to clinically validate this challenge paradigm for house dust mite.

Immobilized cells cover a wide area of applications and are essential components of many biotechnological processes. In general it can be distinguished between two immobilization methods: (1) entrapment into polymers and (2) natural adsorption onto porous and inert support materials. The immobilization by adsorption is discussed by the following criteria: biomass loading, strength of adhesion, enzymatic stability/specific activity of the biocatalyst, effectivity/reaction engineering and operational stability.

We describe the purification and properties of a catabolic aromatic amino acid transaminase from a Pseudomonas putida strain, which was screened from soil samples for high phenylpyruvate transamination activity. The enzyme has a molecular size of 72000 dalton and is composed of two subunits of 36000 dalton. It appears not to be fully saturated with pyridoxalphosphate in vivo, which otherwise is tightly bound. The best substrates are the aromatic amino acids and l -aspartate or the corresponding keto-acids. Prephenate, however, is not used as a substrate. l -Glutamate and l -histidine are substrates with distinctly lower affinity. d -Amino acids are not transaminated. For the enzymatic transamination from l -aspartate to phenylpyruvate, the spontaneous decarboxylation of the second product oxaloacetate appears to be the conversion limiting reaction step.