Pathogen-specific antibody profiles in patients with severe systemic infections.

Infections are often caused by pathobionts, endogenous bacteria that belong to the microbiota. Trauma and surgical intervention can allow bacteria to overcome host defences, ultimately leading to sepsis if left untreated. One of the main defence strategies of the immune system is the production of highly specific antibodies. In the present proof-of-concept study, plasma antibodies against 9 major pathogens were measured in sepsis patients, as an example of severe systemic infections. The binding of plasma antibodies to bacterial extracellular proteins was quantified using a semi-automated immunoblot assay. Comparison of the pathogen-specific antibody levels before and after infection showed an increase in plasma IgG in 20 out of 37 tested patients. This host-directed approach extended the results of pathogen-oriented microbiological and PCR diagnostics: a specific antibody response to additional bacteria was frequently observed, indicating unrecognised poly-microbial invasion. This might explain some cases of failed, seemingly targeted antibiotic treatment.

Author Correction: Proteomic and Transcriptomic Changes in Hibernating Grizzly Bears Reveal Metabolic and Signaling Pathways that Protect against Muscle Atrophy.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Proteomic and Transcriptomic Changes in Hibernating Grizzly Bears Reveal Metabolic and Signaling Pathways that Protect against Muscle Atrophy.

Muscle atrophy is a physiological response to disuse and malnutrition, but hibernating bears are largely resistant to this phenomenon. Unlike other mammals, they efficiently reabsorb amino acids from urine, periodically activate muscle contraction, and their adipocytes differentially responds to insulin. The contribution of myocytes to the reduced atrophy remains largely unknown. Here we show how metabolism and atrophy signaling are regulated in skeletal muscle of hibernating grizzly bear. Metabolic modeling of proteomic changes suggests an autonomous increase of non-essential amino acids (NEAA) in muscle and treatment of differentiated myoblasts with NEAA is sufficient to induce hypertrophy. Our comparison of gene expression in hibernation versus muscle atrophy identified several genes differentially regulated during hibernation, including Pdk4 and Serpinf1. Their trophic effects extend to myoblasts from non-hibernating species (including C. elegans), as documented by a knockdown approach. Together, these changes reflect evolutionary favored adaptations that, once translated to the clinics, could help improve atrophy treatment.

Regulation of Biofilm Aging and Dispersal in Bacillus subtilis by the Alternative Sigma Factor SigB.

Bacterial biofilms are important in natural settings, biotechnology, and medicine. However, regulation of biofilm development and its persistence in different niches is complex and only partially understood. One key step during the biofilm life cycle is dispersal, when motile cells abandon the mature biofilm to spread out and colonize new niches. Here, we show that in the model bacterium Bacillus subtilis the general stress transcription factor SigB is essential for halting detrimental overgrowth of mature biofilm and for triggering dispersal when nutrients become limited. Specifically, SigB-deficient biofilms were larger than wild-type biofilms but exhibited accelerated cell death, significantly greater sensitivity to different stresses, and reduced dispersal. Interestingly, the signal detected by SigB to limit biofilm growth was transduced through the RsbP-dependent metabolic arm of the SigB regulatory cascade, which in turn positively controlled expression of SinR, the master regulator of biofilm formation and cell motility. This novel SigB-SinR regulatory circuit might be important in controlling the fitness of biofilms (either beneficial or harmful) in diverse environments.IMPORTANCE Biofilms are crucial for bacterial survival, adaptation, and dissemination in natural, industrial, and medical systems. Sessile cells embedded in the self-produced extracellular matrix of the biofilm benefit from a division of labor and are protected from environmental insults. However, as the biofilm ages, cells become stressed because of overcrowding, starvation, and accumulation of waste products. How does the sessile biofilm community sense and respond to stressful conditions? Here, we show that in Bacillus subtilis, the transcription factors SigB and SinR control whether cells remain in or leave a biofilm when metabolic conditions become unfavorable. This novel SigB-SinR regulatory circuit might be important for controlling the fitness of biofilms (either beneficial or harmful) in diverse environments.

Quantitative analysis of the intra- and inter-subject variability of the whole salivary proteome.

BACKGROUND AND OBJECTIVE: Interest in human saliva is increasing for disease-specific biomarker discovery studies. However, protein composition of whole saliva can grossly vary with physiological and environmental factors over time and it comprises human as well as bacterial proteins. MATERIAL AND METHODS: We compared intra- and inter-subject variabilities using complementary gel-based (two-dimensional difference gel electrophoresis, 2-D DIGE) and gel-free (liquid chromatography tandem mass spectrometry, LC-MS/MS) proteomics profiling of saliva. Unstimulated whole saliva of four subjects was examined at three different time-points (08.00 h, 12.00 h and 17.00 h) and variability of the saliva proteome was analyzed on two successive days by LC-MS/MS. RESULTS: In the 2-D DIGE experiment, the median coefficient of variation (CV) for intra-subject variability was significantly lower (CV of 0.39) than that for inter-subject variability (CV of 0.57; CV of technical replicates 0.17). LC-MS/MS data confirmed the significantly lower variation within subjects over time (CV of 0.37) than the inter-subject variability (CV of 0.53; CV of technical replicates 0.11), and that the inter-subject variability was not time-dependent. CONCLUSION: Both techniques revealed similar trends of variations on technical, intra- and inter-subject level but provided peptide and protein focused information and should thus be used as complementary approaches. The data presented indicate that 2-D DIGE as well as LC-MS/MS approaches are suitable for biomarker screening in saliva.

Regulation of interferon-inducible proteins by doxorubicin via interferon γ-Janus tyrosine kinase-signal transducer and activator of transcription signaling in tumor cells.

Activation of the immune system is a way for host tissue to defend itself against tumor growth. Hence, treatment strategies that are based on immunomodulation are on the rise. Conventional cytostatic drugs such as the anthracycline doxorubicin can also activate immune cell functions of macrophages and natural killer cells. In addition, cytotoxicity of doxorubicin can be enhanced by combining this drug with the cytokine interferon-γ (IFNγ). Although doxorubicin is one of the most applied cytostatics, the molecular mechanisms of its immunomodulation ability have not been investigated thoroughly. In microarray analyses of HeLa cells, a set of 19 genes related to interferon signaling was significantly over-represented among genes regulated by doxorubicin exposure, including signal transducer and activator of transcription (STAT) 1 and 2, interferon regulatory factor 9, N-myc and STAT interactor, and caspase 1. Regulation of these genes by doxorubicin was verified with real-time polymerase chain reaction and immunoblotting. An enhanced secretion of IFNγ was observed when HeLa cells were exposed to doxorubicin compared with untreated cells. IFNγ-neutralizing antibodies and inhibition of Janus tyrosine kinase (JAK)-STAT signaling [aurintricarboxylic acid (ATA), (E)-2-cyano-3-(3,4-dihydrophenyl)-N-(phenylmethyl)-2-propenamide (AG490), STAT1 small interfering RNA] significantly abolished doxorubicin-stimulated expression of interferon signaling-related genes. Furthermore, inhibition of JAK-STAT signaling significantly reduced doxorubicin-induced caspase 3 activation and desensitized HeLa cells to doxorubicin cytotoxicity. In conclusion, we demonstrate that doxorubicin induces interferon-responsive genes via IFNγ-JAK-STAT1 signaling and that this pathway is relevant for doxorubicin's cytotoxicity in HeLa cells. Immunomodulation is a promising strategy in anticancer treatment, so this novel mode of action of doxorubicin may help to further improve the use of this drug among different types of anticancer treatment strategies.

Antibody responses in furunculosis patients vaccinated with autologous formalin-killed Staphylococcus aureus.

Autologous vaccines (short: autovaccines) have been used since the beginning of the 20th century to treat chronic staphylococcal infections, but their mechanisms of action are still obscure. This prospective pilot study involved four patients with furunculosis who were vaccinated with autologous formalin-killed Staphylococcus aureus cells. Vaccines were individually prepared from the infecting S. aureus strain and repeatedly injected subcutaneously in increasing doses over several months. We characterized the virulence gene repertoire and spa genotype of the infecting and colonising S. aureus strains. Serum antibody responses to secreted and surface-bound bacterial antigens were determined by two-dimensional immunoblotting and flow-cytometry based assays (Luminex). All patients reported clinical improvement. Molecular characterization showed that all strains isolated from one patient over time belonged to the same S. aureus clone. Already before treatment, there was robust antibody binding to a broad range of staphylococcal antigens. Autovaccination moderately boosted the IgG response to extracellular antigens in two patients, while the antibody response of the other two patients was not affected. Similarly, vaccination moderately enhanced the antibody response against some staphylococcal surface proteins, e.g. ClfA, ClfB, SdrD and SdrE. In summary, autovaccination only slightly boosted the pre-existing serum antibody response, predominantly to bacterial surface antigens.