Murine macrophage inflammatory cytokine production and immune activation in response to Vibrio parahaemolyticus infection

Vibrio parahaemolyticus is the most common cause of bacterial, seafood‐related illness in the USA. Currently, there is a dearth of published reports regarding immunity to infection with this pathogen. Here, production of both pro‐ and anti‐inflammatory cytokines by V. parahaemolyticus‐infected RAW 264.7 murine macrophages was studied. It was determined that this infection results in increased concentrations of IL‐1α, IL‐6, TNF‐α and IL‐10. Additionally, decreases in cell surface TLR2 and TLR4 and increases in T‐cell co‐stimulatory molecules CD40 and CD86 were discovered. The data presented here begin to identify the immune variables required to eliminate V. parahaemolyticus from infected host tissues.     

Development of a core outcome set for clinical trials in rosacea: study protocol for a systematic review of the literature and identification of a core outcome set using a Delphi survey

BackgroundRosacea is a chronic inflammatory disorder affecting millions of individuals worldwide. Diagnosis is based on signs and symptoms with management and treatment aimed to suppress inflammatory lesions, erythema, and telangiectasia. While many clinical trials of rosacea exist, the lack of consensus in outcome reporting across all trials poses a concern. Proper evaluation and comparison of treatment modalities is challenging. In order to address the inconsistencies present, this project aims to determine a core set of outcomes which should be evaluated in all clinical trials of rosacea.Methods/designThis project will utilize a methodology similar to previous core outcome set research. A long list of outcomes will be extracted over four phases: (1) systematic literature review, (2) patient interviews, (3) other published sources, and (4) stakeholder involvement. Potential outcomes will be examined by the Steering Committee to provide further insight. The Delphi process will then be performed to prioritize and condense the list of outcomes generated. Two homogenous groups of physicians and patients will participate in two consecutive rounds of Delphi surveys. A consensus meeting, composed of physicians, patients, and stakeholders, will be conducted after the Delphi exercise to further select outcomes, taking into account participant scores. By the end of the meeting, members will vote and decide on a final recommended set of core outcomes. For the duration of the study, we will be in collaboration with both the Core Outcome Measures in Effectiveness Trials (COMET) and Cochrane Skin Group - Core Outcome Set Initiative (CSG-COUSIN).DiscussionThis study aims to develop a core outcome set to guide assessment in clinical trials of rosacea. The end-goal is to improve the reliability and consistency of outcome reporting, thereby allowing sufficient evaluation of treatment effectiveness and patient satisfaction.

Electronic supplementary material

The online version of this article (doi:10.1186/s13063-016-1554-3) contains supplementary material, which is available to authorized users.     

Peptidase N encoded by Salmonella enterica serovar Typhimurium modulates systemic infection in mice

The cytosolic protein degradation pathway, involving ATP-dependent proteases and ATP-independent peptidases, is important for modulating several cellular responses. The involvement of pathogen-encoded ATP-dependent proteases is well established during infection. However, the roles of ATP-independent peptidases in this process are not well studied. The functional role of Peptidase N (PepN), an ATP-independent enzyme belonging to the M1 family, during systemic infection of mice by Salmonella enterica serovar Typhimurium (Salmonella typhimurium) was investigated. In a systemic model of infection, the number of CFU of S. typhimurium containing a targeted deletion in peptidase N (DeltapepN), compared with wild type, was significantly higher in the lymph node and spleen. In addition, S. typhimurium replicated in the thymus and greatly reduced the number of immature CD4(+)CD8(+) thymocytes in a dose- and time-dependent manner. Strains lacking or overexpressing pepN were used to show that the reduction in the number of thymocytes, but not lymph node cells, depends on a critical number of CFU. These findings establish a role for PepN in reducing the in vivo CFU of S. typhimurium during systemic infection. The implications of these results, in the context of the roles of proteases and peptidases, during host-pathogen interactions are discussed.     

Characterization and physical mapping of ribosomal RNA gene families in Plantago

• Background and Aims The organization of rRNA genes incultivated Plantago ovata Forsk. and several of its wild allieswas analysed to gain insight into the phylogenetic relationshipsof these species in the genus which includes some 200 species. • Methods Specific primers were designed to amplify theinternal transcribed spacer (ITS1 and ITS2) regions from sevenPlantago species and the resulting fragments were cloned andsequenced. Similarly, using specific primers, the 5S rRNA genesfrom these species were amplified and subsequently cloned. Fluorescencein-situ hybridization (FISH) was used for physical mapping of5S and 45S ribosomal RNA genes. • Results The ITS1 region is 19–29 bp longer thanthe ITS2 in different Plantago species. The 5S rRNA gene-repeatingunit varies in length from 289 to 581 bp. Coding regions arehighly conserved across species, but the non-transcribed spacers(NTS) do not match any database sequences. The clone from thecultivated species P. ovata was used for physical mapping ofthese genes by FISH. Four species have one FISH site while threehave two FISH sites. In P. lanceolata and P. rhodosperma, the5S and 45S (18S-5·8S-25S) sites are coupled. • Conclusions Characterization of 5S and 45S rRNA geneshas indicated a possible origin of P. ovata, the only cultivatedspecies of the genus and also the only species with x = 4, froma species belonging to subgenus Psyllium. Based on the studiesreported here, P. ovata is closest to P. arenaria, althoughon the basis of other data the two species have been placedin different subgenera. FISH mapping can be used as an efficienttool to help determine phylogenetic relationships in the genusPlantago and show the interrelationship between P. lanceolataand P. lagopus.     

Synergistic Amyloid Switch Triggered by Early Heterotypic Oligomerization of Intrinsically Disordered α-Synuclein and Tau

Amyloidogenic intrinsically disordered proteins, α-synuclein and tau are linked to Parkinson's disease and Alzheimer's disease, respectively. A body of evidence suggests that α-synuclein and tau, both present in the presynaptic nerve terminals, co-aggregate in many neurological ailments. The molecular mechanism of α-synuclein-tau hetero-assembly is poorly understood. Here we show that amyloid formation is synergistically facilitated by heterotypic association mediated by binding-induced misfolding of both α-synuclein and tau K18. We demonstrate that the intermolecular association is largely driven by the electrostatic interaction between the negatively charged C-terminal segment of α-synuclein and the positively charged tau K18 fragment. This heterotypic association results in rapid formation of oligomers that readily mature into hetero-fibrils with a much shorter lag phase compared to the individual proteins. These findings suggested that the critical intermolecular interaction between α-synuclein and tau can promote facile amyloid formation that can potentially lead to efficient sequestration of otherwise long-lived lethal oligomeric intermediates into innocuous fibrils. We next show that a well-known familial Parkinson's disease mutant (A30P) that is known to aggregate slowly via accumulation of highly toxic oligomeric species during the long lag phase converts into amyloid fibrils significantly faster in the presence of tau K18. The early intermolecular interaction profoundly accelerates the fibrillation rate of A30P α-synuclein and impels the disease mutant to behave similar to wild-type α-synuclein in the presence of tau. Our findings suggest a mechanistic underpinning of bypassing toxicity and suggest a general strategy by which detrimental amyloidogenic precursors are efficiently sequestered into more benign amyloid fibrils.     

Heteroglucan-dendrimer glycoconjugate: a modulated construct with augmented immune responses and signaling phenomena

Background

Newer strategies for augmenting immune responses of pharmacologically active glucans may serve to improve the medicinal potential of these biomolecules. With this aim, the present work was focused on generating targeted high molecular size glucan particles with magnified immune response activity.

Methods

Heteroglucans were conjugated with PAMAM dendrimers using a Schiff base reductive amination reaction to generate a polytethered molecule with multiple glucan motifs. The modulated construct was characterized by FTIR, TEM, ¹H NMR and dynamic light scattering (DLS) methods. Effects of conjugated glucans were examined in RAW 264.7 macrophage cells as well as in S-180 murine tumor models.

Results

Dendrimer-conjugated glucans were found to exhibit a two-fold increase in immune stimulation in comparison to unconjugated glucans. This may be corroborated by the predominant enhancement in immunological functions such as nitric oxide production, ROS generation and immune directed tumor inhibition in murine models. Immune cell surface markers (CD4, CD8, CD19, MHC-II) and cytokine levels were also found to be highly up-regulated in the splenocytes of mice subjected to particulate glucan administration. Our study also demonstrated that conjugated glucan treatment to RAW 264.7 cells strongly enhanced the phosphorylation of two downstream signalling molecules of the mitogen activated protein kinase (MAPKs) family: p38 and MEK1/2 relative to single glucans thereby relating molecular mechanisms with enhanced immune stimulation.

Conclusions and general significance

The results obtained thus support that particulate format of soluble heteroglucan will thereby improve its functionality and identify leads in therapeutic competence.     

Comparative analysis of caveolins in mouse and tammar wallaby: Role in regulating mammary gland function

Recent studies using the mouse showed an inverse correlation between the Caveolin 1 gene expression and lactation, and this was regulated by prolactin. However, current study using mammary explants from pregnant mice showed that while insulin (I), cortisol (F) and prolactin (P) resulted in maximum induction of the β-casein gene, FP and IFP resulted in the downregulation of Caveolin 1. Additionally, IF, FP and IFP resulted in the downregulation of Caveolin 2. Immunohistochemistry confirmed localisation of Caveolin 1 specific to myoepithelial cells and adipocytes. Comparative studies with the tammar wallaby showed Caveolin 1 and 2 had 70–80% homology with the mouse proteins. However, in contrast to the mouse, Caveolin 1 and 2 genes showed a significantly increased level of expression in the mammary gland during lactation. The regulation of tammar Caveolin 1 and 2 gene expression was examined in mammary explants from pregnant tammars, and no significant difference was observed either in the absence or in the presence of IFP.