Delivery of a Chlamydial Adhesin N-PmpC Subunit Vaccine to the Ocular Mucosa Using Particulate Carriers

Trachoma, caused by the intracellular bacterium Chlamydia trachomatis (Ct), remains the world’s leading preventable infectious cause of blindness. Recent attempts to develop effective vaccines rely on modified chlamydial antigen delivery platforms. As the mechanisms engaged in the pathology of the disease are not fully understood, designing a subunit vaccine specific to chlamydial antigens could improve safety for human use. We propose the delivery of chlamydia-specific antigens to the ocular mucosa using particulate carriers, bacterial ghosts (BGs). We therefore characterized humoral and cellular immune responses after conjunctival and subcutaneous immunization with a N-terminal portion (amino acid 1–893) of the chlamydial polymorphic membrane protein C (PmpC) of Ct serovar B, expressed in probiotic Escherichia coli Nissle 1917 bacterial ghosts (EcN BGs) in BALB/c mice. Three immunizations were performed at two-week intervals, and the immune responses were evaluated two weeks after the final immunization in mice. In a guinea pig model of ocular infection animals were immunized in the same manner as the mice, and protection against challenge was assessed two weeks after the last immunization. N-PmpC was successfully expressed within BGs and delivery to the ocular mucosa was well tolerated without signs of inflammation. N-PmpC-specific mucosal IgA levels in tears yielded significantly increased levels in the group immunized via the conjunctiva compared with the subcutaneously immunized mice. Immunization with N-PmpC EcN BGs via both immunization routes prompted the establishment of an N-PmpC-specific IFNγ immune response. Immunization via the conjunctiva resulted in a decrease in intensity of the transitional inflammatory reaction in conjunctiva of challenged guinea pigs compared with subcutaneously and non-immunized animals. The delivery of the chlamydial subunit vaccine to the ocular mucosa using a particulate carrier, such as BGs, induced both humoral and cellular immune responses. Further investigations are needed to improve the immunization scheme and dosage.     

Intracellular Cytokine Staining and Flow Cytometry: Considerations for Application in Clinical Trials of Novel Tuberculosis Vaccines

Intracellular cytokine staining combined with flow cytometry is one of a number of assays designed to assess T-cell immune responses. It has the specific advantage of enabling the simultaneous assessment of multiple phenotypic, differentiation and functional parameters pertaining to responding T-cells, most notably, the expression of multiple effector cytokines. These attributes make the technique particularly suitable for the assessment of T-cell immune responses induced by novel tuberculosis vaccines in clinical trials. However, depending upon the particular nature of a given vaccine and trial setting, there are approaches that may be taken at different stages of the assay that are more suitable than other alternatives. In this paper, the Tuberculosis Vaccine Initiative (TBVI) TB Biomarker Working group reports on efforts to assess the conditions that will determine when particular assay approaches should be employed. We have found that choices relating to the use of fresh whole blood or peripheral blood mononuclear cells (PBMC) and frozen PBMC; use of serum-containing or serum-free medium; length of stimulation period and use of co-stimulatory antibodies can all affect the sensitivity of intracellular cytokine assays. In the case of sample material, frozen PBMC, despite some loss of sensitivity, may be more advantageous for batch analysis. We also recommend that for multi-site studies, common antibody panels, gating strategies and analysis approaches should be employed for better comparability.     

CD4+CD25hiFOXP3+ Regulatory T Cells and Cytokine Responses in Human Schistosomiasis before and after Treatment with Praziquantel

Background

Chronic schistosomiasis is associated with T cell hypo-responsiveness and immunoregulatory mechanisms, including induction of regulatory T cells (Tregs). However, little is known about Treg functional capacity during human Schistosoma haematobium infection.

Methodology

CD4⁺CD25^hiFOXP3⁺ cells were characterized by flow cytometry and their function assessed by analysing total and Treg-depleted PBMC responses to schistosomal adult worm antigen (AWA), soluable egg antigen (SEA) and Bacillus Calmette-Guérin (BCG) in S. haematobium-infected Gabonese children before and 6 weeks after anthelmintic treatment. Cytokines responses (IFN-γ, IL-5, IL-10, IL-13, IL-17 and TNF) were integrated using Principal Component Analysis (PCA). Proliferation was measured by CFSE.

Principal Findings

S. haematobium infection was associated with increased Treg frequencies, which decreased post-treatment. Cytokine responses clustered into two principal components reflecting regulatory and Th2-polarized (PC1) and pro-inflammatory and Th1-polarized (PC2) cytokine responses; both components increased post-treatment. Treg depletion resulted in increased PC1 and PC2 at both time-points. Proliferation on the other hand, showed no significant difference from pre- to post-treatment. Treg depletion resulted mostly in increased proliferative responses at the pre-treatment time-point only.

Conclusions

Schistosoma-associated CD4⁺CD25^hiFOXP3⁺Tregs exert a suppressive effect on both proliferation and cytokine production. Although Treg frequency decreases after praziquantel treatment, their suppressive capacity remains unaltered when considering cytokine production whereas their influence on proliferation weakens with treatment.     

Arsenic Exposure Increases Monocyte Adhesion to the Vascular Endothelium,a Pro-Atherogenic Mechanism

Epidemiological studies have shown that arsenic exposure increases atherosclerosis, but the mechanisms underlying this relationship are unknown. Monocytes, macrophages and platelets play an important role in the initiation of atherosclerosis. Circulating monocytes and macrophages bind to the activated vascular endothelium and migrate into the sub-endothelium, where they become lipid-laden foam cells. This process can be facilitated by platelets, which favour monocyte recruitment to the lesion. Thus, we assessed the effects of low-to-moderate arsenic exposure on monocyte adhesion to endothelial cells, platelet activation and platelet-monocyte interactions. We observed that arsenic induces human monocyte adhesion to endothelial cells in vitro. These findings were confirmed ex vivo using a murine organ culture system at concentrations as low as 10 ppb. We found that both cell types need to be exposed to arsenic to maximize monocyte adhesion to the endothelium. This adhesion process is specific to monocyte/endothelium interactions. Hence, no effect of arsenic on platelet activation or platelet/leukocyte interaction was observed. We found that arsenic increases adhesion of mononuclear cells via increased CD29 binding to VCAM-1, an adhesion molecule found on activated endothelial cells. Similar results were observed in vivo, where arsenic-exposed mice exhibit increased VCAM-1 expression on endothelial cells and increased CD29 on circulating monocytes. Interestingly, expression of adhesion molecules and increased binding can be inhibited by antioxidants in vitro and in vivo. Together, these data suggest that arsenic might enhance atherosclerosis by increasing monocyte adhesion to endothelial cells, a process that is inhibited by antioxidants.     

Relationship between endoplasmic reticulum- and Golgi-associated calcium homeostasis and 4-NQO-induced DNA repair in Saccharomyces cerevisiae

Calcium (Ca²⁺) is an important ion that is necessary for the activation of different DNA repair mechanisms. However, the mechanism by which DNA repair and Ca²⁺ homeostasis cooperate remains unclear. We undertook a systems biology approach to verify the relationship between proteins associated with Ca²⁺ homeostasis and DNA repair for Saccharomyces cerevisiae. Our data indicate that Pmr1p, a Ca²⁺ transporter of Golgi complex, interacts with Cod1p, which regulates Ca²⁺ levels in the endoplasmic reticulum (ER), and with Rad4p, which is a nucleotide excision repair (NER) protein. This information was used to construct single and double mutants defective for Pmr1p, Cod1p, and Rad4p followed by cytotoxic, cytostatic, and cell cycle arrest analyses after cell exposure to different concentrations of 4-nitroquinoline 1-oxide (4-NQO). The results indicated that cod1Δ, cod1Δrad4Δ, and cod1Δpmr1Δ strains have an elevated sensitivity to 4-NQO when compared to its wild-type (WT) strain. Moreover, both cod1Δpmr1Δ and cod1Δrad4Δ strains have a strong arrest at G₂/M phases of cell cycle after 4-NQO treatment, while pmr1Δrad4Δ have a similar sensitivity and cell cycle arrest profile when compared to rad4Δ after 4-NQO exposure. Taken together, our results indicate that deletion in Golgi- and ER-associated Ca²⁺ transporters affect the repair of 4-NQO-induced DNA damage.     

The Cytosolic Tail Dipeptide Ile-Met of the Pea Receptor BP80 Is Required for Recycling from the Prevacuole and for Endocytosis

Pea (Pisum sativum) BP80 is a vacuolar sorting receptor for soluble proteins and has a cytosolic domain essential for its intracellular trafficking between the trans-Golgi network and the prevacuole. Based on mammalian knowledge, we introduced point mutations in the cytosolic region of the receptor and produced chimeras of green fluorescent protein fused to the transmembrane domain of pea BP80 along with the modified cytosolic tails. By analyzing the subcellular location of these chimera, we found that mutating Glu-604, Asp-616, or Glu-620 had mild effects, whereas mutating the Tyr motif partially redistributed the chimera to the plasma membrane. Replacing both Ile-608 and Met-609 by Ala (IMAA) led to a massive redistribution of fluorescence to the vacuole, indicating that recycling is impaired. When the chimera uses the alternative route, the IMAA mutation led to a massive accumulation at the plasma membrane. Using Arabidopsis thaliana plants expressing a fluorescent reporter with the full-length sequence of At VSR4, we demonstrated that the receptor undergoes brefeldin A–sensitive endocytosis. We conclude that the receptors use two pathways, one leading directly to the lytic vacuole and the other going via the plasma membrane, and that the Ileu-608 Met-609 motif has a role in the retrieval step in both pathways.     

The MMS22L-TONSL complex mediates recovery from replication stress and homologous recombination

Genome integrity is jeopardized each time DNA replication forks stall or collapse. Here we report the identification of a complex composed of MMS22L (C6ORF167) and TONSL (NFKBIL2) that participates in the recovery from replication stress. MMS22L and TONSL are homologous to yeast Mms22 and plant Tonsoku/Brushy1, respectively. MMS22L-TONSL accumulates at regions of ssDNA associated with distressed replication forks or at processed DNA breaks, and its depletion results in high levels of endogenous DNA double-strand breaks caused by an inability to complete DNA synthesis after replication fork collapse. Moreover, cells depleted of MMS22L are highly sensitive to camptothecin,?a topoisomerase I poison that impairs DNA replication progression. Finally, MMS22L and TONSL are necessary for the efficient formation of RAD51 foci after DNA damage, and their depletion impairs homologous recombination. These results indicate that MMS22L and TONSL are genome caretakers that stimulate the recombination-dependent repair of stalled or collapsed replication forks.     

Septal and lateral wall localization of PBP5, the major D,D‐carboxypeptidase of Escherichia coli,requires substrate recognition and membrane attachment

The distribution of PBP5, the major D,D‐carboxypeptidase in Escherichia coli, was mapped by immunolabelling and by visualization of GFP fusion proteins in wild‐type cells and in mutants lacking one or more D,D‐carboxypeptidases. In addition to being scattered around the lateral envelope, PBP5 was also concentrated at nascent division sites prior to visible constriction. Inhibiting PBP2 activity (which eliminates wall elongation) shifted PBP5 to midcell, whereas inhibiting PBP3 (which aborts divisome invagination) led to the creation of PBP5 rings at positions of preseptal wall formation, implying that PBP5 localizes to areas of ongoing peptidoglycan synthesis. A PBP5(S44G) active site mutant was more evenly dispersed, indicating that localization required enzyme activity and the availability of pentapeptide substrates. Both the membrane bound and soluble forms of PBP5 converted pentapeptides to tetrapeptides in vitro and in vivo, and the enzymes accepted the same range of substrates, including sacculi, Lipid II, muropeptides and artificial substrates. However, only the membrane‐bound form localized to the developing septum and restored wild‐type rod morphology to shape defective mutants, suggesting that the two events are related. The results indicate that PBP5 localization to sites of ongoing peptidoglycan synthesis is substrate dependent and requires membrane attachment.     

Would SYNTAX have been a positive trial if XIENCE V had been used instead of TAXUS?: A meta-analysis of a first-generation vs. a second-generation drug-eluting stent system

Treatment options for coronary revascularisation include percutaneous coronary intervention (PCI) and coronary artery bypass grafting (CABG). In the ‘synergy between PCI with TAXUS and cardiac surgery (SYNTAX)’ trial, PCI and CABG using state-of-the-art techniques (using paclitaxel-eluting stents and arterial grafts, respectively) were compared in the treatment of complex coronary artery disease. In Syntax, PCI was inferior to CABG at one year, entirely due to an increased repeat intervention rate. We hypothesised that the use of a superior drug-eluting stent system could reduce the need for repeat intervention. (Neth Heart J 2010;18:451-3.)     

Foetal and adult cardiomyocyte progenitor cells have different developmental potential

In the past years, cardiovascular progenitor cells have been isolated from the human heart and characterized. Up to date, no studies have been reported in which the developmental potential of foetal and adult cardiovascular progenitors was tested simultaneously. However, intrinsic differences will likely affect interpretations regarding progenitor cell potential and application for regenerative medicine. Here we report a direct comparison between human foetal and adult heart‐derived cardiomyocyte progenitor cells (CMPCs). We show that foetal and adult CMPCs have distinct preferences to differentiate into mesodermal lineages. Under pro‐angiogenic conditions, foetal CMPCs form more endothelial but less smooth muscle cells than adult CMPCs. Foetal CMPCs can also develop towards adipocytes, whereas neither foetal nor adult CMPCs show significant osteogenic differentiation. Interestingly, although both cell types differentiate into heart muscle cells, adult CMPCs give rise to electrophysiologically more mature cardiomyocytes than foetal CMPCs. Taken together, foetal CMPCs are suitable for molecular cell biology and developmental studies. The potential of adult CMPCs to form mature cardiomyocytes and smooth muscle cells may be essential for cardiac repair after transplantation into the injured heart.