Myosin Vb is required for trafficking of the cystic fibrosis transmembrane conductance regulator in Rab11a-specific apical recycling endosomes in polarized human airway epithelial cells

Cystic fibrosis transmembrane conductance regulator (CFTR)-mediated Cl(-) secretion across fluid-transporting epithelia is regulated, in part, by modulating the number of CFTR Cl(-) channels in the plasma membrane by adjusting CFTR endocytosis and recycling. However, the mechanisms that regulate CFTR recycling in airway epithelial cells remain unknown, at least in part, because the recycling itineraries of CFTR in these cells are incompletely understood. In a previous study, we demonstrated that CFTR undergoes trafficking in Rab11a-specific apical recycling endosomes in human airway epithelial cells. Myosin Vb is a plus-end-directed, actin-based mechanoenzyme that facilitates protein trafficking in Rab11a-specific recycling vesicles in several cell model systems. There are no published studies examining the role of myosin Vb in airway epithelial cells. Thus, the goal of this study was to determine whether myosin Vb facilitates CFTR recycling in polarized human airway epithelial cells. Endogenous CFTR formed a complex with endogenous myosin Vb and Rab11a. Silencing myosin Vb by RNA-mediated interference decreased the expression of wild-type CFTR and DeltaF508-CFTR in the apical membrane and decreased CFTR-mediated Cl(-) secretion across polarized human airway epithelial cells. A recombinant tail domain fragment of myosin Vb attenuated the plasma membrane expression of CFTR by arresting CFTR recycling. The dominant-negative effect was dependent on the ability of the myosin Vb tail fragment to interact with Rab11a. Taken together, these data indicate that myosin Vb is required for CFTR recycling in Rab11a-specific apical recycling endosomes in polarized human airway epithelial cells.     

Promoter methylation and functional variants in arachidonate 5-lipoxygenase and forkhead box protein O1 genes associated with coronary artery disease

Coronary artery disease (CAD) is a multifactorial chronic inflammatory disease, which is the most common form of heart disease. This is one of the main causes of death in the United States. Inflammation is one of the key drivers of atherosclerotic plaque development. Forkhead box protein O1 (FOXO1s) family and 5-lipoxygenase make an important contribution to atherosclerosis. The aim of this study was to investigate the methylation pattern and polymorphism analysis of FOXO1 and arachidonate 5-lipoxygenase (ALOX5) promoter genes. We studied 50 patients with CAD and 50 age- and sex-matched healthy controls by high resolution melt technique. Overall, we found significant differences between patients and controls in terms of the promoter methylation of ALOX5 (P > 0.05). But there was no significant difference in FOXO1 promoter methylation between patient and controls. Single nucleotide polymorphisms genotyping of rs12762303 and rs2297627, in ALOX5 and FOXO1 genes were demonstrated a significant correlation between mutant allele and the risk of CAD, respectively. Furthermore, there were significant associations between CT + CC genotype and ALOX5 expression. Our findings demonstrated functional effects of single nucleotide polymorphisms (SNPs) and DNA methylation in ALOX5 on mentioned genes expression and they resulted in CAD progression.     

Regulatory B and T cells in infections

The role of T and B lymphocytes, as antigen-specific effector immune cells playing an essential role in host defense against pathogens, is well recognized. Over the last decade, these lymphocytes have however also emerged as key regulatory components of the immune system, able to prevent various immunopathologies due to excessive inflammatory responses. These regulatory T (Treg) and B (Breg) cells, endowed with anti-inflammatory properties, operate via both antigen-specific and non-specific mechanisms and mainly develop during chronic infections. Here, we discuss the role of Treg and Breg lymphocytes in various infectious diseases, in experimental murine models and in human.     

Immune mobilization of autologous blood progenitor cells: direct influence on the cellular subsets collected

Background aimsA phase I trial examined the ability of immunotherapy to mobilize progenitor and activated T cells.MethodsInterleukin (IL)-2 was administered subcutaneously for 11 days, with granulocyte (G)-colony-stimulating factor (CSF) (5 mcg/kg/day) and granulocyte–macrophage (GM)-CSF (7.5 mcg/kg/day) added for the last 5 days. Leukapheresis was initiated on day 11. Thirteen patients were treated (myeloma n = 11, non-Hodgkin's lymphoma n = 2).ResultsToxicities were minimal. IL-2 was stopped in two patients because of capillary leak (n = 1) and diarrhea (n = 1). Each patient required 2.5 leukaphereses (median; range 1–3) to collect 3.2 × 10⁶ CD34⁺ cells/kg (median; range 1.9–6.6 × 10⁶/kg). Immune mobilization increased the number of CD3⁺ CD8⁺ T cells (P = 0.002), CD56⁺ natural killer (NK) cells (P = 0.0001), CD8⁺ CD56⁺ T cells (P = 0.002) and CD4⁺ CD25⁺ cells (P = 0.0001) compared with cancer patients mobilized with G-CSF alone. There was increased lysis of myeloma cells after 7 days (P = 0.03) or 11 days (P = 0.02). The maximum tolerated dose of IL-2 was 1 × 10⁶ IU/m²/day.ConclusionsImmune mobilization is well tolerated with normal subsequent marrow engraftment. As cells within the graft influence lymphocyte recovery, an increased number of functional lymphocytes may result in more rapid immune reconstitution.     

Preclinical Evaluation of Novel Triphenylphosphonium Salts with Broad-Spectrum Activity

Background

Recently, there has been a surge of interest in developing compounds selectively targeting mitochondria for the treatment of neoplasms. The critical role of mitochondria in cellular metabolism and respiration supports this therapeutic rationale. Dysfunction in the processes of energy production and metabolism contributes to attenuation of response to pro-apoptotic stimuli and increased ROS production both of which are implicated in the initiation and progression of most human cancers.

Methodology/Principal Findings

A high-throughput MTT-based screen of over 10,000 drug-like small molecules for anti-proliferative activity identified the phosphonium salts TP187, 197 and 421 as having IC₅₀ concentrations in the submicromolar range. TP treatment induced cell cycle arrest independent of p53 status, as determined by analysis of DNA content in propidium iodide stained cells. In a mouse model of human breast cancer, TP-treated mice showed significantly decreased tumor growth compared to vehicle or paclitaxel treated mice. No toxicities or organ damage were observed following TP treatment. Immunohistochemical staining of tissue sections from TP187-treated tumors demonstrated a decrease in cellular proliferation and increased caspase-3 cleavage. The fluorescent properties of analog TP421 were exploited to assess subcellular uptake of TP compounds, demonstrating mitochondrial localization. Following mitochondrial uptake cells exhibited decreased oxygen consumption and concomittant increase in mitochondrial superoxide production. Proteomics analysis of results from a 600 target antibody microarray demonstrated that TP compounds significantly affected signaling pathways relevant to growth and proliferation.

Conclusions/Significance

Through our continued interest in designing compounds targeting cancer-cell metabolism, the Warburg effect, and mitochondria we recently discovered a series of novel, small-molecule compounds containing a triphenylphosphine moiety that show remarkable activity in a panel of cancer cell lines as well as in a mouse model of human breast cancer. The mechanism of action includes mitochondrial localization causing decreased oxygen consumption, increased superoxide production and attenuated growth factor signaling.     

Decoration of Outer Membrane Vesicles with Multiple Antigens by Using an Autotransporter Approach

Outer membrane vesicles (OMVs) are spherical nanoparticles that naturally shed from Gram-negative bacteria. They are rich in immunostimulatory proteins and lipopolysaccharide but do not replicate, which increases their safety profile and renders them attractive vaccine vectors. By packaging foreign polypeptides in OMVs, specific immune responses can be raised toward heterologous antigens in the context of an intrinsic adjuvant. Antigens exposed at the vesicle surface have been suggested to elicit protection superior to that from antigens concealed inside OMVs, but hitherto robust methods for targeting heterologous proteins to the OMV surface have been lacking. We have exploited our previously developed hemoglobin protease (Hbp) autotransporter platform for display of heterologous polypeptides at the OMV surface. One, two, or three of the Mycobacterium tuberculosis antigens ESAT6, Ag85B, and Rv2660c were targeted to the surface of Escherichia coli OMVs upon fusion to Hbp. Furthermore, a hypervesiculating ΔtolR ΔtolA derivative of attenuated Salmonella enterica serovar Typhimurium SL3261 was generated, enabling efficient release and purification of OMVs decorated with multiple heterologous antigens, exemplified by the M. tuberculosis antigens and epitopes from Chlamydia trachomatis major outer membrane protein (MOMP). Also, we showed that delivery of Salmonella OMVs displaying Ag85B to antigen-presenting cells in vitro results in processing and presentation of an epitope that is functionally recognized by Ag85B-specific T cell hybridomas. In conclusion, the Hbp platform mediates efficient display of (multiple) heterologous antigens, individually or combined within one molecule, at the surface of OMVs. Detection of antigen-specific immune responses upon vesicle-mediated delivery demonstrated the potential of our system for vaccine development.     

Differential Effects of Ornithine Enantiomers on the Activity of Anti‐oxidant Enzymes,Polyamines Content,and Growth of Tobacco Cells under Osmotic Stresses

Ornithine (Orn) plays an essential role in the metabolism of plant cells through incorporation in polyamines biosynthesis, the urea cycle and nitrogen metabolism. Herein, we show that Orn enantiomers have different effects on anti‐oxidant enzymes activities, polyamines and proline biosynthesis and also an alleviation effect of osmotic stresses on tobacco cells. The type of stress has a significant impact on the function of L‐ and D‐Orn for improvement of the stress effect on the cells. Under saline conditions, both enantiomers restored cell growth, though D‐Orn was more beneficial to some extent. This was accompanied with a higher biosynthesis of putrescine, proline, and up‐regulated activity of certain anti‐oxidant enzymes by D‐Orn. Under drought stress conditions, a distinct differential behavior emerged and only L‐Orn showed an alleviative effect on the cell growth. Regulation of hydrogen peroxide content via the activity of catalase/peroxidase and production of osmolytes, e.g., proline and fructans, was dependent on the type of enantiomers. Activity of anti‐oxidant enzymes and production of malondialdehyde from cell membranes were differently regulated following treatment with either Orn enantiomer. The results suggest that management of H₂O₂ content is a determining feature of the function of Orn enantiomers in tobacco cells under salinity and drought stress conditions. Chirality 25:583–588, 2013. © 2013 Wiley Periodicals, Inc.     

Complete Genome Sequence and Genome Analysis of Eggplant mottled dwarf virus‐Iranian Isolate

The full‐length nucleotide sequence of the Iranian isolate of Eggplant mottled dwarf virus (EMDV), a phytorhabdovirus, was determined using the random polymerase chain reaction method (rPCR) followed by PCR with specific primers to fill in the gaps. The negative‐sense RNA genome of the Iranian isolate of EMDV contains 13154 nucleotides and seven open‐reading frames (ORFs) in the order 3′‐leader‐N‐X‐P‐Y‐M‐G‐L‐trailer‐5′. These ORFs encode the nucleocapsid, X protein (of unknown function), phosphoprotein, Y protein (putative movement protein), matrix protein, glycoprotein and RNA‐dependent RNA polymerase, respectively. EMDV has a 199 nt 3′ leader RNA and a 151 nt 5′ trailer, and the ORFs are separated by conserved intergenic sequences. Phylogenetic analyses indicate that EMDV is most closely related to Potato yellow dwarf virus, which has a distinctly different geographical distribution.     

Effect of attenuation of Treg during BCG immunization on anti-mycobacterial Th1 responses and protection against Mycobacterium tuberculosis

Background

The functional equilibrium between natural regulatory T cells (Treg) and effector T cells can affect the issue of numerous infections. In unvaccinated mice, the influence of Treg in the control of primary infection with mycobacteria remains controversial.

Methodology

Here, we evaluated the role of Treg during prophylactic vaccination with Mycobacterium bovis BCG (Bacillus Calmette-Guérin) on the induction of T cell responses and on the protective effect against subsequent M. tuberculosis challenge in mice.

Principal Findings

We demonstrated that, subsequent to BCG injection, Treg were recruited to the draining lymph nodes and negatively control anti-mycobacterial CD4⁺ — but not CD8⁺ — T-cell responses. Treatment of BCG-immunized mice with an anti-CD25 mAb (PC61) induced an increase IFN-γ response against both subdominant and immunodominant regions of the protective immunogen TB10.4. In Treg-attenuated, BCG-immunized mice, which were then infected with M. tuberculosis, the lung mycobacterial load was significantly, albeit moderately, reduced compared to the control mice.

Conclusions

Our results provide the first demonstration that attenuation of Treg subset concomitant to BCG vaccination has a positive, yet limited, impact on the protective capacity of this vaccine against infection with M. tuberculosis. Thus, for rational design of improved BCG, it should be considered that, although the action of Treg does not represent the major cause of the limited efficiency of BCG, the impact of this cell population on the subsequent control of M. tuberculosis growth is significant and measurable.