Matched sizes of activating and inhibitory receptor/ligand pairs are required for optimal signal integration by human natural killer cells

It has been suggested that receptor-ligand complexes segregate or co-localise within immune synapses according to their size, and this is important for receptor signaling. Here, we set out to test the importance of receptor-ligand complex dimensions for immune surveillance of target cells by human Natural Killer (NK) cells. NK cell activation is regulated by integrating signals from activating receptors, such as NKG2D, and inhibitory receptors, such as KIR2DL1. Elongating the NKG2D ligand MICA reduced its ability to trigger NK cell activation. Conversely, elongation of KIR2DL1 ligand HLA-C reduced its ability to inhibit NK cells. Whereas normal-sized HLA-C was most effective at inhibiting activation by normal-length MICA, only elongated HLA-C could inhibit activation by elongated MICA. Moreover, HLA-C and MICA that were matched in size co-localised, whereas HLA-C and MICA that were different in size were segregated. These results demonstrate that receptor-ligand dimensions are important in NK cell recognition, and suggest that optimal integration of activating and inhibitory receptor signals requires the receptor-ligand complexes to have similar dimensions.     

A Comparative Study Between the Antibacterial Effect of Nisin and Nisin-Loaded Chitosan/Alginate Nanoparticles on the Growth of Staphylococcus aureus in Raw and Pasteurized Milk Samples

The aim of this study was to evaluate the antibacterial effect of nisin-loaded chitosan/alginate nanoparticles as a novel antibacterial delivery vehicle. The nisin-loaded nanoparticles were prepared using colloidal dispersion of the chitosan/alginate polymers in the presence of nisin. After the preparation of the nisin-loaded nanoparticles, their physicochemical properties such as size, shape, and zeta potential of the formulations were studied using scanning electron microscope and nanosizer instruments, consecutively. FTIR and differential scanning calorimetery studies were performed to investigate polymer–polymer or polymer–protein interactions. Next, the release kinetics and entrapment efficiency of the nisin-loaded nanoparticles were examined to assess the application potential of these formulations as a candidate vector. For measuring the antibacterial activity of the nisin-loaded nanoparticles, agar diffusion and MIC methods were employed. The samples under investigation for total microbial counts were pasteurized and raw milks each of which contained the nisin-loaded nanoparticles and inoculated Staphylococcus aureus (ATCC 19117 at 10⁶ CFU/mL), pasteurized and raw milks each included free nisin and S. aureus (10⁶ CFU/mL), and pasteurized and raw milks each had S. aureus (10⁶ CFU/mL) in as control. Total counts of S. aureus were measured after 24 and 48 h for the pasteurized milk samples and after the time intervals of 0, 6, 10, 14, 18, and 24 h for the raw milk samples, respectively. According to the results, entrapment efficiency of nisin inside of the nanoparticles was about 90–95%. The average size of the nanoparticles was 205 nm, and the average zeta potential of them was ?47 mV. In agar diffusion assay, an antibacterial activity (inhibition zone diameter, at 450 IU/mL) about 2 times higher than that of free nisin was observed for the nisin-loaded nanoparticles. MIC of the nisin-loaded nanoparticles (0.5 mg/mL) was about four times less than that of free nisin (2 mg/mL). Evaluation of the kinetic of the growth of S. aureus based on the total counts in the raw and pasteurized milks revealed that the nisin-loaded nanoparticles were able to inhibit more effectively the growth of S. aureus than free nisin during longer incubation periods. In other words, the decrease in the population of S. aureus for free nisin and the nisin-loaded nanoparticles in pasteurized milk was the same after 24 h of incubation while lessening in the growth of S. aureus was more marked for the nisin-loaded nanoparticles than the samples containing only free nisin after 48 h of incubation. Although the same growth reduction profile in S. aureus was noticed for free nisin and the nisin-loaded nanoparticles in the raw milk up to 14 h of incubation, after this time the nisin-loaded nanoparticles showed higher growth inhibition than free nisin. Since, generally, naked nisin has greater interactions with the ingredients present in milk samples in comparison with the protected nisin. Therefore, it is concluded that the antibacterial activity of nisin naturally decreases more during longer times of incubation than the protected nisin with the chitosan/alginate nanoparticles. Consequently, this protection increases and keeps antibacterial efficiency of nisin in comparison with free nisin during longer times of storage. These results can pave the way for further research and use of these nanoparticles as new antimicrobial agents in various realms of dairy products.     

Reprograming of gut microbiome energy metabolism by the FUT2 Crohn's disease risk polymorphism

Fucosyltransferase 2 (FUT2) is an enzyme that is responsible for the synthesis of the H antigen in body fluids and on the intestinal mucosa. The H antigen is an oligosaccharide moiety that acts as both an attachment site and carbon source for intestinal bacteria. Non-secretors, who are homozygous for the loss-of-function alleles of FUT2 gene (sese), have increased susceptibility to Crohn''s disease (CD). To characterize the effect of FUT2 polymorphism on the mucosal ecosystem, we profiled the microbiome, meta-proteome and meta-metabolome of 75 endoscopic lavage samples from the cecum and sigmoid of 39 healthy subjects (12 SeSe, 18 Sese and 9 sese). Imputed metagenomic analysis revealed perturbations of energy metabolism in the microbiome of non-secretor and heterozygote individuals, notably the enrichment of carbohydrate and lipid metabolism, cofactor and vitamin metabolism and glycan biosynthesis and metabolism-related pathways, and the depletion of amino-acid biosynthesis and metabolism. Similar changes were observed in mice bearing the FUT2^−/− genotype. Metabolomic analysis of human specimens revealed concordant as well as novel changes in the levels of several metabolites. Human metaproteomic analysis indicated that these functional changes were accompanied by sub-clinical levels of inflammation in the local intestinal mucosa. Therefore, the colonic microbiota of non-secretors is altered at both the compositional and functional levels, affecting the host mucosal state and potentially explaining the association of FUT2 genotype and CD susceptibility.     

SRPK1 inhibition in vivo: modulation of VEGF splicing and potential treatment for multiple diseases

SRPK1 (serine-arginine protein kinase 1) is a protein kinase that specifically phosphorylates proteins containing serine-arginine-rich domains. Its substrates include a family of SR proteins that are key regulators of mRNA AS (alternative splicing). VEGF (vascular endothelial growth factor), a principal angiogenesis factor contains an alternative 3' splice site in the terminal exon that defines a family of isoforms with a different amino acid sequence at the C-terminal end, resulting in anti-angiogenic activity in the context of VEGF165-driven neovascularization. It has been shown recently in our laboratories that SRPK1 regulates the choice of this splice site through phosphorylation of the splicing factor SRSF1 (serine/arginine-rich splicing factor 1). The present review summarizes progress that has been made to understand how SRPK1 inhibition may be used to manipulate the balance of pro- and anti-angiogenic VEGF isoforms in animal models in vivo and therefore control abnormal angiogenesis and other pathophysiological processes in multiple disease states.     

Ameliorating the Effect of Pioglitazone on LPS-Induced Inflammation of Human Oligodendrocyte Progenitor Cells

Oligodendrocyte progenitor cells (OPCs) are appropriate model cells for studying the progress of neurodegenerative disorders and evaluation of pharmacological efficacies of small molecules for treatment of these disorders. Here, we focused on the therapeutic role of Pioglitazone, which is a selective agonist of peroxisome proliferator-activated receptor gamma (PPARγ), a respective nuclear receptor in inflammatory responses. Human embryonic stem cell-derived OPCs were pretreated by Pioglitazone at differing concentrations. Pretreated OPCs were further examined after induction of inflammation by LPS. Interestingly, Pioglitazone reversed the inflammatory conditions and enhanced OPC viability. Data showed that Pioglitazone reduced Nitric Oxide (NO) production. Moreover, Pioglitazone enhanced cell viability through distinct mechanisms including reduction of apoptosis and regulation of cell cycle markers. This study demonstrated that NO induces apoptosis through FOXO1 and degradation of β-catenin, while the presence of Pioglitazone inhibited these effects in rescuing human OPCs from apoptosis. Also, Pioglitazone did not show a significant influence on mRNA levels of TLR2, TRL4, and TNFα. Furthermore, simultaneous treatment of Pioglitazone with CHIR, a GSKβ inhibitor, facilitated anti-apoptotic responses of OPCs. Taken together, therapy with Pioglitazone represents a novel potential drug in alleviating the loss of OPCs in neurodegenerative conditions.     

Application of a quartz crystal nanobalance to the molecularly imprinted recognition of phenylalanine in solution

A quartz crystal nanobalance (QCN) biosensor was developed for the selective determination of phenylalanine (Phe) in aqueous solutions. A Phe imprinted copolymer was synthesized using polyacrylonitrile and acrylic acid [poly(AN-co-AA)]. The copolymer was then coated on quartz crystal electrode to form complementary structures for the template recognition of Phe. The composite electrode was then used to determine Phe levels in solution. Determinations were based on frequency shifts of molecularly imprinted polymer (MIP) modified quartz crystal electrode caused by Phe adsorption. The frequency shifts were linearly dependent on Phe concentration over the range 50∼500 mgL⁻¹. The results obtained show that the imprinted poly(AN-co-AA) modified biosensor had higher sensitivity (0.5839 Hz/mgL⁻¹) than a non-molecularly imprinted copolymer (0.2724 Hz/mgL⁻¹). Furthermore, good reproducibility, R.S.D. = 1.84% (n = 7) was observed, and the detection limit was 45 mgL⁻¹. The selectivity of the imprinted poly(AN-co-AA) modified biosensor was examined using a number of analytes similar to Phe, i.e., dopamine (DA), ascorbic acid (AscA), vanillylmandelic acid (VMA), uric acid (UA), tryptophan (Trp), and tyrosine (Tyr), and the results obtained showed a size dependent selective effect.     

Comparison between MDCK and MDCK-SIAT1 cell lines as preferred host for cell culture-based influenza vaccine production

Objectives

To evaluate MDCK and MDCK-SIAT1 cell lines for their ability to produce the yield of influenza virus in different Multiplicities of Infection.

Results

Yields obtained for influenza virus H1N1 grown in MDCK-SIAT1 cell was almost the same as MDCK; however, H3N2 virus grown in MDCK-SIAT1 had lower viral titers in comparison with MDCK cells. The optimized MOIs to infect the cells on plates and microcarrier were selected 0.01 and 0.1 for H1N1 and 0.001 and 0.01 for H3N2, respectively.

Conclusions

MDCK-SIAT1 cells may be considered as an alternative mean to manufacture cell-based flu vaccine, especially for the human strains (H1N1), due to its antigenic stability and high titer of influenza virus production.

     

Targeting human glutathione transferase A3-3 attenuates progesterone production in human steroidogenic cells

hGSTA3-3 (human Alpha-class glutathione transferase 3-3) efficiently catalyses steroid Delta(5)-Delta(4) double-bond isomerization in vitro, using glutathione as a cofactor. This chemical transformation is an obligatory reaction in the biosynthesis of steroid hormones and follows the oxidation of 3beta-hydroxysteroids catalysed by 3beta-HSD (3beta-hydroxysteroid dehydrogenase). The isomerization has commonly been ascribed to a supplementary function of 3beta-HSD. The present study is the first to provide evidence that hGSTA3-3 contributes to this step in steroid hormone biosynthesis in complex cellular systems. First, we find glutathione-dependent Delta(5)-Delta(4) isomerase activity in whole-cell extracts prepared from human steroidogenic cells. Secondly, effective inhibitors of hGSTA3-3 dramatically decrease the conversion of Delta(5)-androstene-3,17-dione into Delta(4)-androstene-3,17-dione in cell lysates. Thirdly, we show that RNAi (RNA interference) targeting hGSTA3-3 expression decreases by 30% the forskolin-stimulated production of the steroid hormone progesterone in a human placental cell line. This effect is achieved at low concentrations of two small interfering RNAs directed against distinct regions of hGSTA3-3 mRNA, and is weaker in unstimulated cells, in which hGSTA3-3 expression is low. The results concordantly show that hGSTA3-3 makes a significant contribution to the double-bond isomerization necessary for steroid hormone biosynthesis and thereby complements the indispensable 3beta-hydroxysteroid oxidoreductase activity of 3beta-HSD. The results indicate that the lower isomerase activity of 3beta-HSD is insufficient for maximal rate of cellular sex hormone production and identify hGSTA3-3 as a possible target for pharmaceutical intervention in steroid hormone-dependent diseases.     

Interactions of human serum albumin with bioactive 3<Emphasis Type="Italic">H</Emphasis>-imidazo[4,5-<Emphasis Type="Italic">a</Emphasis>]acridines: Insights from fluorescence spectroscopic studies

Several 3H-imidazo[4,5-a]acridine derivatives were conveniently synthesized by the reaction of imidazo[4,5-a]acridones in boiling POCl3. The imidazoacridones were obtained by rearrangement of 3H-imidazo[4',5':3,4]benzo[c]isoxazoles in concentrated sulfuric acid containing nitrous acid at room temperature. The structures of all newly synthesized compounds were confirmed by IR, ¹H NMR, and mass spectral data. The interactions of 3H-imidazo[4,5-a]acridines with human serum albumin (HSA) were studied by fluorescence spectroscopy. The binding of 3H-imidazo[4,5-a]acridines quenches the HSA fluorescence, revealing a 1: 1 interaction with a binding constant of about 2.34 × 10⁵–3.16 × 10⁶ M^–1. A decrease in fluorescence intensity at 339 nm, when excited at 280 nm, is attributed to changes in the environment of the protein fluorophores caused by the presence of the ligand. The differences in interactions of 3H-imidazo[4,5-a]acridines with HSA were observed using spectrofluorimetry technique.     

The Antibacterial Activity of Acetic Acid against Biofilm-Producing Pathogens of Relevance to Burns Patients

Introduction

Localised infections, and burn wound sepsis are key concerns in the treatment of burns patients, and prevention of colonisation largely relies on biocides. Acetic acid has been shown to have good antibacterial activity against various planktonic organisms, however data is limited on efficacy, and few studies have been performed on biofilms.

Objectives

We sought to investigate the antibacterial activity of acetic acid against important burn wound colonising organisms growing planktonically and as biofilms.

Methods

Laboratory experiments were performed to test the ability of acetic acid to inhibit growth of pathogens, inhibit the formation of biofilms, and eradicate pre-formed biofilms.

Results

Twenty-nine isolates of common wound-infecting pathogens were tested. Acetic acid was antibacterial against planktonic growth, with an minimum inhibitory concentration of 0.16–0.31% for all isolates, and was also able to prevent formation of biofilms (at 0.31%). Eradication of mature biofilms was observed for all isolates after three hours of exposure.

Conclusions

This study provides evidence that acetic acid can inhibit growth of key burn wound pathogens when used at very dilute concentrations. Owing to current concerns of the reducing efficacy of systemic antibiotics, this novel biocide application offers great promise as a cheap and effective measure to treat infections in burns patients.