Zebrafish modeling mimics developmental phenotype of patients with RAPGEF1 mutation.

RAPGEF1 is a guanine nucleotide exchange factor responsible for transmitting extracellular signals to the Ras family of GTPase located at the inside of membrane. Here, we report for the first time a homozygous mutation of RAPGEF1 in a consanguineous family with two siblings affected by neuropsychiatric disorder. To confirm the correlation of the mutation and the phenotype, we utilized in silico analysis and established a zebrafish model. Survival rate was reduced in the rapgef1a-knockdown model, and the zebrafish showed global morphological abnormalities, particularly of brain and blood vessels. Co-application of human RAPGEF1 wildtype mRNA effectively rescued the abnormal phenotype, while that of RAPGEF1 mRNA carrying the human mutation did not. This work is the first report of a human Mendelian disease associated with RAPGEF1 and the first report of a zebrafish model built for this gene. The phenotype of zebrafish model provides further evidence that defective RAPGEF1 may lead to global developmental delay in human patients.

A Recombinant Fragment of Human Surfactant Protein D Suppresses Basophil Activation and T-Helper Type 2 and B-Cell Responses in Grass Pollen-induced Allergic Inflammation.

RATIONALE: Recombinant fragment of human surfactant protein D (rfhSP-D) has been shown to suppress house dust mite- and Aspergillus fumigatus-induced allergic inflammation in murine models. OBJECTIVES: We sought to elucidate the effect of rfhSP-D on high-affinity IgE receptor- and CD23-mediated, grass pollen-induced allergic inflammatory responses. METHODS: rfhSP-D, containing homotrimeric neck and lectin domains, was expressed in Escherichia coli BL21(λDE3)pLysS cells. Peripheral blood mononuclear cells and sera were obtained from individuals with grass pollen allergy (n = 27). The effect of rfhSP-D on basophil activation and histamine release was measured by flow cytometry. IgE-facilitated allergen binding and presentation were assessed by flow cytometry. T-helper cell type 2 (Th2) cytokines were measured in cell culture supernatants. The effect of rfhSP-D on IgE production by B cells when stimulated with CD40L, IL-4, and IL-21 was also determined. MEASUREMENTS AND MAIN RESULTS: rfhSP-D bound to Phleum pratense in a dose- and calcium-dependent manner. Allergen-induced basophil responsiveness and histamine release were inhibited in the presence of rfhSP-D, as measured by CD63, CD203c (P = 0.0086, P = 0.04205), and intracellularly labeled diamine oxidase (P = 0.0003, P = 0.0148). The binding of allergen-IgE complexes to B cells was reduced by 51% (P = 0.002) in the presence of rfhSP-D. This decrease was concomitant with reduction in CD23 expression on B cells (P < 0.001). rfhSP-D suppressed allergen-driven CD27-CD4+CRTh2+ T-cell proliferation (P < 0.01), IL-4, and IL-5 levels (all P < 0.01). Moreover, rfhSP-D inhibited CD40L/IL-4- and IL-21-mediated IgE production (77.12%; P = 0.02) by B cells. CONCLUSIONS: For the first time, to our knowledge, we show that rfhSP-D inhibited allergen-induced basophil responses at a single-cell level and suppressed CD23-mediated facilitated allergen presentation and Th2 cytokine production. In addition, rfhSP-D inhibited IgE synthesis by B cells, which is also a novel observation.

Biallelic truncating mutations in FMN2, encoding the actin-regulatory protein Formin 2, cause nonsyndromic autosomal-recessive intellectual disability.

Dendritic spines represent the major site of neuronal activity in the brain; they serve as the receiving point for neurotransmitters and undergo rapid activity-dependent morphological changes that correlate with learning and memory. Using a combination of homozygosity mapping and next-generation sequencing in two consanguineous families affected by nonsyndromic autosomal-recessive intellectual disability, we identified truncating mutations in formin 2 (FMN2), encoding a protein that belongs to the formin family of actin cytoskeleton nucleation factors and is highly expressed in the maturing brain. We found that FMN2 localizes to punctae along dendrites and that germline inactivation of mouse Fmn2 resulted in animals with decreased spine density; such mice were previously demonstrated to have a conditioned fear-learning defect. Furthermore, patient neural cells derived from induced pluripotent stem cells showed correlated decreased synaptic density. Thus, FMN2 mutations link intellectual disability either directly or indirectly to the regulation of actin-mediated synaptic spine density.

Identification of a novel homozygous TRAPPC9 gene mutation causing non-syndromic intellectual disability, speech disorder, and secondary microcephaly.

TRAPPC9 gene mutations have been linked recently to autosomal recessive mental retardation 13 (MRT13; MIM#613192) with only eight families reported world-wide. We assessed patients from two consanguineous pedigrees of Pakistani descent with non-syndromic intellectual disability and postnatal microcephaly through whole exome sequencing (WES) and cosegregation analysis. Here we report six further patients from two pedigrees with homozygous TRAPPC9 gene mutations, the novel nonsense mutation c.2065G>T (p.E689*) and the previously identified nonsense mutation c.1423C>T (p.R475*). We provide an overview of previously reported clinical features and highlight common symptoms and variability of MRT13. Common findings are intellectual disability and absent speech, and frequently microcephaly, motor delay and pathological findings on MRI including diminished cerebral white matter volume are present. Mutations in TRAPPC9 should be considered in non-syndromic autosomal recessive intellectual disability with severe speech disorder.

Serum indoleamine 2,3-dioxygenase activity is associated with reduced immunogenicity following vaccination with MVA85A.

BACKGROUND: There is an urgent need for improved vaccines to protect against tuberculosis. The currently available vaccine Bacille Calmette-Guerin (BCG) has varying immunogenicity and efficacy across different populations for reasons not clearly understood. MVA85A is a modified vaccinia virus expressing antigen 85A from Mycobacterium tuberculosis which has been in clinical development since 2002 as a candidate vaccine to boost BCG-induced protection. A recent efficacy trial in South African infants failed to demonstrate enhancement of protection over BCG alone. The immunogenicity was lower than that seen in UK trials. The enzyme Indoleamine 2,3-dioxygenase (IDO) catalyses the first and rate-limiting step in the breakdown of the essential amino acid tryptophan. T cells are dependent on tryptophan and IDO activity suppresses T-cell proliferation and function. METHODS: Using samples collected during phase I trials with MVA85A across the UK and South Africa we have investigated the relationship between vaccine immunogenicity and IDO using IFN-γ ELISPOT, qPCR and liquid chromatography mass spectrometry. RESULTS: We demonstrate an IFN-γ dependent increase in IDO mRNA expression in peripheral blood mononuclear cells (PBMC) following MVA85A vaccination in UK subjects. IDO mRNA correlates positively with the IFN-γ ELISPOT response indicating that vaccine specific induction of IDO in PBMC is unlikely to limit the development of vaccine specific immunity. IDO activity in the serum of volunteers from the UK and South Africa was also assessed. There was no change in serum IDO activity following MVA85A vaccination. However, we observed higher baseline IDO activity in South African volunteers when compared to UK volunteers. In both UK and South African serum samples, baseline IDO activity negatively correlated with vaccine-specific IFN-γ responses, suggesting that IDO activity may impair the generation of a CD4+ T cell memory response. CONCLUSIONS: Baseline IDO activity was higher in South African volunteers when compared to UK volunteers, which may represent a potential mechanism for the observed variation in vaccine immunogenicity in South African and UK populations and may have important implications for future vaccination strategies. TRIAL REGISTRATION: Trials are registered at ClinicalTrials.gov; UK cohort NCT00427830, UK LTBI cohort NCT00456183, South African cohort NCT00460590, South African LTBI cohort NCT00480558.

Anti-hyperglycemic activity of rutin in streptozotocin-induced diabetic rats: an effect mediated through cytokines, antioxidants and lipid biomarkers.

Administration of rutin (50 and 100 mg/kg) and pioglitazone (10 mg/kg) orally for 3 weeks treatment significantly improved body weight, reduced plasma glucose and glycosylated hemoglobin, pro-inflammatory cytokines (IL-6 and TNF-alpha), restored the depleted liver antioxidant status and serum lipid profile in high fat diet + streptozotocin induced type 2 diabetic rats. Rutin treatment also improved histo-architecture of beta islets and reversed hypertrophy of hepatocytes. Rutin exhibited significant antidiabetic activity, presumably by inhibiting inflammatory cytokines, improving antioxidant and plasma lipid profiles in High fat diet + streptozotocin induced type 2 diabetic model and may be useful as a diabetic modulator along with standard antidiabetic drugs. However, such effects need to be confirmed on human subjects in clinical condition.

Mapping the interaction sites of human and avian influenza A viruses and complement factor H.

The complement system is an innate immune mechanism against microbial infections. It involves a cascade of effector molecules that is activated via classical, lectin and alternative pathways. Consequently, many pathogens bind to or incorporate in their structures host negative regulators of the complement pathways as an evasion mechanism. Factor H (FH) is a negative regulator of the complement alternative pathway that protects "self" cells of the host from non-specific complement attack. FH has been shown to bind viruses including human influenza A viruses (IAVs). In addition to its involvement in the regulation of complement activation, FH has also been shown to perform a range of functions on its own including its direct interaction with pathogens. Here, we show that human FH can bind directly to IAVs of both human and avian origin, and the interaction is mediated via the IAV surface glycoprotein haemagglutinin (HA). HA bound to common pathogen binding footprints on the FH structure, complement control protein modules, CCP 5-7 and CCP 15-20. The FH binding to H1 and H3 showed that the interaction overlapped with the receptor binding site of both HAs, but the footprint was more extensive for the H3 HA than the H1 HA. The HA - FH interaction impeded the initial entry of H1N1 and H3N2 IAV strains but its impact on viral multicycle replication in human lung cells was strain-specific. The H3N2 virus binding to cells was significantly inhibited by preincubation with FH, whereas there was no alteration in replicative rate and progeny virus release for human H1N1, or avian H9N2 and H5N3 IAV strains. We have mapped the interaction between FH and IAV, the in vivo significance of which for the virus or host is yet to be elucidated.

Effect of estrogen on growth and apoptosis in esophageal adenocarcinoma cells.

The epidemiology of esophageal adenocarcinoma demonstrates a strong gender bias with a sex ratio of 8-9:1 in favor of males. A potential explanation for this is that estrogen might protect against esophageal adenocarcinoma. Estrogen has previously been shown to stimulate apoptosis in esophageal squamous cancer cells. However, the effect of estrogen on esophageal adenocarcinoma cells has not been determined. We used immunoblotting analysis to determine the expression of estrogen receptors, cell adhesion marker E-cadherin, and proliferation marker Ki-67 in cell lines derived from esophageal adenocarcinoma (OE-19, OE-33) and Barrett's esophagus (QhTRT, ChTRT, GihTRT). Estrogen and selective estrogen receptor modulator (SERM)-dependent effects on cell growth were determined by the CellTiter-96 Aqueous Proliferation Assay. Apoptosis was determined by Annexin V/Propidium Iodide cell labeling and flow cytometry. We detected that physiological and supra-physiological concentrations of 17ß-estradiol and SERM decreased cell growth in esophageal adenocarcinoma cells. In Barrett's esophagus cells (QhTRT, ChTRT), decreased growth was also detected in response to estrogen/SERM. The level of estrogen receptor expression in the cell lines correlated with the level of anti-growth effects induced by the receptor agonists. Flow cytometry analysis confirmed estrogen/SERM stimulated apoptosis in esophageal adenocarcinoma cells. Estrogen/SERM treatments were associated with a decrease in the expression of Ki-67 and an increase in E-cadherin expression in esophageal adenocarcinoma cells. This study suggests that esophageal adenocarcinoma and Barrett's esophagus cells respond to treatment with selective estrogen receptor ligands, resulting in decreased cell growth and apoptosis. Further research to explore potential therapeutic applications is warranted.

Bioanalytical method development, validation and quantification of dorsomorphin in rat plasma by LC-MS/MS.

The present investigation describes the development and validation of a sensitive liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) method for the estimation of dorsomorphin in rat plasma. A sensitive LC-MS/MS method was developed using multiple reaction monitoring mode, with the transition of m/z (Q1/Q3) 400.2/289.3 for dorsomorphin and m/z (Q1/Q3) 306.2/236.3 for zaleplon. Chromatographic separation was achieved on a reverse phase Agilent XDB C18 column (100 × 4.6 mm, 5 µm). The mobile phase consisted of acetonitrile and 5 mm ammonium acetate buffer (pH 6.0) 90:10 v/v, at a flow rate of 0.8 mL/min. The effluence was ionized in positive ion mode by electrospray ionization (ESI) and quantitated by mass spectrometry. The retention times of dorsomorphin and internal standard were found to be 2.13 and 1.13 min, respectively. Mean extraction recovery of dorsomorphin and internal standard in rat plasma was above 80%. Dorsomorphin calibration curve in rat plasma was linear (r(2) ≥ 0.99) ranging from 0.005 to 10 µg/mL. Inter-day and intra-day precision and accuracy were found to be within 85-115% (coefficient of variation). This method was successfully applied for evaluation of the oral pharmacokinetic profile of dorsomorphin in male Wistar rats.

Recombinant modified vaccinia virus Ankara expressing antigen 85A boosts BCG-primed and naturally acquired antimycobacterial immunity in humans.

Protective immunity against Mycobacterium tuberculosis depends on the generation of a T(H)1-type cellular immune response, characterized by the secretion of interferon-gamma (IFN-gamma) from antigen-specific T cells. The induction of potent cellular immune responses by vaccination in humans has proven difficult. Recombinant viral vectors, especially poxviruses and adenoviruses, are particularly effective at boosting previously primed CD4(+) and CD8(+) T-cell responses against a number of intracellular pathogens in animal studies. In the first phase 1 study of any candidate subunit vaccine against tuberculosis, recombinant modified vaccinia virus Ankara (MVA) expressing antigen 85A (MVA85A) was found to induce high levels of antigen-specific IFN-gamma-secreting T cells when used alone in bacille Calmette-Guerin (BCG)-naive healthy volunteers. In volunteers who had been vaccinated 0.5-38 years previously with BCG, substantially higher levels of antigen-specific IFN-gamma-secreting T cells were induced, and at 24 weeks after vaccination these levels were 5-30 times greater than in vaccinees administered a single BCG vaccination. Boosting vaccinations with MVA85A could offer a practical and efficient strategy for enhancing and prolonging antimycobacterial immunity in tuberculosis-endemic areas.

Genetic Causes of Oculocutaneous Albinism in Pakistani Population.

Melanin pigment helps protect our body from broad wavelength solar radiation and skin cancer. Among other pigmentation disorders in humans, albinism is reported to manifest in both syndromic and nonsyndromic forms as well as with varying inheritance patterns. Oculocutaneous albinism (OCA), an autosomal recessive nonsyndromic form of albinism, presents as partial to complete loss of melanin in the skin, hair, and iris. OCA has been known to be caused by pathogenic variants in seven different genes, so far, according to all the currently published population studies. However, the detection rate of alleles causing OCA varies from 50% to 90%. One of the significant challenges of uncovering the pathological variant underlying disease etiology is inter- and intra-familial locus heterogeneity. This problem is especially pertinent in highly inbred populations. As examples of such familial locus heterogeneity, we present nine consanguineous Pakistani families with segregating OCA due to variants in one or two different known albinism-associated genes. All of the identified variants are predicted to be pathogenic, which was corroborated by several in silico algorithms and association with diverse clinical phenotypes. We report an individual affected with OCA carries heterozygous, likely pathogenic variants in TYR and OCA2, raising the question of a possible digenic inheritance. Altogether, our study highlights the significance of exome sequencing for the complete genetic diagnosis of inbred families and provides the ramifications of potential genetic interaction and digenic inheritance of variants in the TYR and OCA2 genes.

Safety and immunogenicity of a new tuberculosis vaccine, MVA85A, in Mycobacterium tuberculosis-infected individuals.

RATIONALE: An effective new tuberculosis (TB) vaccine regimen must be safe in individuals with latent TB infection (LTBI) and is a priority for global health care. OBJECTIVES: To evaluate the safety and immunogenicity of a leading new TB vaccine, recombinant Modified Vaccinia Ankara expressing Antigen 85A (MVA85A) in individuals with LTBI. METHODS: An open-label, phase I trial of MVA85A was performed in 12 subjects with LTBI recruited from TB contact clinics in Oxford and London or by poster advertisements in Oxford hospitals. Patients were assessed clinically and had blood samples drawn for immunological analysis over a 52-week period after vaccination with MVA85A. Thoracic computed tomography scans were performed at baseline and at 10 weeks after vaccination. Safety of MVA85A was assessed by clinical, radiological, and inflammatory markers. The immunogenicity of MVA85A was assessed by IFNgamma and IL-2 ELISpot assays and FACS. MEASUREMENTS AND MAIN RESULTS: MVA85A was safe in subjects with LTBI, with comparable adverse events to previous trials of MVA85A. There were no clinically significant changes in inflammatory markers or thoracic computed tomography scans after vaccination. MVA85A induced a strong antigen-specific IFN-gamma and IL-2 response that was durable for 52 weeks. The magnitude of IFN-gamma response was comparable to previous trials of MVA85A in bacillus Calmette-Guérin-vaccinated individuals. Antigen 85A-specific polyfunctional CD4(+) T cells were detectable prior to vaccination with statistically significant increases in cell numbers after vaccination. CONCLUSIONS: MVA85A is safe and highly immunogenic in individuals with LTBI. These results will facilitate further trials in TB-endemic areas. Clinical trial registered with www.clinicaltrials.gov (NCT00456183).

Effects of 1alpha,25-dihydroxyvitamin D3 on transporters and enzymes of the rat intestine and kidney in vivo.

1alpha,25-Dihydroxyvitamin D3 (1,25(OH)2D3), the natural ligand of the vitamin D receptor (VDR), was found to regulate bile acid related transporters and enzymes directly and indirectly in the rat intestine and liver in vivo. The kidney is another VDR-rich target organ in which VDR regulation on xenobiotic transporters and enzymes is ill-defined. Hence, changes in protein and mRNA expression of nuclear receptors, transporters and enzymes of the rat intestine and kidney in response to 1,25(OH)2D3 treatment (0 to 2.56 nmol/kg/day intraperitoneally in corn oil for 4 days) were studied. In the intestine, protein and not mRNA levels of Mrp2, Mrp3, Mrp4 and PepT1 in the duodenum and proximal jejunum were induced, whereas Oat1 and Oat3 mRNA were decreased in the ileum after 1,25(OH)2D3 treatment. In the kidney, VDR, Cyp24, Asbt and Mdr1a mRNA and protein expression increased significantly (2- to 20-fold) in 1,25(OH)2D3-treated rats, and a 28-fold increase of Cyp3a9 mRNA but not of total Cy3a protein nor Cyp3a1 and Cyp3a2 mRNA was observed, implicating that VDR played a significant, renal-specific role in Cyp3a9 induction. Additionally, renal mRNA levels of PepT1, Oat1, Oat3, Ostalpha, and Mrp4, and protein levels of PepT1 and Oat1 were decreased in a dose-dependent manner, and the approximately 50% concomitant reduction in FXR, SHP, HNF-1alpha and HNF-4alpha mRNA expression suggests the possibility of cross-talk among the nuclear receptors. It is concluded that the effects of 1,25(OH)2D3 changes are tissue-specific, differing between the intestine and kidney which are VDR-rich organs.

The relationship between human effector and memory T cells measured by ex vivo and cultured ELISPOT following recent and distal priming.

Maintenance of T-cell responses is an essential feature in protection from many infectious diseases that must be harnessed in vaccination. The relationship between effector T-cell responses and more durable and highly proliferative T-cell memory, particularly in humans, is not well understood. In this study, effector T-cell responses were measured by overnight ex vivo interferon-gamma (IFN-gamma) enzyme-linked immunosorbent spot-forming cell assay (ELISPOT), whereas memory T cells were measured by 10-day culture followed by IFN-gamma ELISPOT (cultured ELISPOT). We observed a significant correlation between IFN-gamma responses to CD4-stimulatory, but not to CD8-stimulatory, recall antigens measured by these assays, suggesting a divergence in regulation. In vaccine trial participants who received a prime-boost vaccination regimen comprising malaria antigens delivered by poxviruses, there was a correlation between ex vivo and cultured responses on day 7, but not 3 months post-vaccination, with the ratio of cultured : ex vivo response increasing over time. To compare responses revealed by cultured ELISPOT in more detail, tetramers comprising viral recall antigens were used to ascribe effector-memory and central-memory T-cell phenotypes through CCR7 and CD62L costaining. For CD8(+) responses the effector phenotype decreased during the initial culture period and memory populations remained high within the resulting 20-fold to 50-fold increased IFN-gamma-secreting or tetramer(+) population. This was less marked for CD4(+) responses, which had higher starting memory phenotype. Depletion of these central-memory T-cell populations generally ablated responses in cultured ELISPOT and reduced ex vivo responses. This study highlights differences between CD4(+) and CD8(+) effector and memory T cells, and the more complex phenotype of CD4(+) T cells.

Expression and regulation of the bile acid transporter, OSTalpha-OSTbeta in rat and human intestine and liver.

The regulation of the OSTalpha and OSTbeta expression was studied in the rat jejunum, ileum, colon and liver and in human ileum and liver by ligands for the farnesoid X receptor (FXR), pregnane X receptor (PXR), vitamin D receptor (VDR) and glucocorticoid receptor (GR) using precision cut tissue slices. The gradient of protein and mRNA expression in segments of the intestine for rOSTalpha and rOSTbeta paralleled that of rASBT. OSTalpha and OSTbeta mRNA expression, quantified by qRT-PCR, in rat jejunum, ileum, colon and liver, and in human ileum and liver was positively regulated by FXR and GR ligands. In contrast, the VDR ligand, 1,25(OH)2D3 decreased the expression of rOSTalpha-rOSTbeta in rat intestine, but had no effect on human ileum, and rat and human liver slices. Lithocholic acid (LCA) decreased the expression of rOSTalpha and rOSTbeta in rat ileum but induced OSTalpha-OSTbeta expression in rat liver slices, and human ileum and liver slices. The PXR ligand, pregnenolone-16alpha carbonitrile (PCN) had no effect. This study suggest that, apart from FXR ligands, the OSTalpha and OSTbeta genes are also regulated by VDR and GR ligands and not by PXR ligands. This study show that VDR ligands exerted different effects on OSTalpha-OSTbeta in the rat and human intestine and liver compared with other nuclear receptors, FXR, PXR, and GR, pointing to species- and organ-specific differences in the regulation of OSTalpha-OSTbeta genes.

1alpha,25-Dihydroxyvitamin D(3) triggered vitamin D receptor and farnesoid X receptor-like effects in rat intestine and liver in vivo.

1alpha,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), a natural ligand of the vitamin D receptor (VDR), was found to increase the rat ileal Asbt and bile acid absorption. The effects of VDR, whose expression is low in liver, on hepatic transporters and enzymes are unknown. Protein and mRNA levels of target genes in the small intestine, colon and liver after intraperitoneal dosing of 1,25(OH)(2)D(3) (0-2.56 nmol/kg/day for 4 days) to the rat were determined by Western blotting and qPCR, respectively. The 1,25(OH)(2)D(3) treatment increased total Cyp3a protein and Cyp3a1 mRNA expressions in the proximal small intestine, and the short heterodimer partner (SHP), the fibroblast growth factor 15 (FGF15), organic solute transporter (Ostalpha and Ostbeta) mRNA and Asbt protein expressions in the ileum. About 50% higher portal bile acid concentration (65.1+/-14.9 vs 41.9+/-7.8 microm, p<0.05) and elevated expressions of the hepatic farnesoid X receptor (FXR) and SHP mRNA resulted with 1,25(OH)(2)D(3) treatment. Increased Bsep and Ostalpha mRNA expressions in liver and a>50% reduction in the Cyp7a1 protein level (p<0.05) and cholesterol metabolism in rat liver microsomes (p=0.002), likely consequences of the bile acid-FXR-SHP cascade and activation of the signaling pathway for Cyp7a1 inhibition by FGF15, were found. Increased hepatic multidrug resistance-associated protein (Mrp3) and multidrug resistance protein 1a (Mdr1a) mRNA and P-gp protein were also observed. It was concluded that the changes in hepatic transporters and enzymes in the rat were indirect, secondary effects of the liver FXR-SHP cascade due to increased intestinal absorption of bile acids and elevated levels of FGF15, events that led to the activation of FXR.

Full-length human surfactant protein A inhibits influenza A virus infection of A549 lung epithelial cells: A recombinant form containing neck and lectin domains promotes infectivity.

Hydrophilic lung surfactant proteins have emerged as key immunomodulators which are potent at the recognition and clearance of pulmonary pathogens. Surfactant protein A (SP-A) is a surfactant-associated innate immune molecule, which is known to interact with a variety of pathogens, and display anti-microbial effects. SP-A, being a carbohydrate pattern recognition molecule, has a wide range of innate immune functions against respiratory pathogens, including influenza A virus (IAV). Some pandemic pH1N1 strains resist neutralization by SP-A due to differences in the N-glycosylation of viral hemagglutinin (HA). Here, we provide evidence, for the first time, that a recombinant form of human SP-A (rfhSP-A), composed of α-helical neck and carbohydrate recognition domains, can actually promote the IAV replication, as observed by an upregulation of M1 expression in lung epithelial cell line, A549, when challenged with pH1N1 and H3N2 IAV subtypes. rfhSP-A (10 µg/ml) bound neuraminidase (NA) (∼60 kDa), matrix protein 1 (M1) (∼25 kDa) and M2 (∼17 kDa) in a calcium dependent manner, as revealed by far western blotting, and direct binding ELISA. However, human full length native SP-A downregulated mRNA expression levels of M1 in A549 cells challenged with IAV subtypes. Furthermore, qPCR analysis showed that transcriptional levels of TNF-α, IL-12, IL-6, IFN-α and RANTES were enhanced following rfhSP-A treatment by both IAV subtypes at 6 h post-IAV infection of A549 lung epithelial cells. In the case of full length SP-A treatment, mRNA expression levels of TNF-α and IL-6 were downregulated during the mid-to-late stage of IAV infection of A549 cells. Multiplex cytokine/chemokine array revealed enhanced levels of both IL-6 and TNF-α due to rfhSP-A treatment in the case of both IAV subtypes tested, while no significant effect was seen in the case of IL-12. Enhancement of IAV infection of pH1N1 and H3N2 subtypes by truncated rfhSP-A, concomitant with infection inhibition by full-length SP-A, appears to suggest that a complete SP-A molecule is required for protection against IAV. This is in contrast to a recombinant form of trimeric lectin domains of human SP-D (rfhSP-D) that acts as an entry inhibitor of IAV.

Secretion of functionally active complement factor H related protein 5 (FHR5) by primary tumour cells derived from Glioblastoma Multiforme patients.

The complement system is an important humoral immune surveillance mechanism against tumours. However, many malignant tumours are resistant to complement mediated lysis. Here, we report secretion of complement factor H related protein 5 (FHR5) by primary tumour cells derived from Glioblastoma multiforme (GBM) patients. We investigated whether the secreted FHR5 exhibited functional activity similar to factor H, including inhibition of complement mediated lysis, acting as a co-factor for factor I mediated cleavage of C3b, and decay acceleration of C3 convertase. Immunoblotting analysis of primary GBM cells (B30, B31 and B33) supernatant showed the active secretion of FHR5, but not of Factor H. ELISA revealed that the secretion of soluble GBM-FHR5 by cultured GBM cells increased in a time-dependent manner. Primary GBM-FHR5 inhibited complement mediated lysis, possessed co-factor activity for factor I mediated cleavage and displayed decay acceleration of C3 convertase. In summary, we detected the secretion of FHR5 by primary GBM cells B30, B31 and B33. The results demonstrated that GBM-FHR5 shares biological function with FH as a mechanism primary GBM cells potentially use to resist complement mediated lysis.