Concentrated Culture Supernatant Research Articles

Preventive effect of culture supernatant of epithelial-like peritoneal mesothelial cells on peritoneal fibrosis.

Peritoneal fibrosis (PF) is a primary reason for discontinuing peritoneal dialysis, which involves characteristic changes of peritoneal mesothelial cells (PMCs). We previously reported preventive effects of implanting human epithelial-like PMCs (P-Epi) for mouse PF caused by mechanical peritoneum scrapings. In the present study, we analysed the preventive effects of culture supernatant of P-Epi in PF. Concentrated culture supernatant of P-Epi or human fibroblast-like PMCs (P-Fibro) or vehicles was injected into nude mice that had undergone mechanical scraping of the parietal and visceral peritoneum, and thickness and amount of adhesions were analysed. Although increased peritoneal adhesions and peritoneum thickening were observed in the vehicle-injected positive control group compared to the sham operation group, fewer number of adhesions and less thickness were observed in the mice treated with culture supernatant of P-Epi, but not P-Fibro, compared to the vehicle-injected positive controls. Immunofluorescent analysis revealed that the expression of extracellular matrix, type I collagen and fibronectin, was lower in the mice treated with culture supernatant of P-Epi than in the vehicle-injected positive controls. In addition, exosomes from P-Epi significantly reduced transforming growth factor-β (TGF-β)-induced expressions of type I collagen and fibronectin in 3T3 fibroblast cells. Collectively, culture supernatant of P-Epi has preventive effects on PF, thus cell therapy is not necessarily required. Further exploration of substances secreted by P-Epi and their protective mechanisms could lead to the development of therapeutic strategies to limit PF.

Translation of circHGF RNA encodes an HGF protein variant promoting glioblastoma growth through stimulation of c-MET

IntroductionHGF/c-MET signaling is a significant driver of glioblastoma (GBM) growth and disease progression. Unfortunately, c-MET targeted therapies have been found to be largely ineffective suggesting additional redundant mechanisms of c-MET activation.MethodsUtilizing RNA-sequencing (RNA-seq) and ribosome profiling analyses of circular RNAs, circ-HGF (hsa_circ_0080914) was identified as markedly upregulated in primary GBM and found to potentially encode an HGF protein variant (C-HGF) 119 amino acids in length. This candidate HGF variant was characterized and evaluated for its ability to mediate c-MET activation and regulate PDX GBM cell growth, motility and invasive potential in vitro and tumor burden in intracranial xenografts in mice.ResultsAn internal ribosome entry site (IRES) was identified within the circ-HGF RNA which mediated translation of the cross-junctional ORF encoding C-HGF and was observed to be highly expressed in GBM relative to normal brain tissue. C-HGF was also found to be secreted from GBM cells and concentrated cell culture supernatants or recombinant C-HGF activated known signaling cascades downstream of c-MET. C-HGF was shown to interact directly with the c-MET receptor resulting in its autophosphorylation and activation in PDX GBM lines. Knockdown of C-HGF resulted in suppression of c-MET signaling and marked inhibition of cell growth, motility and invasiveness, whereas overexpression of C-HGF displayed the opposite effects. Additionally, modulation of C-HGF expression regulated tumor growth in intracranial xenografted PDX GBM models.ConclusionsThese results reveal an alternative mechanism of c-MET activation via a circular RNA encoded HGF protein variant which is relevant in GBM biology. Targeting C-HGF may offer a promising approach for GBM clinical management.

Evaluation of the application of <i>Wickerhamomyces anomalus</i> supernatant for the control of relevant spoilage yeasts in wines

SO2 is traditionally used to limit or nullify the proliferation of spoilage yeasts in must/wine, but its use has become controversial due to its negative effects on human health. An alternative strategy for the control of spoilage yeasts is the use of biocontrol yeasts. Wickerhamomyces anomalus is an outstanding biocontroller yeast that has been examined by our group and that is a good biocontrol agent against Brettanomyces bruxellensis and Zygosaccharomyces rouxii. The challenge regarding the application of the results of microbial biocontrol studies lies in exploring technologically simple and economical ways to make use of microbial biocontrol activity. The introduction of foreign microbiota adds complexity to the fermentation medium and can cause technological difficulties and have oenological consequences inherent to the implementation of mixed cultures. Therefore, the objective of this study was to characterise the concentrated culture supernatant of W. anomalus and evaluate its biocontrol action in liquid medium on two main wine spoilage yeasts. W. anomalus BWa156 supernatant demonstrated inhibitory killer protein characteristics. The production of the inhibitory supernatant by the biocontroller yeast was independent of the presence or absence of the spoilage yeast. Supernatant can be produced faster under aerobic conditions than in traditional fermentation (i.e., with around 24 h), thus increasing its potential for technological development. The treatment with the supernatant of BWa156 was effective against the two spoilage populations Zygosaccharomyces rouxii and Brettanomyces bruxellensis, which are considered problematic for the wine industry. The supernatant of the biocontrol yeast can be considered to be a relevant additional stress factor for the spoilage population, which together with other factors such as ethanol, competition for nutrients, oxygen and pH, contributes to the elimination of the polluting population. This technology would allow a simple future application, through its production in a bioreactor parallel to the fermentation and subsequent inoculation in the must/wine.

Vaccination against Borna Disease: Overview, Vaccine Virus Characterization and Investigation of Live and Inactivated Vaccines.

(1) Background: Vaccination of horses and sheep against Borna disease (BD) was common in endemic areas of Germany in the 20th century but was abandoned in the early 1990s. The recent occurrence of fatal cases of human encephalitis due to Borna disease virus 1 (BoDV-1) has rekindled the interest in vaccination. (2) Methods: The full genomes of the BD live vaccine viruses "Dessau" and "Giessen" were sequenced and analyzed for the first time. All vaccination experiments followed a proof-of-concept approach. Dose-titration infection experiments were performed in rabbits, based on both cell culture- and brain-derived viruses at various doses. Inactivated vaccines against BD were produced from concentrated cell culture supernatants and investigated in rabbits and horses. The BoDV-1 live vaccine "Dessau" was administered to horses and antibody profiles were determined. (3) Results: The BD live vaccine viruses "Dessau" and "Giessen" belong to clusters 3 and 4 of BoDV-1. Whereas the "Giessen" virus does not differ substantially from field viruses, the "Dessau" virus shows striking differences in the M gene and the N-terminal part of the G gene. Rabbits infected with high doses of cell-cultured virus developed neutralizing antibodies and were protected from disease, whereas rabbits infected with low doses of cell-cultured virus, or with brain-derived virus did not. Inactivated vaccines were administered to rabbits and horses, following pre-defined vaccination schemes consisting of three vaccine doses of either adjuvanted or nonadjuvanted inactivated virus. Their immunogenicity and protective efficacy were compared to the BD live vaccine "Dessau". Seventy per cent of horses vaccinated with the BD live vaccine "Dessau" developed neutralizing antibodies after vaccination. (4) Conclusion: Despite a complex evasion of immunological responses by bornaviruses, some vaccination approaches can protect against clinical disease. For optimal effectiveness, vaccines should be administered at high doses, following vaccination schemes consisting of three vaccine doses as basic immunization. Further investigations are necessary in order to investigate and improve protection against infection and to avoid side effects.

416.5: Pregnancy-Induced Sensitization in Female Transplant Candidates: HLA-Specific Memory B Cells and Paternal HLA Epitope Mismatch

Background: Access to kidney transplantation (KT) may be limited by HLA antibodies (HLA-Ab) induced by sensitizing events. Pregnancy is a frequent sensitizing event in female candidates, through which they are exposed to foreign paternal HLA antigens. Consequently, they can generate specific HLA-Ab and memory B cells (mBc), which can be re-stimulated upon antigen reencounter after KT. We aimed to study the existence of mBc able to produce specific HLA-Ab against paternal antigens in female KT candidates sensitised by previous pregnancies, to compare them with HLA-Ab found in their serum and to analyse these HLA-Ab in the context of the molecular HLA mismatch between mother and father. Methods: We selected 8 HLA sensitised women awaiting KT with at least one successful pregnancy from a single partner, detectable serum HLA-Ab (MFI≥1000) and available paternal DNA for full HLA typing. All women and their partners were typed by next-generation sequencing for HLA–A, B, C, DRB1, DRB3/4/5, DQB1/A1, DPB1. To study mBc, we used peripheral blood mononuclear cells cryopreserved before KT and cultured them with a polyclonal stimulation cocktail consisting of a toll-like receptor agonist and an interleukin for ten days. We examined concentrated culture supernatants (SN) derived from activated mBc, purified IgG and identified HLA-Abs using single antigen beads on Luminex®. Then, we compared the HLA-Ab profiles of SN and serum. We also studied the paternal molecular HLA mismatch with HLAMatchmaker® software. Results: After 10-day polyclonal stimulation, mBc increased from 29.9% (day 0) to 65.3% (day- 10), and antibody-secreting cells (ASC) from 0.1% (day-0) to 29.4% (day-10) (Figure 1). SAB analysis revealed 211 HLA-Abs in serum and 116 in SN: 98/211 (46,5%) HLA-Ab were serum-exclusive (79,6% class I and 20,4% class II) and only 3/116 (2,3%) HLA-Ab were SN-exclusive, all class II. Finally, we found 113/327 (34,6%) HLA-Ab shared by serum and SN: 65/113 (57,5%) class I and 48/113 (42,5%) class II.Pregnancy-Induced Antibodies (PIA) were present in serum in 6/8 women (21 HLA-Abs: 10 class I, 11 class II) and in 4/8 women in SN (8 HLA-Abs: 2 class I, 6 class II). The HLAMatchmaker analysis showed that those specific HLA-ab were reactive against incompatible paternal epitopes: in 3/6 women for class I & 3/6 women for class II in serum; in 2/4 women for class I & 3/4 women for class II in SN (Table 1).Conclusion: We describe the study of the memory B cell compartment and the HLA-Abs produced with Luminex technology in KT female candidates with previous pregnancies. We found that the HLA-Abs secreted by memory B cells followed a restricted pattern compared with serum HLA-Ab. The study of paternal HLA molecular mismatch explains many of these HLA-Abs decades after pregnancies. The entire value of this tool for risk stratification in HLA-sensitised patients awaiting TK is currently a matter of study.

Genomic and pathogenic characterization of RTX toxin producing Rodentibacter sp. that is closely related to Rodentibacter haemolyticus.

Rodentibacter spp. are opportunistic pathogens that are often isolated from the upper respiratory tracts of laboratory rodents. In particular, R. pneumotropicus and R. heylii require considerable caution in rodent colonies, as they cause lethal pneumonia in rodents. A new species, R. haemolyticus, has recently been classified in the genus, and a very closely related strain, Rodentibacter sp. strain JRC, has been isolated in Japan. This study focused on strain JRC by performing genomic and pathogenic analyses. Draft genome sequencing of strain JRC identified several genes coding for putative virulent proteins, including hemolysin and adhesin. Furthermore, we found a new RTX (repeats-in-structural toxin) toxin gene in the genome, which was predicted to produce a critical virulence factor (RTXIA) similar to Enterobacteriaceae. The concentrated culture supernatant containing RTX toxin (RTXIA) showed cytotoxicity toward RAW264.7 cells. Pre-incubation with anti-CD11a attenuated the cytolysis, suggesting that the concentrated culture supernatant containing RTXIA is cell surface LFA-1 mediated cytolysin. Experimental infection of strain JRC intranasally with 5 female BALB/c-Rag2-/- mice showed 60% lethality and was not significantly different from those of R. pneumotropicus ATCC 35149T using the log-rank test. Combined with our finding that RTXIA has an almost identical amino acid sequence (98% identity) to that of R. haemolyticus 1625/19T, these results strongly suggest that RTXIA-producing strain JRC (and related R. haemolyticus) is pathogenic to immunodeficient rodents, and both agents should be excluded in laboratory rodent colonies.

FC 112: HLA-Specific Memory B Cells and Paternal HLA Molecular Mismatch in Female Transplant Candidates Sensitized by Pregnancy

Abstract BACKGROUND AND AIMS Access to kidney transplantation (KT) may be limited by HLA sensitization. It is clinically relevant to know both level of HLA-immunization and potential sensitizing events in the history transplant candidates [1–3]. Pregnancy is a frequent sensitizing event in female transplant candidates. Women exposed to foreign paternal HLA can generate specific HLA antibodies (HLA-Ab) and memory B cells (mBc) [4, 5], which can be re-stimulated upon antigen reencounter after TR. We aimed to study the existence of mBc able to produce HLA-Ab in female KT candidates with previous pregnancies, to compare them with HLA-Ab in their serum and to analyse them in the context of molecular HLA mismatch between mother and father. METHOD We selected eight HLA sensitized women awaiting KT, who fulfilled these criteria: minimum of one successful pregnancy before KT (12–44 years before) from a single partner, HLA-Ab (HLA class I or II) and availability of a DNA sample from the father of their children. All women in the study cohort and the father of their children were HLA typed by next-generation sequencing (NGS®) for HLA–A, B, C, DRB1, DRB3/4/5, DQB1/A1 and DPB1. We used peripheral blood mononuclear cells cryopreserved before KT. We cultured them with a polyclonal stimulation cocktail consisting of a Toll-like receptor 7/8 agonist and a proleukin for 10 days. We examined concentrated culture supernatants (SN) derived from activated cells, purified IgG and identified HLA-Abs using single antigen beads (SAB) on Luminex. Then, we compared the HLA-Ab profiles of SN and serum. We also studied the paternal molecular HLA incompatibilities with the HLAMatchmaker software. RESULTS After 10-day polyclonal stimulation, mBc increased from 29.90% (day 0) to 65.29% (day 10) (Figure 1), and antibody-secreting cells (ASC) from 0.08% (day 0) to 29.37% (day 10). SAB analysis revealed 211 HLA-Abs in serum and 116 HLA-Abs in SN: 98/211 (46.5%) of total HLA-Ab were serum-exclusive [78/98 (52.9%) class I and 25/98 (34.3%) class II] and only 3/116 (2.59%) HLA-Ab were SN-exclusive, all class II. Finally, we found 113/327 (34.6%) HLA-Ab shared by serum and SN: 65/113 (57.5%) class I and 48/113 (42.5%) class II. Seven of eight women had Pregnancy-Induced Antibodies (PIA) in serum (20 HLA-Abs: 10 HLA class I and 10 HLA class II) and 4/8 women had PIA in SN (8 HLA-Abs: 2 HLA class I and 6 HLA class II) (Table 1). The HLAMatchmaker analysis showed that antibodies were reacting against incompatible paternal epitopes: in 6/8 women for HLA class I and 5/8 women for class HLA II in serum, and in 2/8 women for HLA class I and 4/8 women for HLA class II in SN. CONCLUSION We describe the study of the memory B cell compartment and the specific HLA-Abs produced by them with Luminex technology in KT female candidates with previous pregnancies. We found that the HLA-Abs secreted by these memory B cells followed a restricted pattern in number and intensity compared with serum HLA-Ab. The study of paternal HLA molecular mismatch incompatibilities explains many of these HLA-Abs decades after pregnancies. The entire value of this tool for risk stratification in HLA-sensitized patients awaiting TK is currently a matter of study.

Donor-specific B Cell Memory in Alloimmunized Kidney Transplant Recipients: First Clinical Application of a Novel Method.

HLA-specific memory B cells may contribute to the serum HLA antibody pool upon antigen reexposure. The aim of this pilot study was to investigate the presence of concurrent donor-specific memory B cell-derived HLA antibodies (DSA-M) in renal allograft recipients with pretransplant donor-specific HLA antibodies (DSA) and its association with occurrence of antibody-mediated rejection (AMR) using a recently developed method. Twenty patients with Luminex single antigen bead (SAB) assay-defined DSA but negative complement-dependent cytotoxicity crossmatches were enrolled. Plasma samples and peripheral blood mononuclear cells were collected at 3 timepoints (pretransplant, mo 6, mo 12). We analyzed IgG-purified and concentrated culture supernatants from polyclonally activated peripheral blood mononuclear cells using SAB assays and compared HLA antibody profiles with same day plasma results. Plasma SAB analysis revealed 35 DSA in 20 patients pretransplant. DSA-M were detected in 9 of 20 (45%) patients and for 10 of 35 specificities (29%). While median mean fluorescence intensity values of DSA with concurrent DSA-M (5877) were higher than those of DSA without DSA-M (1476), 3 of 6 patients with AMR and low mean fluorescence intensity DSA (<3000) had DSA-M. Overall, pretransplant DSA/DSA-Mpos allograft recipients showed a higher incidence of biopsy-proven (sub)clinical AMR (P = 0.032) and a higher extent (g≥1 + ptc≥1) of microvascular inflammation (67% vs 9%, P = 0.02). In 17 patients (28 DSA) with posttransplant analyses, persisting DSA posttransplant had more often DSA-M (6/12; 50%) than nonpersisting DSA (2/16; 13%). Assessment of DSA-M might be a novel tool to supplement serum HLA antibody analysis for pretransplant risk stratification in patients with DSA.

Characterization of the hemolytic activity of Riemerella anatipestifer.

Riemerella anatipestifer infection causes serious economic losses in the duck industry worldwide. Acute septicemia and high blood bacterial loading in R. anatipestifer infected ducks indicate that R. anatipestifer may be able to obtain iron and other nutrients by lysing duck erythrocytes to support its rapid growth and proliferation in the blood. However, so far, little is known about the hemolytic activity of R. anatipestifer to duck erythrocytes. In this study, 29 of 52 R. anatipestifer strains showed hemolytic activity on duck blood agar, whereas all the tested dba+ (with hemolytic activity on duck blood agar) and dba- strains created pores in the duck red blood cells, with 4.35-9.03% hemolytic activity in a liquid hemolysis assay after incubation for 24 h. The concentrated culture supernatants of all the tested R. anatipestifer strains and the extracted outer membrane proteins (OMPs) from dba+R. anatipestifer strains showed hemolytic activity on duck blood agar. These results, together with the median lethal dose (LD50) of some dba+ and dba-R. anatipestifer strains in ducklings, suggested that there was no direct relationship between the hemolytic capacity of R. anatipestifer on duck blood agar and its virulence.

Characterization of Virulent netB+/tpeL+Clostridium perfringens Strains from Necrotic Enteritis-Affected Broiler Chicken Farms.

Clostridium perfringens (CP) type A and newly created type G strains are the key etiological factors in the induction of necrotic enteritis (NE), an important enteric disease that is responsible for the annual loss of $6 billion in the worldwide poultry industry. Several CP toxin genes were found to be critical in NE pathogenesis in chickens, but limited information is available on the CP lethal toxin tpeL gene. In this study, 19 CP strains isolated from NE-affected chicken farms were characterized microbiologically and molecularly and evaluated for their pathogenicity in commercial broiler chickens. Toxin typing by PCR revealed that all strains tested were positive for the netB toxin gene, but only five strains were positive for the tpeL toxin gene (LLY-TpeL 13, -TpeL 15, -TpeL 17, -TpeL 18, and -TpeL 19, simplified as TpeL 13, TpeL 15, TpeL 17, TpeL 18, and TpeL 19). High levels of TpeL proteins were detected in the concentrated culture supernatant from strains TpeL 13, 15, 17, and 19 by western blotting. Quantitative PCR showed that strains TpeL 13, 15, 17, 18, and 19 harbored a high number of copies of tpeL genes, while TpeL 18 had the highest number of copies of the tpeL gene among all CP strains tested when normalized with copy numbers of 16S rRNA gene as a housekeeping gene marker. The in vivo NE challenge test using multiple oral CP inoculations combined with a high-protein diet showed that TpeL 17 was the most virulent in inducing typical NE lesions, followed by TpeL 19 as the next most virulent, when tested in commercial broiler chickens. Infection with TpeL 17 reduced the growth rate significantly, as shown by reduced relative body weight gain percentage at day 5 postinfection. Availability of the virulent netB+tpeL+ CP strains is essential for the development of a CP-alone NE challenge model that could provide significant tools for understanding CP pathogenesis and for development of alternative to antibiotics.

Complex Relationships between the Blue Pigment Marennine and Marine Bacteria of the Genus Vibrio.

Marennine, the water-soluble blue pigment produced by the marine diatom Haslea ostrearia, is known to display antibacterial activities. Previous studies have demonstrated a prophylactic effect of marennine on bivalve larvae challenged with a pathogenic Vibrio splendidus, suggesting that the blue Haslea is a good candidate for applications in aquaculture as a source of a natural antimicrobial agent. Indeed, the genus Vibrio is ubiquitous in aquaculture ecosystems, and regular events of pathogenic invasion cause some of the biggest losses worldwide. To better characterize the effects of marennine on Vibrios, a panel of 30 Vibrio strains belonging to 10 different species was tested, including bivalve pathogenic species (e.g., Vibrio crassostreae and Vibrio harveyi). Vibrio strains were first exposed to 10 and 25 µg mL−1 of Blue Water (BW), a concentrated culture supernatant of H. ostrearia containing marennine. This screening evidenced a great diversity in responses, from growth stimulation to a total inhibition, at both the interspecific or intraspecific level. In a second series of experiments, 10 Vibrio strains were exposed to BW at concentrations ranging from 5 to 80 µg mL−1. The highest concentrations of BW did not systematically result in the highest growth inhibition as hormetic responses—opposite effects regarding the concentration—were occasionally evidenced. The relationships between marennine and Vibrio strains appear more complex than expected and justify further study—in particular, on the mechanisms of action—before considering applications as a natural prophylactic or antibiotic agent in aquaculture.

A Symmetric Molecule Produced by Mycobacteria Generates Cell-Length Asymmetry during Cell-Division and Thereby Cell-Length Heterogeneity.

Diadenosine polyphosphates, Ap(2-7)A, which contain two adenosines in a 5',5' linkage through phosphodiester bonds involving 2-7 phosphates, regulate diverse cellular functions in all organisms, from bacteria to humans, under normal and stress conditions. We had earlier reported consistent occurrence of asymmetric constriction during division (ACD) in 20-30% of dividing mycobacterial cells in culture, irrespective of different growth media, implying exogenous action of some factor of mycobacterial origin. Consistent with this premise, concentrated culture supernatant (CCS), but not the equivalent volume-wise concentrated unused medium, dramatically enhanced the ACD proportion to 70-90%. Mass spectrometry and biochemical analyses of the bioactive fraction from CCS revealed the ACD-effecting factor to be Ap6A. Synthetic Ap6A showed a mass spectrometry profile, biochemical characteristics, and bioactivity identical to native Ap6A in the CCS. Thus, the present work reveals a novel role for Ap6A in generating cell-length asymmetry during mycobacterial cell-division and thereby cell-length heterogeneity in the population.

Nidovirus-Associated Proliferative Pneumonia in the Green Tree Python (Morelia viridis).

In 2014 we observed a noticeable increase in the number of sudden deaths among green tree pythons (Morelia viridis). Pathological examination revealed the accumulation of mucoid material within the airways and lungs in association with enlargement of the entire lung. We performed a full necropsy and histological examination on 12 affected green tree pythons from 7 different breeders to characterize the pathogenesis of this mucinous pneumonia. By histology we could show a marked hyperplasia of the airway epithelium and of faveolar type II pneumocytes. Since routine microbiological tests failed to identify a causative agent, we studied lung tissue samples from a few diseased snakes by next-generation sequencing (NGS). From the NGS data we could assemble a piece of RNA genome whose sequence was <85% identical to that of nidoviruses previously identified in ball pythons and Indian pythons. We then employed reverse transcription-PCR to demonstrate the presence of the novel nidovirus in all diseased snakes. To attempt virus isolation, we established primary cultures of Morelia viridis liver and brain cells, which we inoculated with homogenates of lung tissue from infected individuals. Ultrastructural examination of concentrated cell culture supernatants showed the presence of nidovirus particles, and subsequent NGS analysis yielded the full genome of the novel virus Morelia viridis nidovirus (MVNV). We then generated an antibody against MVNV nucleoprotein, which we used alongside RNA in situ hybridization to demonstrate viral antigen and RNA in the affected lungs. This suggests that in natural infection MVNV damages the respiratory tract epithelium, which then results in epithelial hyperplasia, most likely as an exaggerated regenerative attempt in association with increased epithelial turnover.IMPORTANCE Novel nidoviruses associated with severe respiratory disease were fairly recently identified in ball pythons and Indian pythons. Herein we report on the isolation and identification of a further nidovirus from green tree pythons (Morelia viridis) with fatal pneumonia. We thoroughly characterized the pathological changes in the infected individuals and show that nidovirus infection is associated with marked epithelial proliferation in the respiratory tract. We speculate that this and the associated excess mucus production can lead to the animals' death by inhibiting normal gas exchange in the lungs. The virus was predominantly detected in the respiratory tract, which renders transmission via the respiratory route likely. Nidoviruses cause sudden outbreaks with high rates of mortality in breeding collections, and most affected snakes die without prior clinical signs. These findings, together with those of other groups, indicate that nidoviruses are a likely cause of severe pneumonia in pythons.

Histophilus somni Stimulates Expression of Antiviral Proteins and Inhibits BRSV Replication in Bovine Respiratory Epithelial Cells.

Our previous studies showed that bovine respiratory syncytial virus (BRSV) followed by Histophilus somni causes more severe bovine respiratory disease and a more permeable alveolar barrier in vitro than either agent alone. However, microarray analysis revealed the treatment of bovine alveolar type 2 (BAT2) epithelial cells with H. somni concentrated culture supernatant (CCS) stimulated up-regulation of four antiviral protein genes as compared with BRSV infection or dual treatment. This suggested that inhibition of viral infection, rather than synergy, may occur if the bacterial infection occurred before the viral infection. Viperin (or radical S-adenosyl methionine domain containing 2—RSAD2) and ISG15 (IFN-stimulated gene 15—ubiquitin-like modifier) were most up-regulated. CCS dose and time course for up-regulation of viperin protein levels were determined in treated bovine turbinate (BT) upper respiratory cells and BAT2 lower respiratory cells by Western blotting. Treatment of BAT2 cells with H. somni culture supernatant before BRSV infection dramatically reduced viral replication as determined by qRT PCR, supporting the hypothesis that the bacterial infection may inhibit viral infection. Studies of the role of the two known H. somni cytotoxins showed that viperin protein expression was induced by endotoxin (lipooligosaccharide) but not by IbpA, which mediates alveolar permeability and H. somni invasion. A naturally occurring IbpA negative asymptomatic carrier strain of H. somni (129Pt) does not cause BAT2 cell retraction or permeability of alveolar cell monolayers, so lacks virulence in vitro. To investigate initial steps of pathogenesis, we showed that strain 129Pt attached to BT cells and induced a strong viperin response in vitro. Thus colonization of the bovine upper respiratory tract with an asymptomatic carrier strain lacking virulence may decrease viral infection and the subsequent enhancement of bacterial respiratory infection in vivo.

Generation of virus-like particle as a vaccine strategy against bovine leukemia virus

Bovine leukemia virus (BLV) is a causative agent of the enzootic bovine leucosis (EBL) with the increasing rate of infection and incidence in cattle. A vaccine against BLV is needed but individual differences in sensitivity to the disease make it difficult to assess the efficiency of a candidate vaccine. We previously determined that a bovine MHC (BoLA) class II allele correlated to high proviral load and sensitivity to develop lymphoma. Based on the finding, we designed a peptide antigen “Gag p12-4R1” in silico that is a CD4 epitope optimized to fit BoLA-DR of cattle sensitive to EBL and exhibited the peptide had an effect on reducing proviral load of BLV-infected cattle. In this study, we focused on virus-like particle (VLP) vaccine strategy to enhance the antigenicity of the peptide vaccine. Here, we evaluated expression of Gag containing a p12-4R1 epitope and generation of VLP in mammalian cells, insect cells or a silkworm. Wild-type or p12-4R1-mutant gag was cloned under the control of the CMV promoter and they were transfected into mammalian cells. Recombinant baculovirus AcNPV or BmNPV, encoding wild-type or p12-4R1-mutant Gag fused with HA or FLAG-tag, was generated and infected to S.frugiperda cell lines or a silkworm, respectively. As a result, Gag precursor pr44 was highly expressed in all the tested species introduced with wild-type and p12-4R1-mutant gag. Furthermore, VLP formation in mammalian and insect cells was indicated because of detection of pr44 in the pellet of concentrated culture supernatant by ultracentrifugation. VLP vaccine strategy is useful to produce antigens with deleterious mutations such as some mutations in BLV p12 protein that reduce viral RNA incorporation into virion. The physical property of the VLP, its antigenicity in mouse and vaccine efficiency in cattle remain to be assessed in the future study.

타액선 질환 치료에서의 A형 보툴리눔 독소 주입

Background and Objectives: Temporalis fascia and cartilage are well-known materials for the restoration of defective tympanic membrane. Common organisms of postoperative infection in tympanoplasty are S. aureus and P. aeruginosa. This study was done to understand the resistance of cartilage and fascia to extracellular bacterial proteolytic enzyme of the pathogens. Materials and Methods: S. aureus, P. aeruginosa, E. col were put in LB broth and Nutrient broth, incubated each other during 24 hours under 37 °C. We concentrated culture supernatants on ammonium sulphate precipitation, and dialyzing it with normal saline. Extracellular enzyme with same sized cartilage and temporalis fascia tissue were nourished during 48 hours. The amounts of degradated proteins were measured, then proteins on molecular weight enforced by SDS-PAGE were classified. The activities of proteolytic enzyme by enforcement of zymography were detected. Results: We showed the proteolytic activities of cartilage and temporalis fascia by the reaction of extracellular enzyme. S. aureus had the most potent destructive power and the cartilage was more resistant to bacterial extracellular proteolysis in S. aureus than fascia. Conclusions: It could suggest that the cartilage is good for the graft material in aspect of the resistance to extracellular bacterial proteolysis. (J Clinical Otolaryngol 2014;25:150–155)

Human pDCs preferentially sense enveloped hepatitis A virions.

Unlike other picornaviruses, hepatitis A virus (HAV) is cloaked in host membranes when released from cells, providing protection from neutralizing antibodies and facilitating spread in the liver. Acute HAV infection is typified by minimal type I IFN responses; therefore, we questioned whether plasmacytoid dendritic cells (pDCs), which produce IFN when activated, are capable of sensing enveloped virions (eHAV). Although concentrated nonenveloped virus failed to activate freshly isolated human pDCs, these cells produced substantial amounts of IFN-α via TLR7 signaling when cocultured with infected cells. pDCs required either close contact with infected cells or exposure to concentrated culture supernatants for IFN-α production. In isopycnic and rate-zonal gradients, pDC-activating material cosedimented with eHAV but not membrane-bound acetylcholinesterase, suggesting that eHAV, and not viral RNA exosomes, is responsible for IFN-α induction. pDC activation did not require virus replication and was associated with efficient eHAV uptake, which was facilitated by phosphatidylserine receptors on pDCs. In chimpanzees, pDCs were transiently recruited to the liver early in infection, during or shortly before maximal intrahepatic IFN-stimulated gene expression, but disappeared prior to inflammation onset. Our data reveal that, while membrane envelopment protects HAV against neutralizing antibody, it also facilitates an early but limited detection of HAV infection by pDCs.

Reduced MiR-675 in Exosome in H19 RNA-Related Melanogenesis via MITF as a Direct Target

H19 non-coding RNA downregulation stimulates melanogenesis in melasma patients. However, its mechanism is unclear. In this study, the potential role of a H19 microRNA, miR-675, in melanogenesis was examined. Real-time PCR using cultured normal human skin keratinocytes, melanocytes, and fibroblasts with or without H19 knockdown showed accompanying changes between expression levels of H19 and those of miR-675 in keratinocytes. MiR-675 was also detected in concentrated culture supernatants and showed expression levels parallel with those of cell lysates. In addition to RNase resistance, FACS analysis showed anti-CD63-positive exosomes in culture supernatants, suggesting miR-675 could be released extracellularly and delivered to neighboring cells without degradation. In western blot analysis, the miR-675 mimic reduced the expression of microphthalmia-associated transcription factor (MITF) and phosphorylation of cAMP-responsive element-binding protein, extracellular signal-regulated kinase and apoptosis signal-regulating kinase, whereas these expressions were increased by the miR-675 inhibitor. Although H19 was not a miR-675 target, luciferase reporter assay showed a direct binding of miR-675 to 3'-untranslated region of MITF. In addition, localized in vivo miR-675 overexpression in mouse using a cationic polymer transfection reagent showed reduced mRNA expression levels of MITF, tyrosinase, tyrosine-related protein-1 (Trp-1), and Trp-2. Collectively, the results suggest that miR-675 derived from keratinocytes could be involved in H19-stimulated melanogenesis using MITF as a target of miR-675.

Home-made 요구르트와 시판 중인 요구르트에서 분리한 젖산균의 기능적 특성 조사

본 연구는 home-made 요구르트에서 분리한 Lactobacillus casei SK-7과 시중에서 판매되고 있는 요구르트에서 분리한 Lactobacillus bulgaricus YK-11의 기능성을 비교 분석하기 위해 실시하였다. 먼저 분리세균인 SK-7과 YK-11의 생리화학적 특성조사를 진행하였다. 이들 균주는 16S rRNA 염기서열을 이용한 계통유전학적 분석방법으로 동정하여, 각각 L. casei SK-7와 L. bulgaricus YK-11로 명명하였다. 16S rRNA 염기서열을 바탕으로 SK-7과 YK-11의 유전적 계통수를 작성하였다. 72시간 동안 SK-7과 YK-11 배양에서 lactic acid와 acetic acid의 생산, 그리고 pH 변화를 조사하였다. 배양기간 동안 L. casei SK-7과 L. bulgaricus YK-11의 몇가지 기능성을 조사하였다. L. casei SK-7과 L. bulgaricus YK-11 배양상등액은 주어진 nitrite를 각각 93.9%와 88.2% 소거하였다. SK-7 and YK-11 배양상등액의 항산화능은 각각 62.6%와 54.9%였으며, <TEX>${\beta}$</TEX>-galactosidase 활성은 14.9 units/mg과 13.1 units/mg로 측정되었다. 항미생물능력은 SK-7과 YK-11 배양의 20배 배양농축액으로 조사되었으며, SK-7은 이 조사에 사용된 모든 미생물에서 명백한 것으로 관찰되었으나, YK-11에서는 관찰되지 않았다. 부가적인 기능성 연구가 이루어져야 되겠지만, 본 연구에서는 home-made 요구르트에서 분리된 젖산균의 기능성은 시판요구르트에서의 기능성과 비교하여 우수한 것으로 조사되었다. The objective of this work is to investigate and compare several functional properties of lactic acid bacteria (LAB), Lactobacillus casei SK-7 isolated from home-made yogurt and Lactobacillus bulgaricus YK-11 from commercial yogurt. Initially, physiological and biochemical properties of SK-7 and YK-11 were characterized. Phylogenetic analysis using 16S rRNA sequencing were performed to identify the strains, and the strain could be assigned to Lactobacillus casei and Lactobacillus bulgaricus, designated as L. casei SK-7 and L. bulgaricus YK-11. Phylogenetic tree of SK-7 and YK-11 was plotted based on 16S rRNA sequence comparisons. Production of lactic acid and organic acid, and pH changes in the cultures of SK-7 and YK-11 were monitored during 72 h. During the incubation period, several functional properties of L. casei SK-7 and L. bulgaricus YK-11 were examined. L. casei SK-7 and L. bulgaricus YK-11 cultures eliminated 93.9% and 88.2% of nitrite, respectively. Antioxidant activity of cultural supernatants of SK-7 and YK-11 were 62.6%, 54.9%, and activity of <TEX>${\beta}$</TEX>-galactosidase were 14.9 units/mg and 13.1 units/mg, respectively. The antimicrobial activities were examined with 20-fold concentrated culture supernatants from the cultures of SK-7 and YK-11. The activities of SK-7 supernatants were clearly observed against all microorganisms in this work, whereas no activities were observed in YK-11 supernatants. Although it might be conducted additional functional research, functional properties of LAB isolated from home-made yogurt have been shown to be better than those of commercial yogurt in this work.

Production, Partial Purification and Characterization of an Extracellular Psychrotrophic Lipase from Pseudomonas Sp. ADT3

Psychrotrophic Pseudomonas ADT3 (NCBI GenBank Acc.no.JX914667) is capable of growth on lipid as the sole carbon source. In this paper, we report the purification and characterization of an extracellular lipase from psychrotroph, isolated from soil sample of Ny- Alesund, Svalbard, Arctic region. The Pseudomonas ADT3 isolate produces lipase enzyme in the extracellular minimal media with only 1% olive oil. The lipase was purified from the concentrated culture supernatant. The crude enzyme was partially purified by saturated ammonium sulphate precipitation followed by extensive dialysis. Enzyme activity was found to be induced 6-folds in presence of 1.2 mM lead ion but strongly inhibited by heavy metals Hg2+ as well as EDTA and β-mercaptoethanol. The purified lipase has activity at two pH optima of pH i.e. pH 3.5 and 8.5. Optimum temperature for lipase activity was recorded at 22cC. The purified active fraction of lipase exhibits specific activity of 527.8 U/mg. The Vmax and Km was 144.93 U/mg/min and 0.260 mM respectively determined using Lineweaver-Burk plot. Zymogram analysis revealed prominent lipase band at 13.9 kDa range in the 80% saturated ammonium sulphate purified enzyme fraction.