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Industrial Biotechnology and Applied Microbiology is an optional 128h-course for Chemistry and Biology students at the Faculty of Sciences, University of Buenos Aires, Argentina. This course is usually attended by 25 students, working in teams of two. The curriculum, with 8 lab exercises, includes an oil bioremediation practice covering an insight of bioremediation processes: the influence of pollutants on autochthonous microbiota, biodegrader isolation and biosurfactant production for bioavailability understanding. The experimental steps are: (A) evaluation of microbial tolerance to pollutants by constructing pristine soil microcosms contaminated with diesel or xylene and (B) isolation of degraders and biosurfactant production analysis. To check microbial tolerance, microcosms are incubated during one week at 25-28ºC. Samples are collected at 0, 4 and every 48 h for CFU/g soil testing. An initial decrease of total CFU/g related to toxicity is noticed. At the end of the experiment, a recovery of the CFU number is observed, evidencing enrichment in biodegraders. Some colonies from the CFU counting plates are streaked in M9-agar with diesel as sole carbon source. After a week, isolates are inoculated on M9-Broth supplemented with diesel to induce biosurfactant production. Surface tension and Emulsification Index are measured in culture supernatants to visualize tensioactive effect of bacterial products. Besides the improvement in the good microbiological practices, the students show enthusiasm in different aspects, depending on their own interests. While biology students explore and learn new concepts on solubility, emulsions and bioavailability, chemistry students show curiosity in bacterial behavior and manipulation of microorganisms for environmental benefits. Fil: Raiger Iustman, Laura Judith. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: López, Nancy Irene. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Ruzal, Sandra Mónica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Vullo, Diana Lia. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina. Universidad Nacional de General Sarmiento; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Analysing culture supernatants of yeast and hyphal cells of Candida albicans, we found two close homologues of pathogenesis-related (PR-) 1 proteins, Rbe1p and Rbt4p, in the secretome. Due to seque ...

Luminex® technology provides a powerful methodology for multiplex cytokine detection but remains constrained by high costs and a minimum of 25-50μL sample volume requirement per assay-well often hindering analysis of limited biological samples. Here we compare the results of Luminex-based cytokine multiplexing assay performed using conventional 96-well microtiter plates and a particular 96-well wall-less plate based on Droparray® technology ("DA-Bead"). The application of the DA-Bead plate allows 80% reduction of sample and reagent volume, thus an opportunity for significant cost savings in Luminex reagents with no change to the workflow. To compare the DA-Bead method to the conventional method, two different types of samples were tested with two different commercially available Luminex kits and the results for each method were compared. The first type was splenocyte culture supernatants from murine spleens which were harvested from mice immunized with Ascaris suum protein As24 and followed by cell stimulation ex vivo at various time points with this same antigen. Cytokine levels in these supernatants were evaluated using a Bio-Plex® TH1/TH2 8-plex kit. The second sample type was plasma from mice from an experimental autoimmune encephalomyelitis (EAE) study, and these samples were evaluated using a Milliplex® TH17 25-plex kit. The data showed that the DA-Bead method for analysis was comparable to, if not superior to, the conventional method in terms of consistency/precision, accuracy, sensitivity and dynamic range and these results are not specific to sample type, reagents, or commercial vendor.

The paucity of FDA approved adjuvants renders the synthesis, characterization, and use of new compounds as vaccine adjuvants, a necessity. For this purpose, a novel saccharide analog has been synthesized from glucosamine, pyruvylated galactose and 1,4-cyclohexanediol and its biological efficacy was determined in innate immune cells. More specifically, we assessed the production of pro-inflammatory cytokines from the murine monocyte cell line, Raw 264.7 and from C57 BL/6 mouse peritoneal macrophages following exposure to the saccharide analog. Our data conclude that the novel saccharide has immunostimulatory activity on mouse macrophages as indicated by the elevated levels of IL-6 and TNF-α in culture supernatants. This effect was TLR-4-dependent but TLR-2-independent. Our data, suggest TLR-4 agonism; a key feature of vaccine adjuvants.

Mannheimia haemolytica is a major bacterial contributor to bovine respiratory disease complex that costs the livestock industry a billion dollars a year in USA. Commercial vaccines are only partially efficacious under field conditions. Earlier studies found that outer membrane protein preparations and culture supernatants can induce immune responses that enhance resistance to challenge by M. haemolytica strains. The objective of this study was to characterize secretome of two M. haemolytica stains grown under two different media. Bacteria-free concentrated supernatants from M. haemolytica culture was subjected to LC-MS/MS. The secretome of M. haemolytica from both strains yielded 923 proteins. Using bioinformatic tools, 283 were identified as secreted proteins. Further breakdown of 283 proteins showed that 114 (40.2%), 184 (65.0%), 138(48.7%), 151 (53.3%) and 172 (60.7%) were characterized as secreted proteins by SignalP 4.1, SecretomeP 2.0, LipoP, Phobius, and PRED-TAT, respectively. A total of 95 (33.56%) proteins were characterized as being secreted via non-classical pathway as opposed to the majority that were secreted in signal peptide dependent pathway. The demonstrated proteins include all previously immunologically characterized M. haemolytica proteins. The potential of using secretome analysis in the design and development of a multivalent vaccine is discussed.

A total of 1034 samples were collected from different sources and C. difficile was isolated from 18 (9.04%) of 199 human, 9 (4.89%) of 184 cattle, 29 (12.44%) of 233 pig, and from 23 (13.94%) of 165 poultry samples. Variations were observed on the rate of isolation according to age and clinical conditions (diarrhoea). None of the samples from cow, sheep, goat, local chicken, and wild animals yielded any C. difficile . Out of those isolates, 8, 2, 19 and 6 isolates from human, cattle, pig and poultry, respectively were toxigenic. The toxigenic isolates carried both tcd A, and tcd B (A + B + ) and most of the human and the pig isolates were also positive for binary toxin genes ( cdt A and cdt B). The A + B + isolates belonged to three different toxinotypes (0, VI and XXXIII). Human and pig A + B + isolates belonged to three (045, 126 and ACD 019) and four (046, 087, 126 and ACD 011) different ribotypes, respectively and the ribotypes of two cattle isolates were 014 and ACD 010. Six A + B + avian isolates belonged to six different ribotypes (014, 087, SLO 134, SLO 160, ACD 012, ACD 014). The non-toxigenic isolates from human, cattle, pig and poultry were grouped into 7, 4, 4 and 7 different ribotypes, respectively. PFGE analysis could not differentiate similar ribotypes/toxinotypes of toxigenic isolates. All the toxigenic isolates showed cytopathic effect on Vero and Hela cell monolayers at 1:100 dilutions of cell-free culture supernatants within 18–20 h of inoculation.

Terpenes, a large family of natural products with important applications, are commonly associated with plants and fungi. The diterpenoids dehydroabietic and abietic acids are defense metabolites abundant in resin, and are used as biomarkers for conifer plants. We report here for the first time that the two diterpenoid acids are produced by members of several genera of cyanobacteria. Dehydroabietic acid was isolated from two cyanobacterial strains and its identity was confirmed spectroscopically. One or both of the diterpenoids were detected in the cells of phylogenetically diverse cyanobacteria belonging to four cyanobacterial 'botanical orders', from marine, estuarine and inland environments. Dehydroabietic acid was additionally found in culture supernatants. We investigated the natural role of the two resin acids in cyanobacteria using ecologically-relevant bioassays and found that the compounds inhibited the growth of a small coccoid cyanobacterium. The unexpected discovery of dehydroabietic and abietic acids in a wide range of cyanobacteria has implications for their use as plant biomarkers. We thank CEMUP for NMR and MS analyses. We also thank João Morais for the microphotographs of the strains and Aldo Barreiro Felpeto for the collection of subtidal samples. We would also like to acknowledge funding from Fundação para a Ciência e a Tecnologia (FCT) through grants PTDC/MAR-BIO/2818/2012, a FCT Investigator contract to PNL (IF/01358/2014) and fellowship SFRH/BD/80153/2011 to VR. This research was partially supported by the Strategic Funding UID/Multi/04423/2013 from FCT and the European Regional Development Fund (ERDF). We are sincerely grateful to Antarctica New Zealand and Portuguese Polar Program (PROPOLAR) for providing logistics support to Antarctica McMurdo Dry Valleys field campaign

Objective. To determine active TGF-β1 (aTGF-β1) levels in serum, skin, and peripheral blood mononuclear cell (PBMC) culture supernatants and to understand their associations with clinical parameters in systemic sclerosis (SSc) patients.Methods. We evaluated serum samples from 56 SSc patients and 24 healthy controls (HC). In 20 SSc patients, we quantified spontaneous or anti-CD3/CD28 stimulated production of aTGF-β1 by PBMC. The aTGF-β1 levels were measured by ELISA. Skin biopsies were obtained from 13 SSc patients and six HC, and TGFB1 expression was analyzed by RT-PCR.Results. TGF-β1 serum levels were significantly higher in SSc patients than in HC (p< 0.0001). Patients with increased TGF-β1 serum levels were more likely to have diffuse subset (p= 0.02), digital ulcers (p= 0.02), lung fibrosis (p< 0.0001), positive antitopoisomerase I (p= 0.03), and higher modified Rodnan score (p= 0.046). Most of our culture supernatant samples had undetectable levels of TGF-β1. No significant difference in TGFB1 expression was observed in the SSc skin compared with HC skin.Conclusion. Raised active TGF-β1 serum levels and their association with clinical manifestations in scleroderma patients suggest that this cytokine could be a marker of fibrotic and vascular involvement in SSc.

This study examined laccase production by the fungus Trametes versicolor while it was degrading Delor 103, a commercial mixture of polychlorinated biphenyls (PCBs). Delor 103 (10 mg l−1) caused a significant increase in laccase activity in the culture supernatants in nitrogen-limited medium cultures (the maximum activity of 1439 U g−1 was 42 times that of the controls) where the induction effect was suppressed in a malt extract – glucose medium. Similarly, laccase activity stimulation depended on the growth phase of the fungal cultures. Higher laccase activities that developed in Delor 103 treated with non-growing fungal cultures correlated with an up to 2.6-fold greater laccase gene expression, which was analysed by the quantitative polymerase chain reaction (qPCR). Simultaneously, laccases of different pIs were formed in response to Delor 103 treatment. When analysing laccase gene expression by degenerated primers and denaturing gradient gel electrophoresis, differences between transcript profiles of Delor 103-treated and untreated control cultures were observed. Despite all the differences in laccase production, no PCB removal was observed in the non-growing T. versicolor cultures. This makes the connection between laccase production and PCB degradation uncertain.

This study was conducted to extract and titrate the leukotoxin of Fusobacteriumnecrophorum isolates recovered from 75abscesses found in 24 livers of slaughtered cattle inSulaimaniyah region. The culture supernatants of these isolates were subjected to thetetrazolium dye reduction test which revealed that the leukotoxin titer values of 34 F.necrophorum subsp. necrophorum isolates ranged from 128 to 1024 (with a leukotoxin titermean of 516±46), whereas the leukotoxin titer values of the 11 F. necrophorum subsp.funduliforme isolates ranged from 0 to 128 (with a leukotoxin titer mean of 73±12).

Background Previously, we investigated the possibility of using near-infrared (NIR) spectroscopy for the diagnosis of human immunodeficiency virus type-1 (HIV-1) infection. Here, we further analyze NIR spectra using molecular clones of various HIV-1 subtypes. Methods Culture supernatants of pNL4-3- (HIV-1 molecular clone) or pUC18- (empty vector) transfected 293 T cells were used. In addition, culture supernatants obtained using pBal (HIV-1 subtype B molecular clone) or pIndieC (HIV-1 subtype C molecular clone) were used. Near-infrared radiation (NIR) spectra, obtained using the culture supernatants, were subjected to principal component analysis (PCA) to extract and analyze their properties. Results The PCA demonstrated that HIV-1 in medium altered wavelength absorption at around 950 and 1030 nm, suggesting that the HIV-1 altered OH vibration in water. In addition, absorption varied among subtypes at around 950, 1030 and 1060 nm, suggesting that the interaction between HIV-1 and water varies among subtypes. Conclusions These differences in the NIR spectra may make it possible to delineate HIV-1 subtypes spectroscopically.

The utilization of 1-kestose (GF(2)) and nystose (GF(3)), the main components of fructooligosaccharides (FOS), by Lactobacillus and Bacteroides species was examined. Of seven Lactobacillus and five Bacteroides strains that utilized FOS, L. salivarius, L. rhamnosus, L. casei, and L. gasseri utilized only GF(2), whereas L. acidophilus and all the Bacteroides strains utilized both GF(2) and GF(3). Only the strains able to utilize both GF(2) and GF(3) had β-fructosidase activity in the culture supernatants. The culture supernatants of the Lactobacillus strains had higher β-fructosidase activity for GF(2) than for GF(3), whereas those of the Bacteroides strains had higher activity for GF(3) than for GF(2). Furthermore, β-fructosidase activity of the culture supernatants of the Lactobacillus cells grown in the GF(3) medium was much higher than that of the cells grown in the GF(2) medium, whereas the activity of the culture supernatants of the Bacteroides cells grown in the GF(3) medium was almost the same as that of the cells grown in the GF(2) medium. These results indicate that Lactobacillus species metabolize FOS in a different way from that of Bacteroides species.

Enterohemorrhagic Escherichia coli (EHEC) is a foodborne pathogen that causes watery diarrhea and hemorrhagic colitis. In this study, we identified StcE, a secreted zinc metalloprotease that contributes to intimate adherence of EHEC to host cells, in culture supernatants of atypical Shigella boydii 13 (Shigella B13) strains. Further examination of the Shigella B13 strains revealed that this cluster of pathogens does not invade but forms pedestals on HEp-2 cells similar to EHEC and enteropathogenic E. coli. This study also demonstrates that atypical Shigella B13 strains are more closely related to attaching and effacing E. coli and that their evolution recapitulates the progression from ancestral E. coli to EHEC.

The novel proteinaceous protease inhibitor, isolated from the culture supernatants of Streptomyces chromopfuscus 34-1 (SS 34-1) demonstrated a specific and selective anti-influenza virus effect. By the use of complementary virological assays it was demonstrated that the expression of the viral haemagglutinin (HA) on the surface of infected cells, the virus-induced cytopathic effect (CPE) and the infectious virus yields (IVY), used as measures of A/Germany/34, strain Rostock (H7N1) (A/Rostock) virus growth, were all reduced at non-toxic concentrations of SS 34-1 in a dose-dependent way. In a single cycle of viral growth, the penetration of viral particles and the late stages of viral replication were the most susceptible to inhibition. The preparation protected mice from mortality in the experimental A/Aichi/2/68 (H3N2) influenza virus infection. The SS 34-1-treatment (0.16 mg/mouse/day) of virus-infected mice led to a considerable reduction of mortality rate (index of protection = 71.0%) and marked prolongation of mean survival time (+ 5.5 days). The lesions of the lungs due to infection as well as lung infectious virus titres were significantly reduced - delta log(10)TCID(50)/ml up to 4.5. The application of SS 34-1 induced a continuous rise of the anti-protease activity but did not cause substantial changes in the lung protease activity of healthy mice. The infection triggered a slight reduction of protease activity in the lungs at 5 and 48 h p.i. and a marked increase at 24 hand 6 day p.i. Protease inhibition in the lungs was reduced at 24 and 48 h p.i. and an increase was observed at 5 h, 6 and 9 day p.i. SS 34-1-treatment brought both activities to normal levels. It was assumed that SS 34-1 exerted its inhibitory effect indirectly, through an increase of the protease-inhibitory activity. Histopathological examination of the lungs showed that under SS 34-1-treatment pulmonary lesions were less severe than those of untreated controls. The isolated novel protease inhibitor was purified by anion-exchange chromatography and reversed phase-HPLC. Analysis by differential scanning calorimetry (DSC) revealed that at pH 7.5 the denaturation temperature was 75 degrees C and the denaturation itself was irreversible. Analysis by circular dichroism (CD) in the UV region 190-250 nm showed that at pH 7.5 the spectrum presented 2 minima at 208 and 222 nm, typical for an a-helical structure. At pH 2.5 and after heating the spectrum corresponded to an unordered structure.

Aim The aim of this work was to evaluate the efficacy of domestic cooking in inactivating Manila clams experimentally infected with human hepatitis A virus (HAV). Methods and results Electronic temperature probes were positioned to measure the internal temperature of Manila clams during domestic cooking. Two batches were infected with 10(7) and 10(5) TCID50 ml(-1) of HAV. The infected whole-in-shell clams were divided into three replicates and cooked on a conventional stove both singularly and in group and removed from the pan at fixed intervals. Pools of three digestive glands were examined by virus isolation for three blind passages and cell culture supernatant tested with real-time PCR. Conclusion Results showed that 2-min cooking by a traditional domestic method at a temperature close to 100°C, after the opening up of the valves of all the clams, can completely devitalize the HAV in high viral load-infected clams. Significance and impact of the study This is the first study on inactivation of HAV in experimentally infected Manila clams subjected to domestic cooking. At present, labelling all lagoon products as 'requiring cooking before consumption' is highly recommended, but no specifications are given on how long and at what temperature they should be cooked. Considering the high commercial value of Manila clams, our results can provide both the producers and the consumer with useful indications on how to cook clams to prevent the risk of HAV foodborne illness.

PD-L1 (CD274) is a critical membrane-bound costimulatory molecule that inhibits immune responses through its receptor, PD-1. Previous data have showed that this molecule is associated with autoimmune diseases, chronic viral infections and tumor immune escape. However, the existence and role of soluble form of human PD-L1 (sPD-L1) remain unknown. We show here that a novel enzyme-linked immunosorbent assay (ELISA) was developed to detect the sPD-L1 protein. Many culture supernatants of the PD-L1+ cell lines contain high levels of this factor. Interestingly, the sPD-L1 is detectable in human serum and the concentration increases in an age-dependent manner. Human sPD-L1 has a unique protein form in the serum and the matrix metalloproteinase inhibitor (MMPI) could suppress sPD-L1 production. Moreover, the sPD-L1 could specially bind to PD-1. Together, these data demonstrate that the existence of circulating sPD-L1 in human serum might play an important role in immunoregulation.

Highlights ► A super high speed qRT-PCR (SHRT-PCR) was developed. RT reaction and PCR (40 cycles) can be completed in less than 20 min. ► Despite the high speed, the sensitivity and specificity of the SHRT-PCR are comparable to conventional qRT-PCR system. ► The SHRT-PCR can be used to test clinical samples., The development of a rapid and sensitive system for detecting influenza viruses is a high priority for controlling future epidemics and pandemics. Quantitative real-time PCR is often used for detecting various kinds of viruses; however, it requires more than 2 h per run. Detection assays were performed with super high-speed RT-PCR (SHRT-PCR) developed according to a newly designed heating system. The new method uses a high-speed reaction (18 s/cycle; 40 cycles in less than 20 min) for typing influenza viruses. The detection limit of SHRT-PCR was 1 copy/reaction and 10−1 plaque-forming unit/reaction for viruses in culture supernatants during 20 min. Using SHRT-PCR, 86 strains of influenza viruses isolated by the Tokyo Metropolitan Institute of Public Health were tested; the results showed 100% sensitivity and specificity for each influenza A and B virus, and swine-origin influenza virus. Twenty-seven swabs collected from the pharyngeal mucosa of outpatients were also tested, showing positive signs for influenza virus on an immunochromatographic assay; the results between SHRT-PCR and immunochromatography exhibited 100% agreement for both positive and negative results. The rapid reaction time and high sensitivity of SHRT-PCR makes this technique well suited for monitoring epidemics and pre-pandemic influenza outbreaks.

This research was done to verify the effect of Ecklonia cava water extracts (ECWE) on inhibition of allergic reactions using ovalbumin (OVA)-immunized food allergy mouse model. For in vitro test, 10～100 ㎍/mL of ECWE and OVA were added to splenocytes obtained from OVA-immunized mice. The significant reduction of IgE antibody level in culture supernatants of splenocytes was shown in ECWE adding group at all tested concentrations. In addition, ECWE decreased IL-4 and IFN-γ levels in supernatants of splenocytes. To confirm the effect of ECWE in in vivo test, ECWE was injected to peritoneal cavity of OVA-immunized mice. Subsequently, IgE level was measured in serum and cultured supernatants of splenocytes. As a result, the injection of ECWE (5 and 10 ㎎/㎏?BW) significantly attenuated the secretion of IgE antibody in both serum and splenocytes. In conclusion, the present study indicates that ECWE could suppress in a food allergy mouse model through the inhibition of IgE secretion.

Staphylococcal enterotoxins (SEs) are the cause of staphylococcal food poisoning (SFP) outbreaks. Recently, many new types of SEs and SE-like toxins have been reported, but it has not been proved whether these new toxins cause food poisoning. To develop an immunoassay for detection of SE-like J (SElJ), a non-characterized toxin in SFP, a mutant SElJ with C-terminus deletion (SElJ∆C) was expressed and purified in an E. coli expression system. Anti-SElJ antibody was produced in rabbits immunized with the SElJ∆C. Western blotting and sandwich enzyme-linked immunosorbent assay (ELISA) detection systems were established and showed that the antibody specifically recognizes SElJ without cross reaction to other SEs tested. The limit of detection for the sandwich ELISA was 0.078 ng/mL, showing high sensitivity. SElJ production in S. aureus was detected by using the sandwich ELISA and showed that selj-horboring isolates produced a large amount of SElJ in the culture supernatants, especially in that of the strain isolated from a food poisoning outbreak in Japan. These results demonstrate that the immunoassay for detection of SElJ is specific and sensitive and is useful for determining the native SElJ production in S. aureus isolated from food poisoning cases.

Berberine has shown a number of beneficial effects, including anti-tumor, anti-inflammation, and vasodilatory effects. In this work we investigated the effects of berberine on the production of proinflammatory cytokines such as TNF-, IL-, and IL-6 in mice. The supernatants of cultured splenocytes exposed with berberine or berberine plus LPS were harvested to assay TNF-, IL-, and IL-6. The sera from the mice injected with berberine or berberine plus LPS were then isolated to assay these cytokines. The TNF- production in mice splenocyte cultures exposed to berberine was inhibited compared to the PBS control. The sera from LPS plus berberine injected mice showed lower levels of TNF- compared to those of LPS only injected mice. The IL- production in mice splenocyte cultures exposed to berberine was inhibited at a high dose (3.0 ) compared to the PBS control. Also, the increase of IL- by LPS exposure in splenocyte cultures was inhibited by a high dose of berberine. The IL-6 in splenocyte culture supernatants showed lower levels after berberine compared to the PBS control. Also, production of IL-6 after LPS exposure in splenocyte cultures was inhibited by a low dose of berberine (0.3 ). These findings suggest the probability that berberine down-regulates the production of proinflammatory cytokines such as TNF-, IL-, and IL-6.

Osteocalcin was recently identified as an osteoblast-secreted hormone regulating insulin secretion and sensitivity. In mice and humans, osteocalcin can be present in the serum in carboxylated or undercarboxylated forms and it has been shown that it is the undercarboxylated form of osteocalcin which acts as a hormone. The study of osteocalcin different circulating forms in mouse serum, however, has been hampered by the absence of quantitative methodology. Here we described a triple enzyme-linked immunosorbent assay (ELISA) system for quantification of mouse total, carboxylated and uncarboxylated osteocalcin. That carboxylation of osteocalcin was decreased in mouse osteoblasts cultures treated with warfarin, an inhibitor of carboxylation validated this assay. This ELISA could also detect elevated levels of undercarboxylated osteocalcin in the serum of mice treated with warfarin and in the serum of Esp -/- mice, a mouse model known to have more undercarboxylated, i.e., active osteocalcin. These results show that this new ELISA system is a reliable method to assess carboxylation status of osteocalcin in cell culture supernatants as well as in mouse serum. Its use should facilitate the analysis of culture system or mouse model in which the hormonal activity of osteocalcin needs to be evaluated.

A new electrophoretic technique for the qualitative and quantitative analyses of IgM isoforms and fragments has been developed. IgMs which are more complex than many other recombinantly expressed immunoglobulins are characterized by their high molecular weighted active forms and many additional isoforms and fragments in the molecular range between 25 and 1200 kDa. To analyze the multimers, isoforms and fragments simultaneously a high-resolution method, which enables sufficient migration and separation is required. Furthermore, this method should be appropriate to analyze IgMs in crude culture supernatants as well as purified samples. Simple sample preparation avoiding unspecific protein loss has been established. Currently no standard method to analyze all of them accordingly is available. The IgM-SDS-PAGE investigated for this purpose includes all these aspects. The combination of simple sample preparation and the application of precast gels make this electrophoretic method suitable for research but also quality control. The selective quantification of the multimers and the relative isoform distribution were performed by sensitive Sypro Ruby staining obtaining reliable and reproducible data in clone screening and process development which has been demonstrated by recombinantly expressed IgMs with significantly different isoform pattern.

Zusammenfassung Bei einem kolostrumfrei aufgezogenen Kalb wurde nach subkutaner Infektion mit Staph. epidermidis Biotyp 2 nachgewiesen, das dieser Erreger beim Rind Hautveranderungen verursacht, die mit Exsudation und Exfoliation einhergehen. Mit bakterienfreien, konzentrierten Kulturuberstanden von 5 verschiedenen Staph. hyicus-Stammen konnten nach subkutaner Injektion beim Schwein die gleichen Hautveranderungen wie beim lokalen NE hervorgerufen werden. In gleicher Weise gelang es mit entsprechenden Kulturuberstanden von 3 Staph. epidermidis-Biotyp-2-Stammen vom Rind bei kolostrumfrei aufgezogenen Kalbern mit Exsudation und Exfoliation einhergehende Hautreaktionen zu erzeugen. Verschiedene Staph. epidermidis-Stamme anderer Biotypen von Schwein und Rind wiesen in ihren konzentrierten Kulturuberstanden keine Exfoliation auslosenden Substanzen auf. Der Vergleich dieser Exfoliation auslosenden Substanzen mit dem Exfoliatin, einem exfoliativen Toxin, von Staph. aureus des Menschen, das von einem Stamm der Phaggruppe II gewonnen wurde, last vermuten, das es sich bei den Exfoliation auslosenden Substanzen von Staph. hyicus des Schweines und Staph. epidermidis Biotyp 2 des Rindes ebenfalls um exfoliative Toxine handelt. Summary Demonstration of exfoliation-producing substances in cultures of Staph. hyicus of pigs and Staph. epidermidis biotype 2 of cattle After subcutaneous injection of Staph. epidermidis biotype 2 into a calf reared without colostrum the agent produced skin changes with exfoliation and exudation. Bacterium-free, concentrated culture supernatant from 5 different Staph. hyicus strains injected s. c. into pigs produced the same changes as in exudative epidermitis (Nassendes Ekzem). In the same way culture supernatants from 3 Staph. epidermidis biotype 2 strains from cattle provoked similar changes in the skin of calves reared without colostrum. Various Staph. epidermidis strains of other biotypes from pigs and cattle showed no evidence of substances in their concentrated culture supernatants which caused exfoliation. Comparison of these exfoliation-inducing substances with exfoliatin, and exfoliative toxin from human staphylococci, obtained from a strain of phage group 2, suggests that the exfoliation-inducing substances of Staph. hyicus of pigs and Staph. epidermidis biotype 2 from cattle are also exfoliative toxins. Resume Mise en evidence de substances exfoliatrices dans des cultures de Staphylococcus epidermidis biotype 2 du bovin Une infection sous-cutanee chez un veau prive de colostrum avec Staph. epidermidis biotype 2 a provoque des lesions cutanees avec exsudation et exfoliation. Des lesions semblables ont ete constatees apres une injection sous-cou-tanee chez un porc avec le surnageant sans germes des cultures concentrees de 5 souches differentes de Staph. hyicus. On a pu provoquer des reactions cutanees avec exsudation et exfoliation de la meme maniere en inoculant des veaux prives de colostrum avec les surnageants des cultures de 3 souches de Staph. epidermidis biotype 2 de bovin. Differentes souches de Staph. epidermidis d'autres biotypes provenant de porcs et de bovins n'ont pas montre de substances exfoliatrices dans les surnageants concentres. En comparant ces substances exfoliatrices avec l'exfoliatrine, toxine exfoliative isolee d'une souche de Staph. aureus humaine du groupe de phage II, on peut supposer qu'il s'agit egalement d'une toxine exfoliative en ce qui concerne les substances exfoliatrices de Staph. hyicus du porc et Staph. epidermidis biotype 2 du bovin. Resumen Identificacion de substancias desencadenantes de exfoliacion en cultivos de Staphylococcus epidermidis biotipo 2 vacuno En un ternero recriado sin probar calostro se demostro tras infeccion subcutanea con Staph. epidermidis biotipo 2 que este agente etiologico ocasiona en el ganado vacuno lesiones cutaneas acompanadas por exudacion y exfoliacion. Con sobrenadantes culturales concentrados, libres de bacterias, de 5 estirpes diferentes de Staph. hyicus se pudieron producir tras inyeccion subcutanea en el cerdo las mismas lesiones cutaneas que en la impetigo contagiosa local. De la misma forma se logro producir con los sobrenadantes culturales correspondientes de 3 estirpes de Staph. epidermidis biotipo 2 vacuno en terneros criados sin probar calostro reacciones cutaneas ligadas a exudacion y exfoliacion. Diversas estirpes de Staph. epidermidis de otros biotipos porcinos y vacunos no tenian ninguna substancia en sus sobrenadantes culturales concentrados que desencadenase la exfoliacion. La comparacion de estas substancias desencadenantes de exfoliacion con la exfoliatina, una toxina exfoliativa de Staph. aureus humano, obtenida de una cepa del grupo fago II, permite sospechar que en las substancias desencadenantes de exfoliacion de Staph. hyicus porcino y Staph. epidermidis biotipo 2 vacuno se trata tambien de toxinas exfoliativas.

Zusammenfassung Die Stamme Gifu-1 und Cux-1 des Erregers der Kukenanamie (CAA) wurden in Kulturen der lymphoblastoiden Zellinie MDCC-MSB 1 vermehrt und titriert. Maximale Infektiositatstiter wurden in den Zellen 2 Tage p. i. und im zellfreien Kulturmedium 3 Tage p. i. erreicht. Zytopathische Effekte waren nur nach Beimpfung der Kulturen mit CAA in einer Multiplizitat von mindestens 5ID50 pro Zelle eindeutig zu erkennen. Fast alle MSB-Zellen erschienen 30 Stunden nach ausreichend starker Infektion der Kulturen stark aufgeblaht. Der Zerfall infizierter Zellen begann 34 Stunden p. i. und nahm wahrend der folgenden 24 Stunden erheblich zu. Mit Hilfe des indirekten Immunfluoreszenz(FA)-Tests liesen sich ab 34 Stunden p. i. bei uber 50% der vergroserten Zellen CAA-Antigene in Form von kleinen Granula im Zellkern nachweisen. CAA-Titrationen erforderten eine mindestens 7 malige Subkultivierung der beimpften Zellen in Abstanden von 3—4 Tagen und dauerten deshalb jeweils 5—6 Wochen. Die Bestimmung neutralisierender Antikorper-Titer in Huhnerseren war ebenso langwierig. Qualitative SN-Tests mit niedrigen Serum- oder Dotterverdunnungen und hohen CAA-Dosen erforderten nur 2—3 Subkultivierungen mit einer Dauer von 10—14 Tagen bis definitive Ergebnisse vorlagen. Der indirekte FA-Test mit CAA-infizierten, 34—44 Stunden p. i. in Aceton fixierten MSB-Zellen als Antigen envies sich fur den Nachweis von CAA-Antikorpern als etwas weniger empfindlich als der SN-Test. Bei Versuchen zum qualitativen Nachweis von CAA-Antikorpern in Serum- oder Dotterproben ergab sich jedoch eine Ubereinstimmung von mehr als 80% zwischen den Ergebnissen von FA- und SN-Tests. Summary Investigations on chicken infectious anaemia agent (CAA) in vitro: Propagation, titration, serum neutralization tests, and indirect immunofluorescence assays Strains Gifu-1 and Cux-1 of chicken anaemia agent (CAA) were propagated and titrated in cultures of the MDCC-MSB 1 lymphoblastoid cell line. Maximum infectivity titres were reached in the cultured cells at 2 days p. i., and in cell-free culture supernatants at 3 days p. i. Cytopathic effects could only be clearly recognized if cultures were inoculated with CAA at a multiplicity of at least 5ID50 per cell. In sufficiently infected cultures, almost all cells appeared considerably swollen at 30 hours p.i.; cell destruction started by 34 hours p. i. and was increasing during the following 24 hours. Small granules of intranuclear CAA antigens could be demonstrated by indirect immunofluorescence at 34 hours p. i. and thereafter in at least 50% of the swollen MSB 1 cells. Titration of CAA infectivity required 5—6 weeks because inoculated cells had to be subcultured at least 7 times every 3 to 4 days. Determination of neutralizing antibody titres in chicken sera was equally lengthy, but qualitative SN tests employing low serum or yolk dilutions and high concentrations of CAA needed only 2 to 3 subcultures, i. e. 10 to 14 days, to provide definite results. Indirect fluorescent antibody (FA) tests, using CAA-infected cells, fixed in acetone at 34 to 44 hours p. i., as a source of antigen were less sensitive than SN tests. However, there was a more than 80 % concordance between the results of FA and SN tests if employed as qualitative assays to detect the presence of CAA antibody in egg yolks or in chicken sera. Resume Recherches sur l'agent de l'anemie infectieuse des poussins (CAA) in vitro: multiplication, titration, test de seroneutralisation et test d'immunofluorescence indirecte Les souches Gifu-1 et Cux-1 de l'agent de l'anemie du poussin (CAA) ont ete multipliees et titres dans des cultures de lignee cellulaire lymphoblastoide MDCC-MSB 1. Les titres infectieux maximaux ont ete atteints 2 jours p. i. dans les cellules et 3 jours p. i. dans le milieu de culture sans cellules. Des effets cytopathiques furent seulement reconnaissables apres une inoculation des cultures avec CAA dans une multiplication d'au-moins 5ID50 par cellule. Presque toutes les cellules MSB sont apparues gonflees 30 heures apres une infection suffisamment forte des cultures. La destruction des cellules infectees a commence 34 heures p. i. et a augmente fortement durant les 24 heures suivantes. Des antigenes CAA sous la forme de fines granulations dans le noyau cellulaire ont ete mis en evidence des la 34e heure p. i. chez plus du 50% des cellules grossies avec le test d'immunofluorescence (FA) indirecte. Les titrations CAA exigerent au moins une sous-culture en 7 fois des cellules inocuees en l'espace de 3—4 jours et durerent 5—6 semaines. La determination du titre des anticorps neutralisants dans les serums de volaille furent egalement de longue duree. Les tests de seroneutralisation avec des dilutions basses de serum ou de liquide vitellin et des doses Clevies CAA exigerent seulement 2—3 sous-cultures avec une duree de 10—14 jours pour obtenir des resultats definitifs. Le test FA indirect avec des cellules MSB CAA-infectees et fixees a l'acetone 34—44 heures p. i. comme antigene s'est revele moins sensible pour la mise en evidence des anticorps CAA que le test de neutralisation. Une concordance de plus de 80% entre les resultats de tests FA et SN a ete trouvee lors des essais de mise en evidence qualitative des anticorps CAA dans des echantillons seriques et vitellins. Resumen Estudios in vitro sobre el agente etiologico de la anemia infecciosa en pollitos (CAA): multiplicacion, titulacion, prueba de seroneutralizacion y prueba indirecta de la inmunofluorescencia Las estirpes Gifu-1 y Cux-1 del agente etiologico de la anemia de los pollitos (CAA) fueron multiplicadas y tituladas en cultivos de la linea celular linfoblastoide MDCC-MSB 1. Los titulos maximos de infecciosidad se consiguieron en las celulas 2 dias p. i. y en el medio de cultivo libre de cilulas 3 dias p. i. Efectos citopaticos solo se pudieron reconocer de forma clara tras la inoculacion de los cultivos con CAA en una multiplicidad al menos de 5DI50 por celula. Casi todas las celulas MSB aparecieron fuertemente insufladas a las 30 horas tras una infeccion suficientemente intcnsa de los cultivos. La desintegracion de las celulas infectadas comenzo 34 horas p. i. y aumento considerablemente durante las 24 horas siguientes. Con ayuda de la prueba indirecta de inmunofluorescencia (FA) se pudieron poner en evidencia, a partir de las 34 horas p. i., en mas del 50% de las celulas aumentadas de tamano, antigenos CAA en forma de granulos pequenos en el nucleo celular. Las titulaciones CAA precisaron al menos un subcultivo de 7 pases de las celulas inoculadas en intervalos de 3—4 dias y duraba, por tanto, cada vez de 5—6 semanas. La determinacion de los titulos de anticuerpos neutralizantes en sueros de gallinas era igual de engorrosa. Las pruebas cualitativas de SN con diluciones bajas de suero o yema y dosis elevadas de CAA solo precisaban 2—3 subcultivos con una duracion de 10—14 dias hasta que se podia disponer de los resultados definitivos. La prueba FA indirecta con celulas MSB fijadas 34—44 horas p. i. en acetona, infectadas con CAA, como antigeno resulto ser para la puesta en evidencia de anticuerpos CAA algo menos sensible que la prueba de SN. Sin embargo, en ensayos acerca de la puesta en evidencia cualitativa de anticuerpos CAA en muestras de suero y yema se infirio una concordancia superior al 80% entre los resultados de las pruebas de FA y de SN.

目的．肺癌の早期診断に有用なバイオマーカーの確立は治療成績向上のための重要な課題である．我々は血清マーカーを探索する目的で，肺腺癌組織から分泌あるいは漏出する一群の蛋白質を分析した．方法．外科的に切除した12症例の肺腺癌組織および同じ症例の正常な末梢肺組織を各々無血清培地で短期培養した．培養上清中の癌組織および正常組織由来の蛋白質を，異なる蛍光波長を持つ色素化合物（CyDyeTM：Cy2，Cy3 and Cy5）でそれぞれ標識した．標識試料を症例ごとに混合後，各々二次元電気泳動で展開した．結果．二次元ゲル上で検出された蛋白質スポットのうち，34個のスポットは少なくとも5症例から検出され，かつ正常組織に対する癌組織の平均蛍光強度比が2.0以上を示した（p

Background The role and function of T regulatory (Treg) cells have not been fully investigated in patients with systemic sclerosis (SSc). Methods Ten patients with SSc donated 20 ml of peripheral blood. Activity (Valentini) and severity (Medsger) scores for SSc were calculated for all patients. Healthy volunteers (controls) were matched to each patient by gender and age. CD4 + cells were separated using the MACS system. The numbers of Treg cells were estimated by flow cytometry after staining for CD4, CD25, and FoxP3 and calculated as patient-to-control ratio separately for each experiment. Correlations with activity and severity indices of the disease were performed. Twenty-four-hour production of TGF-β and IL-10 by activated CD4 + cells was measured by ELISA in culture supernatants. Results The numbers of Treg cells, expressed as patient-to-control ratio, correlated significantly with both activity and severity indices ( r = 0.71, p = 0.034 and r = 0.67, p = 0.044, respectively). ELISA-measured production of TGF-β and IL-10 by CD4 + cells was similar in patients and controls. Conclusions Increased numbers of Treg cells are present in patients with SSc, correlating with activity and severity of the disease. This expansion of Treg cells was not accompanied, however, by heightened TGF-β or IL-10 production. Further studies to elaborate the causes and functional significance of Treg cell expansion in SSc are needed.

Lipase production of 425 S. aureus strains isolated from patients with different clinical diagnoses and healthy carriers were measured by a specific method, using emulsified trioleoylglycerol substrate. Strains isolated from patients with septicemia showed significantly higher lipase activity than osteomyelitis strains (p = 0.011), impetigo strains (p = 0.002) and strains isolated from healthy relatives of patients with recurrent furunculosis (p = 0.019). Recurrent furunculosis and pyomyositis strains had significantly higher (p = 0.002 and 0.032, respectively) lipase activity than septicemia strains. S. aureus strains isolated from patients with a significant antibody response in an antilipase ELISA did not show a higher lipase activity in culture supernatants than strains from patients without a significant antibody response. The lipase activity was significantly higher in strains isolated from deep or subcutaneous infections, i.e., septicemia, pyomyositis, osteomyelitis, aerobic and anaerobic furunculosis, than in strains from superficial infections, i.e. impetigo, or from nasal mucosa.

Sheep red cell-stimulated cultures containing lymphocytes from sheep red cell immunized rabbits were harvested after different culture periods. Blastoid cells appeared and increased in numbers in the first 4–6 days of incubation, with subsequent reduction. Transformed cells with the ability to form pericellular lytic plaques in sheep red cell monolayers were regularly found after 6–8 days' incubation. Plaque-forming cells were also present after 11 and 14 days' incubation, but usually in reduced numbers. In the second week of culture agglutinins were regularly found in the culture supernatants. In the same period cells morphologically similar to plasma cells were found in the cultures.

Porphyromonas gingivalis produces proteases capable of degrading cytokines, host heme proteins and some antimicrobial peptides. In this study, we show that P. gingivalis culture supernatants fully or partially degrade human neutrophil peptide α-defensins and human β-defensins after 30 min. This observation suggests that proteases from P. gingivalis degrade defensins and this activity could abrogate defensin-related innate immune functions.

Vibrio vulnificus has been known to secrete one form of metalloprotease VvpE (45 kDa) that is cleaved to 34 kDa-VvpE and 11 kDa-C-terminal propeptide via extracellular autoproteolysis. However, we found that extracellular secretion of both the 34 and 45 kDa forms of VvpE began in the early growth phase; moreover, 34 kDa-VvpE existed as the major form in V. vulnificus cell lysates and culture supernatants. In addition, extracellular secretion of both 34 and 45 kDa-VvpE was blocked by mutation of the pilD gene, which encodes for the type IV leader peptidase/N-methyltransferase of the type II general secretion system, and the blocked VvpE secretion was recovered by in trans-complementation of the wild-type pilD gene. These results indicate that 34 kDa-VvpE is the major form secreted along with 45 kDa-VvpE from the early growth phase via the PilD-mediated type II general secretion system.

Objective To investigate the effects of soluble factors secreted by acute myeloid leukemia (AML) cells on the phenotypical and functional properties of DCs derived from normal mononuclear cells.

Summary Equine arteritis virus (EAV), a member of the newly established family Arteriviridae, is a small, positive-stranded RNA virus. It carries two protein complexes in its envelope, gp5/M and the recently described gp2b/gp3/gp4 complex. We report here on several basic features of EAV replication in cell culture and on the protein composition of virus particles. We have also characterized gp2b, gp3, and gp4 expressed using a baculovirus system in insect cells. Finally, we provide evidence that EAV possess hemagglutinating and hemolytic activity. The hemolysis assay might be useful for determining which of the surface proteins carries the receptor-binding and membrane fusion activity of EAV.

The aim of this study was to examine whether human gingival fibroblasts (HGFs) sustain to produce prostaglandin E 2 (PGE 2 ) in the presence of lipopolysaccharide (LPS). HGFs were treated with or without 10 ng/ml of LPS from Porphyromonas gingivalis (PgLPS) for 7 days collecting the culture supernatants for every 24 h, and PGE 2 concentration in the supernatants was measured by ELISA. HGFs produced PGE 2 at low level without PgLPS treatment. In contrast, HGFs continuously produce PGE 2 with PgLPS treatment. At each day, the amount of PGE 2 production by HGFs with PgLPS treatment was higher than that without treatment. These results demonstrate that the characteristic of HGFs, which continuously produce PGE 2 in the presence of LPS, sustains inflammatory response in periodontal disease.

We previously reported a requirement of interferon-gamma (IFN-gamma) production by both T cells and cells other than T or natural killer (NK) cells in the brain for prevention of toxoplasmic encephalitis. In the present study, we examined whether microglia, the resident macrophage system in the brain, produce IFN-gamma in response to infection with Toxoplasma gondii in SCID and wild-type BALB/c mice. IFN-gamma was detected in the culture supernatants of microglia purified from the brains of SCID mice that had developed toxoplasmic encephalitis due to reactivation of infection. A significant increase in numbers of IFN-gamma-expressing microglia was also observed by immunostaining for this cytokine in the brains of SCID and BALB/c mice during the acute stage of acquired infection, and those numbers decreased in the later stage of infection in the BALB/c animals. These results indicate that microglia produce IFN-gamma in the presence and absence of T cells in response to reactivated or acute acquired infection in the brain. Because IFN-gamma is the essential effector molecule to control tachyzoites and because this cytokine is a potent inducer of expression of chemokines and MHC antigens important for recruitment and activation of T cells, IFN-gamma production by microglia might play a critical role in the early stage of tachyzoite proliferation in the brain by limiting parasite growth and initiating subsequent T cell immunity.

Problem Immunoregulatory effects of choriocarcinoma-derived factors on leukocytes have been documented. The present study was designed to investigate the effect of JEG-3 culture supernatants on interferon-γ (IFN-γ), interleukin-17 (IL-17) and IL-1β production in the mixed lymphocyte reactions (MLRs). Method of study A human choriocarcinoma cell line JEG-3 was used to test the effects of its culture supernatants on the proliferation and cytokine production in the MLRs. The cell proliferation was assessed using the BrdU incorporation and the amounts of cytokines were measured using enzyme-linked immunosorbent assays. Results The JEG-3 culture supernatants caused significantly reduced IFN-γ and IL-17 production in the MLRs. However, the supernatants did not influence MLR production of IL-1β. Conclusion IFN-γ and IL-17 are mainly produced by activated T cells but IL-1β is primarily produced by monocytes, thus suggesting that immunoregulatory factors of JEG-3 cells selectively inhibit cytokine production by activated T cells rather than that of the monocytes.

Initial results demonstrating the feasibility of a multiplexed liquid array immunoassay for foot-and-mouth disease viral antigen detection and simultaneous serotype differentiation are presented. Serotype-specific antibodies from rabbit and guinea pig hyperimmunesera were isolated and prepared for use in a multiplexed, bead-based assay. The performance of all of the available antibodies as both capture and detector reagents was evaluated in the multiplexed system to establish a combination exhibiting the highest homotypic responses and lowest heterotypic reactions. The multiplexed assay was evaluated against inactivated cell culture supernatant samples of the same subtype as the virus used to raise the capture and detector antibodies. Distinct serotype differentiation was observed, except in the case of serotype SAT1. Subsequently, cell culture supernatant samples from a larger pool of viral subtypes were analyzed. Distinct serotype differentiation was obtained when analyzing cell culture supernatant samples from viral serotypes C, Asia, and SAT3, irrespective of the subtype. However, limitations of the current antibody pairs were realized in some inconclusive results obtained when analyzing samples from a broader range of O, A, and SAT2 subtypes. The results obtained in this initial study will be used to further optimize the assay using polyvalent or monoclonal antibodies and move toward the analysis of clinical samples.

The aim of this study was to evaluate and compare the biological effects of three resin monomers on three human gingival fibroblast (HGF) cell lines and immortalised human keratinocytes.Primary HGFs and HaCaT keratinocytes were cultured for 24h and grown to sub-confluent monolayers. Resin monomers were dissolved in dimethyl sulphoxide (DMSO) and diluted with culture medium. Cultures were exposed to different concentrations of monomers (10(-2) to 10mM) for 24h. Cell viability measured by Alamar Blue assay, and cell culture supernatant was examined for the presence of human interlukin-1beta (IL-1beta) using sandwich enzyme-linked immunosorbant assay (ELISA). TC50 values were calculated from fitted dose-response curves.All monomers showed toxic effects on the HGFs and HaCaT cells and inhibited chemical reduction of Alamar Blue in high concentrations. Statistical analysis of TC50 values by one-way ANOVA followed by Tukey's analysis showed that there is a significant difference in TC50 values between the cell lines (p0.05), although the rank order of monomer toxicity remained the same for different cell lines. None of these monomers-induced IL-1beta release from HGFs and HaCaT cells.Dental resin monomers are toxic to human gingival fibroblasts and HaCaT keratinocytes. However, they cannot induce IL-1beta release from these cells by themselves. Alamar Blue assay is a sensitive method for the evaluation of cytotoxicity and it can detect different sensitivities of different cell lines to the resin monomers.

A neutral exopolysaccharide produced in large amounts by Lactobacillus fermentum TDS030603 was purified from culture supernatants and its structure determined by monosaccharide analysis, methylation analysis and 1H-NMR spectroscopy. Optimum conditions for the production of this exopolysaccharide by the above bacterial strain, when cultured using cheese whey medium or MRS broth, were also investigated. The exopolysaccharide was found to be composed of D-galactose and D-glucose in the molar ratio of 1:2.5 and to consist of one major as well as one minor saccharide, both of which contained a tetrasaccharide repeating unit consisting of two α and two β anomers. The major saccharide contained non-reducing glucopyranose, O-3 substituted glucopyranose, O-6 substituted galactopyranose and O-2,3 disubstituted glucopyranose. The minor saccharide consisted of non-reducing galactopyranose, O-3 substituted glucopyranose, O-6 substituted galactopyranose and O-2,3 disubstituted glucopyranose. The results suggest that these exopolysaccharides are novel saccharides that differ from previously reported products of Lactobacillus fermentum strains. Their presence may contribute to the ropy properties of the medium.

Background An improved version of the bioMerieux NucliSENS® EasyQ® HIV-1 v2.0 has been introduced to overcome the underquantification observed with previous versions, especially with non-B HIV-1 subtypes. Objectives Comparing bioMerieux NucliSENS® EasyQ® HIV-1 v2.0 versus Roche Cobas CA/CTM v2.0 and Abbott RealTime HIV-1 assays for HIV-1 group M and non-M (N, O, P) viral load measurement. Study design The three assays were tested in parallel on 103HIV-1 group M plasma samples, and on non-group M HIV-1 culture supernatants. Results Values obtained for the 103HIV-1 group M plasma samples tested with bioMerieux assay showed good overall correlation compared to the 2 others. The Roche Cobas assay gave higher values than the bioMerieux assay, while Abbott and bioMerieux both displayed similar results. However, analysis showed a wider dispersion in results when comparing the bioMerieux NucliSENS® EasyQ® HIV-1 v2.0 and the other 2 techniques. All data taken into account, we observed frequent discrepancies in quantification, of the plasma samples and major differences above 1 log in 10/72 (13.8%). The quantification of non-M HIV groups in culture supernatant has shown variable results, with better quantification of HIV-O and of HIV-N respectively with the Abbott assay and the bioMerieux assay. Conclusions The bioMerieux NucliSENS® EasyQ® HIV-1 v2.0 showed improved sensitivity to non-B HIV-M subtypes compared to previous versions. Notwithstanding, we observed frequent discrepancies and a wide dispersion in results when comparing bioMerieux NucliSENS® EasyQ® HIV-1 v2.0 and the other 2 techniques.

Context: It is necessary to advance the field of alternative treatments for chronic wounds that are financially accessible to the least economically developed countries. Previously we demonstrated that topical applications of Lactobacillus plantarum culture supernatants (LAPS) on human-infected chronic wounds reduce the pathogenic bioburden, the amount of necrotic tissue, and the wound area, as well as promote debridement, granulation tissue, and wound healing. Objective: To study LAPS chemically and biologically and to find potential molecules responsible for its pro-healing and anti-pathogenic properties in chronic wounds. Materials and methods: (1) Chemical analysis: extracts were subjected to a column chromatography and the fractions obtained were studied by GCMS. (2) Quantification: dl-lactic acid (commercial kit), phenolic compounds (Folin?Ciocalteu), H2O2 (micro-titration), and cations (flame photometry). (3) Biological analysis: autoinducers type 2 (AI-2) (Vibrio harveyi BB170 bioassay), DNAase activity (Agar DNAase), and Pseudomonas aeruginosa biofilm inhibition (crystal violet technique). Results: According to its biological activity, the most significant molecules found by GCMS were the following: antimicrobials (mevalonolactone, 5-methyl-hydantoine, benzoic acid, etc.); surfactants (di-palmitin, distearin, and 1,5-monolinolein); anesthetics (barbituric acid derivatives), and AI-2 precursors (4,5-dihydroxy-2,3-pentanedione and 2-methyl-2,3,3,4-tetrahydroxytetrahydrofurane). Concentrations measured (µg/mL): dl-lactic acid (11.71 ± 1.53) and H2O2 (36 ± 2.0); phenolic compounds (485.2 ± 15.20); sodium (370 ± 17); potassium 920 ± 24); calcium (20 ± 4); and magnesium (15 ± 3). DNAase from LAPS had activity on genomic DNA from PMNs and P. aeruginosa. Discussion and conclusion: The molecules and biological activities found in LAPS could explain the observed effects in human chronic wounds. Fil: Ramos, Alberto. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Microbiología. Cátedra de Inmunología; Argentina Fil: Sesto Cabral, María Eugenia. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Arena, Mario Eduardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto de Química del Noroeste. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química del Noroeste; Argentina Fil: Arrighi, Carlos Federico. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Arroyo Aguilar, Abel Alfredo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Valdez, Juan C.. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Microbiología. Cátedra de Inmunología; Argentina

Highlights • Rapid isothermal NAT for influenza reveals improved sensitivity and specificity compared to direct antigen testing. • Simple sample processing and short turn-around time render isothermal NAT suitable for sequential near-patient testing. • The turn-around time advantage is lost by the single-test format precluding sample cohorting and testing of larger series., Background Rapid diagnosis of influenza is important for controlling outbreaks and starting antiviral therapy. Direct antigen detection (DAD) is rapid, but lacks sensitivity, whereas nucleic acid amplification testing (NAT) is more sensitive, but also more time-consuming. Objectives To evaluate the performance of a rapid isothermal NAT and two DADs. Study design During February–May 2014, we tested 211 consecutive patients with influenza-like illness using a commercial isothermal NAT (Alere™ Influenza A&B) as well as the DAD Sofia® Influenza A + B and BinaxNOW® Influenza A&B for detection of influenza-A and -B virus. RespiFinder-22® a commercial multiplex NAT served as reference test. Serial 10-fold dilutions of influenza-A and -B cell culture supernatants were examined. Another 225 patient samples were tested during December 2014–February 2015. Results Compared to RespiFinder-22®, the isothermal NAT Alere™ Influenza A&B, and the DAD Sofia® Influenza A + B and BinaxNOW® Influenza A&B had sensitivities of 77.8%, 59.3% and 29.6%, and specificities of 99.5%, 98.9% and 100%, respectively, for the first 211 patient samples. Alere™ Influenza A&B showed 85.7% sensitivity and 100% specificity in the second cohort. Isothermal NAT was 10-100-fold more sensitive compared to DAD for influenza virus culture supernatants with a lower limit of detection of 5000–50,000 copies/mL. The average turn-around time (TAT) of isothermal NAT and DADs was 15 min, but increased to 110 min for Alere™ Influenza A&B, 30 min for BinaxNOW® Influenza A&B, and 45 min for Sofia® Influenza A + B, when analyzing batches of 6 samples. Conclusion Simple sample processing and a TAT of 15 min render isothermal NAT Alere™ Influenza A&B suitable for sequential near-patient testing, but the TAT advantage is lost when testing of larger series.

Monoclonal antibodies (MAbs) have been widely used in immunodiagnostic and clinical tests, whose applications require a high level of purity. As a result, several stages of purification are required in the downstream processing of MAbs. The objective of this study was to recover and purify an anti-interleukin-6 MAb from cell culture supernatant by a combined method involving precipitation and immobilized metal-ion affinity chromatography (IMAC), aiming its subsequent application in diagnostic kits. Experimental results suggest that it is possible to purify MAbs by precipitation with ammonium sulphate at 20–40% saturation, followed by adsorption on IMAC with immobilized Zn(II). The MAbs were obtained with a purity of 100% and a purification factor of 24.2, proving the feasibility of the method.

TNFalpha is a potent osteoclastogenic cytokine that has a fundamental role in the pathogenesis of wear particle-induced osteolysis. Wear particles of one composition and their biological effects are well characterised. In contrast, little is known about the effects of mixed particles with respect to mix ratio and particle concentration. We evaluated the effects of different mix ratios of polyethylene and TiAlV particles on TNFalpha response. We used a human monocytic cell line (THP-1) in this in vitro study. THP-1 monocytes were differentiated to macrophage-like cells and exposed to different mixtures of lipopolysaccharide-detoxified polyethylene and TiAlV particles. TNFalpha was analysed in culture supernatants using ELISAs. Both polyethylene and TiAlV particles induced a dose- and time-related release of TNFalpha, with maximum levels after 6 h. A PE/TiAlV mix ratio of 36:1 at 10(8) particles/ml induced significantly higher TNFalpha concentrations compared to equal particle concentrations of isolated TiAlV (p=0.047) or PE (p=0.044), indicating the synergistic effect of mixed particles. These results provide evidence that TiAlV and polyethylene particles have significant synergistic effects, depending on the mix ratio and particle concentrations. This supra-additive effect can contribute substantially to the pathogenesis of implant particle-induced osteolysis.

Many licensed vaccines are composed of live, attenuated or inactivated whole-cell microorganisms, or they comprise purified components from whole-cell extracts or culture supernatants. For some diseases, pathology is fairly well understood, and there may be known correlates of protection that provide obvious parameters for assessment of vaccine potency. However, this is not always the case, and some effective vaccines are routinely used even though the mechanisms or correlates of protection are unknown. Some more modern vaccine approaches employ purified recombinant proteins, based on molecules that appear on the surface of the pathogen. This is one of the strategies that has been adopted in the quest to develop a malaria vaccine. Use of these parasite antigens as vaccine candidates is supported by substantial epidemiological data, and some have demonstrated the ability to elicit protective responses in animal models of malaria infection. However, there is as yet no immunological correlate of protection and no functional assays or animal models that have demonstrated the ability to predict efficacy in humans. There is little precedence for the most appropriate and practical method for assessing potency of vaccines based on these recombinant molecules for malaria vaccines. This is likely because the majority of malaria vaccine candidates have only recently entered clinical evaluation. The PATH Malaria Vaccine Initiative (MVI) convened a panel with expertise in potency assay design from industry, governmental institutions, and regulatory bodies to discuss and review the rationale, available methods, and best approaches for assessing the potency of recombinant proteins, specifically for their use as malarial vaccines. The aim of this meeting was to produce a discussion document on the practical potency assessment of recombinant protein malaria vaccines, focusing on early phase potency assay development.