Days Of Co-incubation Research Articles

Fabrication of photocurable zwitterionic polymer coatings with enhanced antifouling and lubrication properties for urinary catheter applications

Urinary catheters necessitate a surface with antifouling and lubricating properties in order to mitigate bacterial infection, calculus formation, and tissue trauma. Hydrophilic coating is a promising strategy but suffers from the complications of the manufacturing method as well as the lack of longevity. Herein, we report an antifouling and lubricating hydrophilic coating based on zwitterionic copolymer poly(2-Methacryloyloxyethyl phosphatidylcholine-co-hydroxyethyl methacrylate-co-(Benzoyl)phenyl methacrylate (poly(MPC-co-HEMA-co-BP)) through pre-gel solution spin-coating followed by UV-curing. The poly(MPC-co-HEMA-co-BP (PMHB) coatings possess hygroscopic properties that facilitate the formation of a hydrated layer, thereby leading to friction reduction. Moreover, both antibacterial and protein resistance tests substantiate the exceptional antifouling characteristics exhibited by the PMHB coating. Furthermore, after 7 days of coincubation, the antibacterial rates of PMHB coatings against Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), and Proteus mirabilis (P. mirabilis) are 72.98 %, 75.11 %, and 88.23 %, respectively. Subsequently, PMHB coating reduced the attachment of encrustation in a 7 days artificial urine cycling experiment. This study brings new perspective on designing urinary catheter surfaces with lubricating and antifouling properties.

P317 Phaeohyphomycosis caused by Phialophora verrucosa in a patient with CARD9 mutations

Poster session 2, September 22, 2022, 12:30 PM - 1:30 PMObjectivesTo study an intractable case of phaeohyphomycosis caused by Phialophora verrucosa and the underlying genetic and immunological mechanisms.MethodsClinical and laboratory findings of the patient were studied. The patient's DNA was sequenced for CARD9. Innate and adaptive immunological responses of patient-derived PBMCs were evaluated with ELISA and flow cytometry.ResultsA 29-year-old Chinese male, born to nonconsanguineous parents, developed an enlarging plaque on his right anterior tibia at 25 years and underwent a 15-month treatment of oral itraconazole at the local hospital. Upon discontinuation of the medication, the plaque spread over his face, upper limbs, and back. Physical examination indicated dark brown verrucous plaques and nodules with surface ulcers and scabs on his right leg, upper limbs, and face. Skin biopsy revealed pigmented yeast cells with budding and yeast cell chain in the dermis. Based on tissue culture, we identified P. verrucosa as the causative pathogen, which was further validated by ITS, BT, and TEF gene sequencing. Thus, subcutaneous phaeohyphomycosis was diagnosed, and itraconazole combined with amphotericin B was prescribed. The lesions improved only marginally. We, therefore, gave him oral posaconazole instead, based on in vitro antifungal susceptibility results. After 1 month of posaconazole treatment, the serum BG assay value decreased and the lesions improved markedly.Based on previous reports linking CARD9 deficiency to phaeohyphomycosis, as well as early onset and recalcitrance characters, Sanger sequencing was performed in this patient and identified two compound heterozygous mutations in exon 6 (R373P) and exon 8 (D274GfsX60) of CARD9. We then evaluated the functional effect of the CARD9 mutations by assessing the stimuli-dependent release of proinflammatory cytokines and helper T cell differentiation. Cytokine production was markedly impaired in PBMCs post 24 h of stimulation with LPS, curdlan, TDB, mannan, MDP, β‐glucan, and various fungal ligands. Moreover, the patient showed a significant absence in Th22 and Th17 cell proportions, as well as lower IL-17A and IL-22 production after six days of co-incubation with the stimuli mentioned above.ConclusionOur report highlighted that otherwise healthy patients diagnosed with early-onset, unexplained, and recalcitrant phaeohyphomycosis should be analyzed for CARD9 mutations and immune deficiency. Furthermore, posaconazole may be an alternative worth trying after the failure of other antifungals due to its ideal results achieved in our case.

IN VITRO ANTAGONISTIC EFFECTS OF SOME BACILLUS SPP. ON THE GROWTH AND MYCOTOXIN PRODUCTION OF TOXIGENIC ASPERGILLI

This study aimed to investigate the antagonistic effect of three strains of the genus Bacillus on toxigenic fungi contaminating food commodities, with emphasis on two ochratoxigenic isolates (A. ochraceus, A. westerdijkiae) and two aflatoxigenic isolates (A. flavus and A. parasiticus). In vitro studies were carried out using two different methods for cultivation tested bacilli with fungal isolates – coincubation and dual culture method. The most sensitive isolate was A. ochraceus by both used methods. B. mycoides (5.72 ± 6.4 mm) and B. subtilis (5.08 ± 2.84 mm) were able to inhibit its growth and sporulation during ten days of cultivation (both inhibited the sporulation of A. ochraceus 100%) in coincubation. The most effective in the dual culture method were B. subtilis and B. thuringiensis against A. ochraceus (growth inhibition rate 84.40%; 90.55%) and A. flavus (growth inhibition rate 91.54%; 92.43%). The most effective sporulation inhibitors were B. subtilis and B. thuringiensis, which completely inhibited the sporulation of A. ochraceus and A. parasiticus after ten days of coincubation. Likewise, all tested bacterial strains showed complete inhibition of ochratoxin A synthesis in A. ochraceus and A. westerdijkiae exposure to bacterial volatiles. So, the current study illustrated that strains of the genus Bacillus could significantly inhibit the growth, sporulation, and mycotoxin production of toxigenic aspergilli and showing the great potential as a biocontrol agent of pre- and post-harvest food diseases caused by microscopic filamentous fungi.

Efficacy of lactic acid bacteria supplementation against Fusarium graminearum growth in vitro and inhibition of Zearalenone causing inflammation and oxidative stress in vivo

Fusarium graminearum invasion and Zearalenone (ZEN)-mycotoxin contamination are considered the most global threat to food and feed. This study investigates the effect Lactobacillus plantarum MON03 viable cells (LPVC) and LP free cells supernatant (LPFCS) against Fusarium graminearum growth and ZEN production in vitro and evaluates if treatment with LP viable cells can counteract the negative effect of ZEN on inflammation and oxidative stress in mesenteric lymph nodes and serum biochemical parameters in mice.For the in vitro study, 7 days of LPVC, LPFCS and F. graminearum co-incubation at different concentrations was done in order to determine the antifungal activity and ZEN- production inhibition. Regarding the in vivo study, Balb/c mice were treated as following: Control, ZEN group, LP group and ZEN + LP group for 30 days.In vitro, LPVC showed an excellent antifungal activity after 7 days of co-incubation (103 CFU/ml). LPVC was succeeded also to inhibit ZEN production by the fungi. In vivo, ZEN has shown an important oxidative damage. As a result of the exposure to ZEN, an increase cytokines, as effectors of an inflammatory response, were observed in the mesenteric lymph nodes (MLN) of intoxicated mice. In parallel, a serum biochemical change was also observed. LPVC induced a reduction of ZEN-induced oxidative stress and counteracts also the biochemical parameters damage and the inflammatory markers increased by ZEN.LPVC can be valorized as an anti-cating agent in the vitro and in the gastro-intestinal tract to decrease ZEN-toxic effects.

Synthesis of multifunctional chlorhexidine-doped thin films for titanium-based implant materials.

Our goal was to create bio-functional chlorhexidine (CHX)-doped thin films on commercially pure titanium (cpTi) discs using the glow discharge plasma approach. Different plasma deposition times (50, 35 and 20min) were used to create bio-functional surfaces based on silicon films with CHX that were compared to the control groups [no CHX and bulk cpTi surface (machined)]. Physico-chemical and biological characterizations included: 1. Morphology, roughness, elemental chemical composition, film thickness, contact angle and surface free energy; 2. CHX-release rate; 3. Antibacterial effect on Streptococcus sanguinis biofilms at 24, 48 and 72h; 4. Cytotoxicity and metabolic activity using fibroblasts cell culture (NIH-F3T3 cells) at 1, 2, 3 and 4days; 5. Protein expression by NIH-F3T3 cells at 1, 2, 3 and 4days; and 6. Co-culture assay of fibroblasts cells and S. sanguinis to assess live and dead cells on the confocal laser scanning microscopy, mitochondrial activity (XTT), membrane leakage (LDH release), and metabolic activity (WST-1 assay) at 1, 2 and 3days of co-incubation. Data analysis showed that silicon films, with or without CHX coated cpTi discs, increased surface wettability and free energy (p<0.05) without affecting surface roughness. CHX release was maintained over a 22-day period and resulted in a significant inhibition of biofilm growth (p<0.05) at 48 and 72h of biofilm formation for 50min and 20min of plasma deposition time groups, respectively. In general, CHX treatment did not significantly affect NIH-F3T3 cell viability (p>0.05), whereas cell metabolism (MTT assay) was affected by CHX, with the 35min of plasma deposition time group displaying the lowest values as compared to bulk cpTi (p<0.05). Moreover, data analysis showed that films, with or without CHX, significantly affected the expression profile of inflammatory cytokines, including IL-4, IL-6, IL-17, IFN-y and TNF-α by NIH-F3T3 cells (p<0.05). Co-culture demonstrated that CHX-doped film did not affect the metabolic activity, cytotoxicity and viability of fibroblasts cells (p>0.05). Altogether, the findings of the current study support the conclusion that silicon films added with CHX can be successfully created on titanium discs and have the potential to affect bacterial growth and inflammatory markers without affecting cell viability/proliferation rates.

Plant Growth-Promoting Methylobacteria Selectively Increase the Biomass of Biotechnologically Relevant Microalgae.

Microalgae, a diverse group of single-celled organisms exhibiting versatile traits, find broad applications in industry. However, high production costs require further efforts to optimize their production and to enhance biomass yields. In the present study, co-occurrence of algae and methylobacteria was observed when naturally occurring microalgae biofilms were subjected to 16S rRNA gene fragment amplicon sequencing. This bacterial group is so far less explored than other microalgae-associated bacteria in terms of mutualistic relationships that might be exploitable for biotechnological applications. In order to assess the potential of four plant growth-promoting strains from the genus Methylobacterium for increased algae biomass production, co-cultivation experiments were conducted with three industrially relevant microalgae (Chlorella vulgaris, Scenedesmus vacuolatus, and Haematococcus lacustris). For S. vacuolatus and H. lacustris, a significant increase in algal biomass formation of 1.3-fold to up to 14-fold was observed after 7 days of co-incubation. Visualization of mixed cultures using confocal laser scanning microscopy revealed a high abundance of methylobacteria in the phycosphere of H. lacustris and S. vacuolatus, visually attached to the algae’s surface forming a biofilm-like assemblage. Genome analyses revealed that features attributable to enhanced algal growth include genes involved in the synthesis of vitamins, siderophores and plant hormones. Our results provide evidence for the constructability of novel symbiotic algae-bacteria relationships with inter-kingdom supportive capacities, underlining the potential of microbial consortia as promising tool for sustainable biotechnology and agriculture.

Contribution of the Twin-Arginine Translocation System to the Intracellular Survival of Salmonella Typhimurium in Dictyostelium discoideum.

The twin-arginine translocation (Tat) system is a specialized secretion pathway required for bacteria to export fully folded proteins through the cytoplasmic membrane. This system is crucial during Salmonella infection of animal hosts. In this study, we show that Salmonella enterica serovar Typhimurium (S. Typhimurium) requires the Tat system to survive and proliferate intracellularly in the social amoeba Dictyostelium discoideum. To achieve this, we developed a new infection assay to assess intracellular bacterial loads in amoeba by direct enumeration of colony forming units (CFU) at different times of infection. Using this assay we observed that a ΔtatABC mutant was internalized in higher numbers than the wild type, and was defective for intracellular survival in the amoeba at all times post infection evaluated. In addition, we assessed the effect of the ΔtatABC mutant in the social development of D. discoideum. In contrast to the wild-type strain, we observed that the mutant was unable to delay the social development of the amoeba at 2 days of co-incubation. This phenotype correlated with defects in intracellular proliferation presented by the ΔtatABC mutant in D. discoideum after 24 h of infection. All phenotypes described for the mutant were reverted by the presence of a plasmid carrying tatABC genes, indicating that abrogation of Tat system attenuates S. Typhimurium in this model organism. Overall, our results indicate that the Tat system is crucial for S. Typhimurium to survive and proliferate intracellularly in D. discoideum and for virulence in this host. To the best of our knowledge, this is the first report on the relevance of the Tat system in the interaction of any bacterial pathogen with the social amoeba D. discoideum.

Biocontrol activity of the marine yeast Debaryomyces hansenii against phytopathogenic fungi and its ability to inhibit mycotoxins production in maize grain (Zea mays L.)

Debaryomyces hansenii is an antagonistic yeast that has been shown high effectiveness against different phytopathogenic fungi in diverse habitats. Both, the antagonistic activity of D. hansenii BCS003 and its mechanism against four pathogenic fungi (Mucor circinelloides, Aspergillus sp., Fusarium proliferatum and Fusarium subglutinans) in maize grains (Zea mays L.) were evaluated. Results indicated an almost entirely decrease (97.2–98.3%) of mycelial growth in a radial inhibition assay against four strains of pathogenic fungi by the D. hansenii action. In diffusible and volatile compounds as well cell-free supernatants in vitro assays D. hansenii showed significant inhibitory activity (p<0.05) against Aspergillus sp., F. proliferatum and F. subglutinans. The four isolated fungi were used to infect maize grains and results showed a partial inhibition of the mycelial growth and a 24h delay in the disease appearance when compared to control (not D. hansenii treated maize grain). Moreover, the application of D. hansenii reduced the production of F. subglutinans fumonisins by 59.8% after seven days of co-incubation without affecting the chemical and mineral composition of maize grain. According to these results, D. hansenii BCS003 could be considered a potential biocontrol agent against toxigenic molds in maize grains, probably due to the synergistic effects of the factors such as the competition for nutrients and space, as well as the production of extracellular soluble and volatile compounds. The potential of marine D. hansenii as biocontrol agent merits further research to improve its efficacy.

Oleacein enhances anti-inflammatory activity of human macrophages by increasing CD163 receptor expression

BackgroundOleacein (dialdehydic form of decarboxymethyl elenolic acid linked to hydroxytyrosol; 3,4-DHPEA-EDA) have been proven to possess antioxidant and anti-inflammatory activity. PurposeIn this study, we examined whether oleacein could increase CD163 and IL-10 receptor expression as well as HO-1 intracellular secretion in human macrophages. MethodsEffect of oleacein (10 and 20 μmol/l) or oleacein together with complexes of haemoglobin (Hb) and haptoglobin 1-1 (Hp11) or haptoglobin 2-2 (Hp22) on expression of IL-10 and CD163 receptor was determined by Flow Cytometry. Expression of CD163mRNA was measured by real-time quantitative RT-PCR. Heme oxygenase 1 (HO-1) intracellular secretion in macrophages was investigated by enzyme-linked immunosorbent assay (ELISA). ResultsOleacein (OC) together with complexes HbHp11 or HbHp22 stimulated the expression of CD163 (30-100-fold), IL-10 (170-300-fold) and HO-1 secretion (60-130-fold) after 5 days of coincubation. The 2-fold (24 h), 4-fold (48 h) increase of CD163 mRNA level and its final (72 h) decrease was also observed. ConclusionOur results suggested that oleacein enhances anti-inflammatory activity of complexes haemoglobin with haptoglobin 1-1 and 2-2 and could play a potential role in the prevention of inflammatory disease related to atherosclerosis.

Compost Teas and Reused Nutrient Solution Suppress Plant Pathogens In Vitro

In vitro testing was conducted to evaluate the inhibition potential of three compost teas (pine bark, manure, and vermicasting), Root Rescue Landscape Powder® (a mix of mycorrhizae and other beneficial microbes), waste diatomaceous earth (DE; from beer brewing), and a greenhouse nutrient solution, which had been reused for 20 years on six plant pathogens: Fusarium foetens, Rhizoctonia solani, Sclerotinia sclerotiorum, Phytophthora cryptogea, Pythium intermedium, and P. ultimum. The test materials showed in vitro inhibition on most of the test pathogens. Pine bark tea suppressed growth of all six pathogens, and inhibition exceeded 50% after 10 days of coincubation. Vermicasting tea showed over 40% inhibition against S. sclerotiorum and F. foetens; manure tea showed 42% inhibition against F. foetens; DE showed 40% inhibition against F. foetens, S. sclerotiorum, and R. solani; whereas reused greenhouse nutrient solution showed 56.7% inhibition against R. solani and 43.4% inhibition against F. foetens; Root Rescue showed 66% inhibition against P. intermedium. The results suggest that the six test materials have potential in the control of these soil- and water-borne pathogens in plant production system.

Interaction of Paramecium caudatum and Picornaviruses

In our paper we have researched the relationship between picornaviruses (poliovirus, foot-and-mouth disease virus and encephalomyocarditis virus) and Ciliata (Paramecium caudatum). We show that the number of Paramecium in medium sharply increased during coincubation with picornaviruses within 2-5days. This cannot be explained only by the fact that viruses were nutrient source for Paramecium because in case of inactivated viruses the number of infusorians in medium increased a little. At the same time the titer of viruses harshly decreased whereas in the control group, which is free of Paramecium, the fall of titer was little. Picornaviruses were eliminated from medium if only living Parameciums were present in medium. After 7-9days of coincubation only a few number of viruses were liberated from destroyed Parameciums. These results will be especially useful for management of reservoirs of picornaviruses in water and prevention of diseases.

Modulation of Amyloid Beta Peptide Aggregation by Apolipoprotein E Isoforms

Apolipoprotein E4 (apoE) is a confirmed risk factor for late onset Alzheimer's disease (AD). Mouse model studies indicate enhanced depostion of amyloid beta (Aβ) peptides in the brain in presence of the e4 isoform of apoE. However, the mechanism of apoE-Aβ interaction and the biophysical nature of these complexes remain largely unclear. In addition, the relative specificity of this interaction with the other two common isoforms (apoE2 and apoE3) not associated with AD is poorly understood. Aβ aggregation followed by turbidity measurements show that the aggregation is accelerated by all three isoforms of apoE and apoE4 showed the greatest and apoE2 the least effects. Fluorescence correlation spectroscopy (FCS) measurements using fluorescently labeled Aβ show that diffusion time of Aβ increases upon incubation with wild type apoE proteins. FCS data confirm that apoE4 has the highest and apoE2 has the lowest affinity for Aβ. However, this interaction appears to be slow and does not reach completion even after several days of co-incubation. The diffusion time of the complexes are found to be large indicating involvement of multimeric forms of Aβ. In addition, a monomeric form of apoE prepared by 4 mutations in the apoE sequence does not bind to Aβ. Thus apoE multimers may be acting as nucleation sites for oligomerization of Aβ. We are currently investigating the molecular basis of the apoE-Aβ complex formations by employing Hydrogen-Deuterium Exchange by mass spectrometry (HDX-MS).

Francisella philomiragia Biofilm Formation and Interaction With the Aquatic Protist Acanthamoeba castellanii

The bacterium Francisella philomiragia has been isolated from environmental samples originating from around the globe. F. philomiragia-related strains cause francisellosis of both farmed and wild fish. In addition, occasional human infections caused by F. philomiragia are found in victims of near-drowning and patients with chronic granulomatous disease. We have shown that F. philomiragia forms in vitro biofilms with increased formation at 25 °C over 37 °C conditions. We found that F. philomiragia can form a biofilm in a co-culture with live Acanthamoeba castellanii, an aquatic amoeba. Interestingly, amoeba-conditioned supernatant has an inhibitory effect on production of biofilm by F. philomiragia, whereas Francisella-conditioned supernatant has no effect on growth of amoebae. We have shown that F. philomiragia can infect A. castellanii after only 5 days of co-incubation and that it infects A. castellanii more quickly than the related species F. novicida does. Our studies point to a potentially overlooked interaction between F. philomiragia and Acanthamoeba. This relationship in the marine lifecycle of F. philomiragia may support the persistence of the bacterium in waterways and its ability to infect fish. An understanding of the persistence of this organism in aquatic systems through biofilm formation and its interaction with Acanthamoeba will be important in developing prevention strategies for this pathogen.

In Vitro Communities Derived from Oral and Gut Microbial Floras Inhibit the Growth of Bacteria of Foreign Origins

The gastrointestinal (GI) tract is home to trillions of microbes. Within the same GI tract, substantial differences in the bacterial species that inhabit the oral cavity and intestinal tract have been noted. While the influence of host environments and nutritional availability in shaping different microbial communities is widely accepted, we hypothesize that the existing microbial flora also plays a role in selecting the bacterial species that are being integrated into the community. In this study, we used cultivable microbial communities isolated from different parts of the GI tract of mice (oral cavity and intestines) as a model system to examine this hypothesis. Microbes from these two areas were harvested and cultured using the same nutritional conditions, which led to two distinct microbial communities, each with about 20 different species as revealed by PCR-based denaturing gradient gel electrophoresis analysis. In vitro community competition assays showed that the two microbial floras exhibited antagonistic interactions toward each other. More interestingly, all the original isolates tested and their closely related species displayed striking community preferences: They persisted when introduced into the bacterial community of the same origin, while their viable count declined more than three orders of magnitude after 4 days of coincubation with the microbial flora of foreign origin. These results suggest that an existing microbial community might impose a selective pressure on incoming foreign bacterial species independent of host selection. The observed inter-flora interactions could contribute to the protective effect of established microbial communities against the integration of foreign bacteria to maintain the stability of the existing communities.Electronic supplementary materialThe online version of this article (doi:10.1007/s00248-010-9711-9) contains supplementary material, which is available to authorized users.

Ex vivo evaluation of prolidase loaded chitosan nanoparticles for the enzyme replacement therapy

Prolidase loaded chitosan nanoparticles were set up in order to suggest an innovative therapeutic approach for Prolidase Deficiency (PD), a rare autosomal inherited disorder of the connective tissue. The satisfactory drug loading efficiency (42.6 ± 2.1%) as well as the suitable physical characteristics (mean diameter of 365.5 ± 35.1 nm and a positive zeta-potential of 17.94 ± 0.12 mV) was achieved. In order to verify the compatibility of the chitosan nanoparticles with cells, the influence of the nanoparticles on the growth and the viability (MTT assay) of cultured skin fibroblasts were determined: the nanoparticles showed a good biocompatibility up to 5 μg of chitosan/10,000 fibroblasts. Uptake of chitosan nanoparticles by fibroblasts was verified by confocal microscopy using FITC-labelled chitosan nanoparticles. The ex vivo experiments were performed by incubating different amounts of prolidase loaded chitosan nanoparticles with skin human fibroblasts from PD patients for scheduled times. The restored prolidase activity was quantitatively monitored by a capillary electrophoretic method and confirmed by cells morphological observations. Standing from the nanoparticles internalization, the enzymatic activity was progressively restored reaching the best value (about 66%) after 5 days of co-incubation. Moreover, prolidase loaded chitosan nanoparticles permitted to restore prolidase activity in PD fibroblasts for a prolonged period of time (8 days).

Progressive increase in conduction velocity across human mesenchymal stem cells is mediated by enhanced electrical coupling

The purpose of the study was to investigate the development of electrical transmission across human adult bone marrow-derived mesenchymal stem cells (hMSCs) during long-term co-incubation with cardiomyocytes (CMCs). Neonatal rat CMCs were cultured in multi-electrode array dishes. A conduction block was induced by creating a central acellular channel, yielding two asynchronously beating CMC fields. Enhanced green fluorescent protein (eGFP)-labeled hMSCs from ischemic heart disease patients (n=8), eGFP-labeled hMSCs having RNA interference-mediated connexin43 (Cx43) knockdown (n=6), 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine (Dil)-labeled CMCs (n=6), or no cells (n=9) were seeded in the channel. Assessment of conduction velocity (CV), Cx expression and localization, gap junctional coupling, and intracellular electrical recordings were performed for up to 14 days. Resynchronization of the two CMC fields occurred within 24 h after seeding of hMSCs. CV across hMSCs increased from 1.4+/-0.4 cm/s at day 7 to 3.5+/-0.1 cm/s (p<0.05) at day 14. CV across seeded CMCs was 16.8+/-0.2 cm/s throughout this period. No resynchronization occurred in the absence of seeded cells. Knockdown of Cx43 in hMSCs abolished conduction across the channel completely. Time-dependent increase of CV across hMSCs was associated with increased Cx43 mRNA and protein expression resulting in increased gap junctional coupling. Intracellular recordings in coupled hMSCs showed increased conducted action potential (AP) amplitude, lower resting membrane potential, and decreased duration of conducted AP (p<0.05, day 14 versus day 1). CV across hMSCs increases progressively after 7 days of co-incubation with CMCs, most likely via improved electrotonic interaction. This is associated with increased Cx43 expression, increased functional gap junctional coupling, and enhanced intercellular electrical coupling between hMSCs and CMCs.

Co-incubation of Clostridium botulinum with protective cultures

Potential neurotoxin production by psychrotrophic Clostridium botulinum is the significant food safety risk in refrigerated foods of extended durability. Protective cultures (PC), nisin-producing Lactococcus lactis (6.3 × 10 5 cfu/ml) and pediocin A-producing Pediococcus pentosaceus (10 7 cfu/ml), were co-incubated with C. botulinum type B (6.2 × 10 2 cfu/ml) in tryptose, peptone, glucose and yeast extract broth for 31 days at 5 °C, 14 days at 10 °C and for 10 days at 15 °C. Bacterial populations, pH, botulinal toxin and bacteriocin production were measured. At 5 °C, the populations of all species did not change during incubation. At 10 and 15 °C, the PCs increased by 1–3log. At 15 °C, C. botulinum increased by 1.5–3log and was not inhibited by the PCs. At 10 °C, this pathogen was not detected after 10 days of co-incubation. The reduction in C. botulinum population coincided with the nisin level produced by L. lactis exceeding 100 IU/ml, but it was not rapid enough to prevent botulinal toxin formation.

Diagnostic dilemma encountered when detecting bovine viral diarrhea virus in IVF embryo production

Routine quality controls in production of bovine embryos by in vitro fertilization (IVF) should include screening all materials of animal origin for the presence of bovine viral diarrhea virus (BVDV). Using a reverse transcription nested polymerase chain reaction (RT-nPCR) assay, we detected BVDV in primary cultures of uterine tubal cells (UTC) that had been used during IVF procedures. The goal of our ensuing investigation was to determine its source and assess risks associated with the identified contaminant. Sequencing of the amplified 5′ nontranslated region (NTR) of the viral genome confirmed a Genotype I BVDV contaminant. This viral contaminant was also identified by RT-nPCR in multiple samples of the same lot of fetal bovine serum (FBS) that was used in transport media by the laboratory that harvested the UTC. Both routine and enhanced roller bottle methods for virus isolation failed to detect BVDV in the FBS. Furthermore, virus neutralization assays did identify antibodies to Genotype I strains of BVDV in the FBS. After 7 days of co-incubation, neither cultured, washed UTC nor exposed, washed embryos were RT-nPCR positive for BVDV. Eight embryos produced in the contaminated system were nonsurgically transferred into eight seronegative cows. None of the embryo recipients seroconverted to BVDV. Thus, contamination of cell culture medium with BVDV did not result in transmission of the virus when IVF embryos were transferred. Failure to transmit disease was likely aided by serendipitous control from anti-BVDV antibodies in the FBS. However, a diagnostic dilemma was created when the RT-nPCR assays used to screen for BVDV were positive, yet attempts to isolate the virus were negative. This case study illustrates that if molecular assays are to be used to confirm the pathogen-free status of IVF embryo production systems, media components of animal origin (e.g. FBS) should be screened with molecular assays for BVDV as well as traditional virus isolation techniques.

In vitro recognition and impairment of pig islet cells by baboon immune cells: similarity to human cellular reactions.

Grafting pig islets into patients with type 1 diabetes requires control of the strong cellular xenogeneic rejection. This in vitro study compared the cellular reaction of baboons and humans to pig islet cells (PICs) to confirm the validity of using these animals for further in vivo preclinical trials. Baboon or human peripheral blood mononuclear cells (PBMCs) or subsets were co-incubated with PICs from specific pathogen-free adult pigs for 7 days to determine the mechanisms and intensity of PBMC proliferation. Interleukin (IL) 10 and interferon (IFN) gamma secretion were assessed by enzyme-linked immunosorbent assay. Because proliferation was not indicative of aggression, a test based on perifusion analysis of the alteration of basal and stimulated insulin releases from PIC incubated with different baboon and human cells was developed. Baboon PBMCs strongly proliferated in response to PICs (stimulation index [SI]=24.8+/-6.9 [n=8] vs. 23.9+/-3.4 [n=34] for human PBMCs), showing considerable variation in intensity among animals (2.3<SI<63) and humans (1.8<SI<97). PBMC proliferation was inhibited in baboons and humans by anti-CD4 (% inhibition of SI: 71+/-10% and 75+/-7%, respectively) and anti-DR (75+/-35% and 80+/-6%) monoclonal antibodies (MoAbs) or by depletion of MHC class II+ cells (99+/-1% and 90+/-6%). Blocking by anti-CD8 or anti-CD16 MoAbs was weaker and variable among both animals and humans. IL-10 production by baboon and human PBMCs in response to PICs increased more than IFN-gamma production after 2 days of coculture, but the IL-10/IFN-gamma ratio was inverted after 5 days of coculture. After 7 days (and even after only 2 days) of coculture with baboon (n=8) or human (n=18) PBMCs, basal and glucose-stimulated insulin secretions from PICs were almost completely abolished (P<0.0001). The drop in insulin release could have mainly resulted from lysis of PICs, because the number of PICs decreased by 78% after 7 days of co-incubation with PBMCs. A decrease of insulin release by PBMCs was reproduced with plastic-adherent cells and was abolished by depletion of MHC class II+ cells or by addition of 100 microg/ml gadolinium (which inhibits macrophages), but not by cyclosporine. In baboons, as in humans, insulin release was also decreased after coculture of PICs with enriched T lymphocytes remixed with antigen-presenting cells (APCs). This study provides the first data on in vitro comparison of baboon and human cell-mediated recognition and impairment of PICs. Proliferation of PBMCs against PICs involves mainly CD4 T cells, with indirect recognition mediated by baboon or human MHC class II+ APCs. The Th2/Th1 profile of cytokines secreted in response to PICs was similar in baboon and human PBMCs. The model based on alteration of insulin secretion indicates that PIC impairment by whole mononuclear cells was strong and rapid and that a crucial role was played by MHC class II+ and plastic-adherent cells. Two mechanisms appear to be responsible for the role of these cells: (1) early and strong direct effect, which is potentially involved in vivo in primary nonfunction of islets aggressed by monocytes and macrophages; and (2) presentation of PIC xenoantigens, which leads to impairment by T lymphocytes possibly involved in in vivo-specific cellular rejection. The mechanisms and intensity of baboon cellular reactions to PICs in vitro were similar to those observed in humans, which suggests that the baboon is a suitable model for the study of cellular mechanisms during preclinical trials of pig islet xenografts.

Laminin inhibition of beta-amyloid protein (Abeta) fibrillogenesis and identification of an Abeta binding site localized to the globular domain repeats on the laminin a chain.

beta-Amyloid protein (Abeta) is a major component of neuritic plaques and cerebrovascular amyloid deposits in the brains of patients with Alzheimer's disease (AD). Inhibitors of Abeta fibrillogenesis are currently sought as potential future therapeutics for AD and related disorders. In the present study, the basement membrane protein laminin was found to bind Abeta 1-40 with a single dissociation constant, K(d) = 2.7 x 10(-9) M, and serve as a potent inhibitor of Abeta fibril formation. 25 microM of Abeta 1-40 was incubated at 37 degrees C for 1 week in the presence of 100 nM of laminin or other basement membrane components, including perlecan, type IV collagen, and fibronectin to determine their effects on Abeta fibril formation as evaluated by thioflavin T fluorometry. Of all the basement membrane components tested, laminin demonstrated the greatest inhibitory effect on Abeta-amyloid fibril formation, causing a ninefold inhibition at 1 and 3 days and a 21-fold inhibition at 1 week. The inhibitory effects of laminin on Abeta fibrillogenesis occurred in a dose-dependent manner and were still effective at lower concentrations. The inhibitory effects of laminin on Abeta 1-40 fibril formation was confirmed by negative stain electron microscopy, whereby laminin caused an almost complete inhibition of Abeta fibril formation and assembly by 3 days, resulting in the appearance of primarily amorphous nonfibrillar material. Laminin also caused partial disassembly of preformed Abeta-amyloid fibrils following 4 days of coincubation. Laminin was not effective as an inhibitor of islet amyloid polypeptide fibril formation, suggesting that laminin's amyloid inhibitory effects were Abeta-specific. To identify a potential Abeta-binding site(s) on laminin, laminin was first digested with V8, trypsin, or elastase. An Abeta-binding elastase digestion product of approximately 120-130 kDa was found. In addition, a approximately 55 kDa fragment derived from V8 and elastase-digested laminin interacted with biotinylated Abeta 1-40. Amino acid sequencing of the approximately 55 kDa fragment identified a conformationally dependent Abeta-binding site within laminin localized to the globular repeats on the laminin A chain. These studies demonstrate that laminin not only binds Abeta with relatively high affinity but is a potent inhibitor of Abeta-amyloid fibril formation. In addition, further identification of an Abeta-binding domain within the globular repeats on the laminin A chain may lead to the design of new therapeutics for the inhibition of Abeta fibrillogenesis.