Rapid Membrane Filtration-Epifluorescent Microscopy Technique for Direct Enumeration of Bacteria in Raw Milk

Membrane filtration and epifluorescent microscopy were used for the direct enumeration of bacteria in raw milk. Somatic cells were lysed by treatment with trypsin and Triton X-100 so that 2 ml of milk containing up to 5 × 10⁶ somatic cells/ml could be filtered. The majority of the bacteria (ca. 80%) remained intact and were concentrated on the membrane. After being stained with acridine organe, the bacteria fluoresced under ultraviolet light and could easily be counted. The clump count of orange fluorescing cells on the membrane correlated well (r = 0.91) with the corresponding plate count for farm, tanker, and silo milks. Differences between counts obtained by different operators and between the membrane clump count and plate count were not significant. The technique is rapid, taking less than 25 min, inexpensive, costing less than 50 cents per sample, and is suitable for milks containing 5 × 10³ to 5 × 10⁸ bacteria per ml.     

Ultracentrifugation in the Concentration and Detection of Enteroviruses

Ultracentrifugation has been evaluated as a method of concentrating enteroviruses from suspensions whose initial titers ranged from 1.7 × 10⁸ to 1.6 × 10^-2 plaque-forming units (PFU) per ml. A technique employing a “trap” of 0.1 ml of 2% gelatin solution at the point at which the pellet forms in tubes for the number 30 and number 50 rotors of the Spinco model L preparative ultracentrifuge has been tested and found to have a number of advantages. Qualitative studies have been performed to determine the sensitivity of the ultracentrifuge technique in detecting the presence of enteroviruses in very dilute suspensions. There was found to be at least a 50% probability of detecting virus present initially at levels as low as 0.12 PFU per ml by means of the number 50 rotor. The input level for similar results with the number 30 rotor was found to be 0.025 PFU per ml.     

Functional Testing of an Inhalable Nanoparticle Based Influenza Vaccine Using a Human Precision Cut Lung Slice Technique

Annual outbreaks of influenza infections, caused by new influenza virus subtypes and high incidences of zoonosis, make seasonal influenza one of the most unpredictable and serious health threats worldwide. Currently available vaccines, though the main prevention strategy, can neither efficiently be adapted to new circulating virus subtypes nor provide high amounts to meet the global demand fast enough. New influenza vaccines quickly adapted to current virus strains are needed. In the present study we investigated the local toxicity and capacity of a new inhalable influenza vaccine to induce an antigen-specific recall response at the site of virus entry in human precision-cut lung slices (PCLS). This new vaccine combines recombinant H1N1 influenza hemagglutinin (HAC1), produced in tobacco plants, and a silica nanoparticle (NP)-based drug delivery system. We found no local cellular toxicity of the vaccine within applicable concentrations. However higher concentrations of NP (≥10³ µg/ml) dose-dependently decreased viability of human PCLS. Furthermore NP, not the protein, provoked a dose-dependent induction of TNF-α and IL-1β, indicating adjuvant properties of silica. In contrast, we found an antigen-specific induction of the T cell proliferation and differentiation cytokine, IL-2, compared to baseline level (152±49 pg/mg vs. 22±5 pg/mg), which could not be seen for the NP alone. Additionally, treatment with 10 µg/ml HAC1 caused a 6-times higher secretion of IFN-γ compared to baseline (602±307 pg/mg vs. 97±51 pg/mg). This antigen-induced IFN-γ secretion was further boosted by the adjuvant effect of silica NP for the formulated vaccine to a 12-fold increase (97±51 pg/mg vs. 1226±535 pg/mg). Thus we were able to show that the plant-produced vaccine induced an adequate innate immune response and re-activated an established antigen-specific T cell response within a non-toxic range in human PCLS at the site of virus entry.     

Reverse regulation of soluble receptor for advanced glycation end products and proinflammatory factor resistin and S100A12 in Kawasaki disease

Introduction

Kawasaki disease (KD), an acute febrile disease, characterized by systemic vasculitis, predominantly affects infants and children under 5 years of age. Coronary artery lesions (CALs) are its most critical complication, and the etiology remains unknown yet. In order to explore the value of resistin, S100A12 and soluble receptor for advanced glycation end products (sRAGE) in the pathophysiology of KD, we studied the serum levels of resistin, S100A12 and sRAGE in different stages of KD.

Methods

Serum levels of resistin, S100A12 and sRAGE were measured by enzyme-linked immunosorbent assay (ELISA) method in 15 healthy children and 40 KD patients at acute, afebrile and subacute stage.

Results

The resistin and S100A12 levels, including the ratio of resistin to sRAGE and S100A12 to sRAGE increased significantly in the acute stage, and decreased progressively in the afebrile and subacute stage. However, the sRAGE levels decreased significantly in the acute stage, and increased progressively in the afebrile and subacute stage. In the acute, afebrile and subacute stage, the resistin levels were higher in intravenous immunoglobulin (IVIG) non-responders (0.64 ± 0.30, 0.48 ± 0.35, 0.28 ± 0.19, × 10² ng/ml) than in IVIG responders (0.35 ± 0.24, 0.21 ± 0.19, 0.12 ± 0.05, × 10² ng/ml). In the acute and subacute stage, the S100A12 levels were higher in IVIG non-responders (7.92 ± 2.61, 4.98 ± 4.75, × 10² ng/ml) than in IVIG responders (5.05 ± 3.22, 2.35 ± 2.26, × 10² ng/ml). In the afebrile and subacute stage, the sRAGE levels were lower in IVIG non-responders (3.51 ± 2.64, 3.65 ± 3.27, × 10² pg/ml) than in IVIG responders (6.00 ± 2.78, 7.19 ± 2.88, × 10² pg/ml). The resistin levels were positively correlated with S100A12 levels. The sRAGE levels were negatively related with S100A12 and resistin levels.

Conclusions

Resistin, S100A12 and sRAGE are involved in the pathophysiology of KD.     

Gene Transfer by Transduction in the Marine Environment

To determine the potential for bacteriophage-mediated gene transfer in the marine environment, we established transduction systems by using marine phage host isolates. Plasmid pQSR50, which contains transposon Tn5 and encodes kanamycin and streptomycin resistance, was used in plasmid transduction assays. Both marine bacterial isolates and concentrated natural bacterial communities were used as recipients in transduction studies. Transductants were detected by a gene probe complementary to the neomycin phosphotransferase (nptII) gene in Tn5. The transduction frequencies ranged from 1.33 × 10⁻⁷ to 5.13 × 10⁻⁹ transductants/PFU in studies performed with the bacterial isolates. With the mixed bacterial communities, putative transductants were detected in two of the six experiments performed. These putative transductants were confirmed and separated from indigenous antibiotic-resistant bacteria by colony hybridization probed with the nptII probe and by PCR amplification performed with two sets of primers specific for pQSR50. The frequencies of plasmid transduction in the mixed bacterial communities ranged from 1.58 × 10⁻⁸ to 3.7 × 10⁻⁸ transductants/PFU. Estimates of the transduction rate obtained by using a numerical model suggested that up to 1.3 × 10¹⁴ transduction events per year could occur in the Tampa Bay Estuary. The results of this study suggest that transduction could be an important mechanism for horizontal gene transfer in the marine environment.     

Ag85B DNA vaccine suppresses airway inflammation in a murine model of asthma

Background

In allergic asthma, Th2 lymphocytes are believed to play important roles in orchestrating airway eosinophilia and inflammation. Resetting the Th1/Th2 imbalance may have a therapeutic role in asthma. The mycobacterium tuberculosis 30-kilodalton major secretory protein (antigen 85B, Ag85B) can protect animals from M. tuberculosis infection by inducing a Th1-dominant response.

Methods

In this study, the Ag85B gene was cloned into pMG plasmids to yield the pMG-Ag85B plasmid. The expression of Ag85B gene in murine bronchial epithelia cells was detected by Western blotting and immunohistochemical staining after intranasal immunization with reconstructed pMG-Ag85B plasmids. The protective effect of pMG-Ag85B plasmids immunization in airway inflammation was evaluated by histological examination and bronchoalveolar lavage (BAL). IL-4 and IFN-γ levels in the BAL and supernatant from splenocyte culture were determined using ELISA kits.

Results

The Ag85B gene was successfully expressed in murine bronchial epithelia cells by intranasal immunization with reconstructed pMG-Ag85B plasmids. Using a murine model of asthma induced by ovalbumin (OVA), pMG-Ag85B immunization significantly inhibited cellular infiltration across the airway epithelium with a 37% decrease in the total number of cells (9.6 ± 2.6 × 10⁵/ml vs. 15.2 ± 3.0 × 10⁵/ml, p < 0.05) and a 74% decrease in the number of eosinophils (1.4 ± 0.2 × 10⁵/ml vs. 5.4 ± 1.1 × 10⁵/ml, p < 0.01) compared with the OVA-sensitized control group. There was no difference in the number of neutrophils in BAL fluid between the pMG-Ag85B group, the OVA-sensitized control group and the empty pMG group. IL-4 production was significantly decreased in the BAL fluid (32.0 ± 7.6 pg/ml vs. 130.8 ± 32.6 pg/ml, p < 0.01) and in the splenocyte supernatant (5.1 ± 1.6 pg/ml vs. 10.1 ± 2.3 pg/ml, p < 0.05) in the pMG-Ag85B group compared with the OVA-sensitized control group, while IFN-γ production was increased in the BAL fluid (137.9 ± 25.6 pg/ml vs. 68.4 ± 15.3 pg/ml, p < 0.05) and in the splenocyte supernatant (20.1 ± 5.4 pg/ml vs. 11.3 ± 3.2 pg/ml, p < 0.05).

Conclusion

In a murine model of asthma induced by OVA, intranasal immunization with pMG-Ag85B significantly reduced allergic airway inflammation with less eosinophil infiltration. This protective effect was associated with decreased IL-4 and increased IFN-γ production in the BAL fluid and in the supernatant of cultured splenocytes.     

Melatonin promoted chemotaxins expression in lung epithelial cell stimulated with TNF-α

BackgroundPatients with asthma demonstrate circadian variations in the airway inflammation and lung function. Pinealectomy reduces the total inflammatory cell number in the asthmatic rat lung. We hypothesize that melatonin, a circadian rhythm regulator, may modulate the circadian inflammatory variations in asthma by stimulating the chemotaxins expression in the lung epithelial cell.MethodsLung epithelial cells (A549) were stimulated with melatonin in the presence or absence of TNF-α(100 ng/ml). RANTES (Regulated on Activation Normal T-cells Expressed and Secreted) and eotaxin expression were measured using ELISA and real-time RT-PCR, eosinophil chemotactic activity (ECA) released by A549 was measured by eosinophil chemotaxis assay.ResultsTNF-α increased the expression of RANTES (307.84 ± 33.56 versus 207.64 ± 31.27 pg/ml of control, p = 0.025) and eotaxin (108.97 ± 10.87 versus 54.00 ± 5.29 pg/ml of control, p = 0.041). Melatonin(10^-10 to 10^-6M) alone didn''t change the expression of RNATES (204.97 ± 32.56 pg/ml) and eotaxin (55.28 ± 6.71 pg/ml). However, In the presence of TNF-α (100 ng/ml), melatonin promoted RANTES (410.88 ± 52.03, 483.60 ± 55.37, 559.92 ± 75.70, 688.42 ± 95.32, 766.39 ± 101.53 pg/ml, treated with 10^-10, 10^-9, 10^-8, 10^-7,10^-6M melatonin, respectively) and eotaxin (151.95 ± 13.88, 238.79 ± 16.81, 361.62 ± 36.91, 393.66 ± 44.89, 494.34 ± 100.95 pg/ml, treated with 10^-10, 10^-9, 10^-8, 10^-7, 10^-6M melatonin, respectively) expression in a dose dependent manner in A549 cells (compared with TNF-α alone, P < 0.05). The increased release of RANTES and eotaxin in A549 cells by above treatment were further confirmed by both real-time RT-PCR and the ECA assay.ConclusionTaken together, our results suggested that melatonin might synergize with pro-inflammatory cytokines to modulate the asthma airway inflammation through promoting the expression of chemotaxins in lung epithelial cell.     

Virulent Bacteriophage for Efficient Biocontrol of Listeria monocytogenes in Ready-To-Eat Foods

Food-borne Listeria monocytogenes is a serious threat to human health, and new strategies to combat this opportunistic pathogen in foods are needed. Bacteriophages are natural enemies of bacteria and are suitable candidates for the environmentally friendly biocontrol of these pathogens. In a comprehensive set of experiments, we have evaluated the virulent, broad-host-range phages A511 and P100 for control of L. monocytogenes strains Scott A (serovar 4b) and WSLC 1001 (serovar 1/2a) in different ready-to-eat (RTE) foods known to frequently carry the pathogen. Food samples were spiked with bacteria (1 × 10³ CFU/g), phage added thereafter (3 × 10⁶ to 3 × 10⁸ PFU/g), and samples stored at 6°C for 6 days. In liquid foods, such as chocolate milk and mozzarella cheese brine, bacterial counts rapidly dropped below the level of direct detection. On solid foods (hot dogs, sliced turkey meat, smoked salmon, seafood, sliced cabbage, and lettuce leaves), phages could reduce bacterial counts by up to 5 log units. Variation of the experimental conditions (extended storage over 13 days or storage at 20°C) yielded similar results. In general, the application of more phage particles (3 × 10⁸ PFU/g) was more effective than lower doses. The added phages retained most of their infectivity during storage in foods of animal origin, whereas plant material caused inactivation by more than 1 log₁₀. In conclusion, our data demonstrate that virulent broad-host-range phages, such as A511 and P100, can be very effective for specific biocontrol of L. monocytogenes in contamination-sensitive RTE foods.     

Highly Pathogenic Avian Influenza Virus Infection of Mallards with Homo- and Heterosubtypic Immunity Induced by Low Pathogenic Avian Influenza Viruses

The potential role of wild birds as carriers of highly pathogenic avian influenza virus (HPAIV) subtype H5N1 is still a matter of debate. Consecutive or simultaneous infections with different subtypes of influenza viruses of low pathogenicity (LPAIV) are very common in wild duck populations. To better understand the epidemiology and pathogenesis of HPAIV H5N1 infections in natural ecosystems, we investigated the influence of prior infection of mallards with homo- (H5N2) and heterosubtypic (H4N6) LPAIV on exposure to HPAIV H5N1. In mallards with homosubtypic immunity induced by LPAIV infection, clinical disease was absent and shedding of HPAIV from respiratory and intestinal tracts was grossly reduced compared to the heterosubtypic and control groups (mean GEC/100 µl at 3 dpi: 3.0×10² vs. 2.3×10⁴ vs. 8.7×10⁴; p<0.05). Heterosubtypic immunity induced by an H4N6 infection mediated a similar but less pronounced effect. We conclude that the epidemiology of HPAIV H5N1 in mallards and probably other aquatic wild bird species is massively influenced by interfering immunity induced by prior homo- and heterosubtypic LPAIV infections.     

Self-Assembling Nanoparticles Containing Dexamethasone as a Novel Therapy in Allergic Airways Inflammation

Nanocarriers can deliver a wide variety of drugs, target them to sites of interest, and protect them from degradation and inactivation by the body. They have the capacity to improve drug action and decrease undesirable systemic effects. We have previously developed a well-defined non-toxic PEG-dendritic block telodendrimer for successful delivery of chemotherapeutics agents and, in these studies, we apply this technology for therapeutic development in asthma. In these proof-of-concept experiments, we hypothesized that dexamethasone contained in self-assembling nanoparticles (Dex-NP) and delivered systemically would target the lung and decrease allergic lung inflammation and airways hyper-responsiveness to a greater degree than equivalent doses of dexamethasone (Dex) alone. We found that ovalbumin (Ova)-exposed mice treated with Dex-NP had significantly fewer total cells (2.78±0.44×10⁵ (n = 18) vs. 5.98±1.3×10⁵ (n = 13), P<0.05) and eosinophils (1.09±0.28×10⁵ (n = 18) vs. 2.94±0.6×10⁵ (n = 12), p<0.05) in the lung lavage than Ova-exposed mice alone. Also, lower levels of the inflammatory cytokines IL-4 (3.43±1.2 (n = 11) vs. 8.56±2.1 (n = 8) pg/ml, p<0.05) and MCP-1 (13.1±3.6 (n = 8) vs. 28.8±8.7 (n = 10) pg/ml, p<0.05) were found in lungs of the Dex-NP compared to control, and they were not lower in the Dex alone group. In addition, respiratory system resistance was lower in the Dex-NP compared to the other Ova-exposed groups suggesting a better therapeutic effect on airways hyperresponsiveness. Taken together, these findings from early-stage drug development studies suggest that the encapsulation and protection of anti-inflammatory agents such as corticosteroids in nanoparticle formulations can improve efficacy. Further development of novel drugs in nanoparticles is warranted to explore potential treatments for chronic inflammatory diseases such as asthma.     

High-Frequency Phage-Mediated Gene Transfer among Escherichia coli Cells, Determined at the Single-Cell Level

Recent whole-genome analysis suggests that lateral gene transfer by bacteriophages has contributed significantly to the genetic diversity of bacteria. To accurately determine the frequency of phage-mediated gene transfer, we employed cycling primed in situ amplification-fluorescent in situ hybridization (CPRINS-FISH) and investigated the movement of the ampicillin resistance gene among Escherichia coli cells mediated by phage at the single-cell level. Phages P1 and T4 and the newly isolated E. coli phage EC10 were used as vectors. The transduction frequencies determined by conventional plating were 3 × 10⁻⁸ to 2 × 10⁻⁶, 1 × 10⁻⁸ to 4 × 10⁻⁸, and <4 × 10⁻⁹ to 4 × 10⁻⁸ per PFU for phages P1, T4, and EC10, respectively. The frequencies of DNA transfer determined by CPRINS-FISH were 7 × 10⁻⁴ to 1 × 10⁻³, 9 × 10⁻⁴ to 3 × 10⁻³, and 5 × 10⁻⁴ to 4 × 10⁻³ for phages P1, T4, and EC10, respectively. Direct viable counting combined with CPRINS-FISH revealed that more than 20% of the cells carrying the transferred gene retained their viabilities. These results revealed that the difference in the number of viable cells carrying the transferred gene and the number of cells capable of growth on the selective medium was 3 to 4 orders of magnitude, indicating that phage-mediated exchange of DNA sequences among bacteria occurs with unexpectedly high frequency.     

Probing the In Vitro Cytotoxicity of the Veterinary Drug Oxytetracycline

The study investigated the effect of oxytetracycline (OTC) on the anti-oxidative defense system, the structure (hemolysis rate and morphology) and function (ATP enzyme activity) of human red blood cells (hRBCs) to investigate the possible toxic mechanism of OTC to hRBCs. The experimental results indicate that OTC can cause a decline in the function of the antioxidant defense system of hRBCs, resulting in oxidative stress. OTC can bring about morphological changes to hRBCs, and further leads to hemolysis, when the concentration of OTC is over 8×10⁻⁵ M (about 164 µg/ml). At a low OTC concentration, below 4×10⁻⁵ M (82 µg/ml), OTC can enhance the activity of ATP enzyme of hRBCs, known as hormesis. However, at a high concentration, above 4×10⁻⁵ M (about 82 µg/ml), the ATP enzymatic activity was inhibited, affecting the function of hRBCs. The estalished mechanism of toxicity of OTC to hRBCs can facilitate a deeper understanding of the toxicity of OTC in vivo.     

An Inexpensive,Accurate, and Precise Wet-Mount Method for Enumerating Aquatic Viruses

Viruses affect biogeochemical cycling, microbial mortality, gene flow, and metabolic functions in diverse environments through infection and lysis of microorganisms. Fundamental to quantitatively investigating these roles is the determination of viral abundance in both field and laboratory samples. One current, widely used method to accomplish this with aquatic samples is the “filter mount” method, in which samples are filtered onto costly 0.02-μm-pore-size ceramic filters for enumeration of viruses by epifluorescence microscopy. Here we describe a cost-effective (ca. 500-fold-lower materials cost) alternative virus enumeration method in which fluorescently stained samples are wet mounted directly onto slides, after optional chemical flocculation of viruses in samples with viral concentrations of <5 × 10⁷ viruses ml⁻¹. The concentration of viruses in the sample is then determined from the ratio of viruses to a known concentration of added microsphere beads via epifluorescence microscopy. Virus concentrations obtained by using this wet-mount method, with and without chemical flocculation, were significantly correlated with, and had precision equivalent to, those obtained by the filter mount method across concentrations ranging from 2.17 × 10⁶ to 1.37 × 10⁸ viruses ml⁻¹ when tested by using cultivated viral isolates and natural samples from marine and freshwater environments. In summary, the wet-mount method is significantly less expensive than the filter mount method and is appropriate for rapid, precise, and accurate enumeration of aquatic viruses over a wide range of viral concentrations (≥1 × 10⁶ viruses ml⁻¹) encountered in field and laboratory samples.     

Experimental Infection of Cynomolgus Macaques (Macaca fascicularis) with Aerosolized Monkeypox Virus

Monkeypox virus (MPXV) infection in humans results in clinical symptoms very similar to ordinary smallpox. Aerosol is a route of secondary transmission for monkeypox, and a primary route of smallpox transmission in humans. Therefore, an animal model for aerosol exposure to MPXV is needed to test medical countermeasures. To characterize the pathogenesis in cynomolgus macaques (Macaca fascicularis), groups of macaques were exposed to four different doses of aerosolized MPXV. Blood was collected the day before, and every other day after exposure and assessed for complete blood count (CBC), clinical chemistry analysis, and quantitative PCR. Macaques showed mild anorexia, depression, and fever on day 6 post-exposure. Lymphadenopathy, which differentiates monkeypox from smallpox, was observed in exposed macaques around day 6 post-exposure. CBC and clinical chemistries showed abnormalities similar to human monkeypox cases. Whole blood and throat swab viral loads peaked around day 10, and in survivors, gradually decreased until day 28 post-exposure. Survival was not dose dependent. As such, doses of 4×10⁴ PFU, 1×10⁵ PFU, or 1×10⁶ PFU resulted in lethality for 70% of the animals, whereas a dose of 4×10⁵ PFU resulted in 85% lethality. Overall, cynomolgus macaques exposed to aerosolized MPXV develop a clinical disease that resembles that of human monkeypox. These findings provide a strong foundation for the use of aerosolized MPXV exposure of cynomolgus macaques as an animal model to test medical countermeasures against orthopoxviruses.     

Infection with Human Metapneumovirus Predisposes Mice to Severe Pneumococcal Pneumonia

Human metapneumovirus (hMPV) is a recently described paramyxovirus that causes respiratory tract infections. Prior clinical studies have highlighted the importance of respiratory viruses, such as influenza virus, in facilitating secondary bacterial infections and increasing host immunopathology. The objective of the present work was to evaluate the effects of initial viral infection with hMPV or influenza A virus followed by Streptococcus pneumoniae superinfection 5 days later in a murine model. Both groups of superinfected mice demonstrated significant weight loss (mean of 15%) and higher levels of airway obstruction (mean enhanced pause value of 2.7) compared to those of mice infected with hMPV, influenza virus, or pneumococcus alone. Bacterial counts increased from 5 × 10² CFU/lung in mice infected with pneumococcus only to 10⁷ and 10⁹ CFU/lung in mice with prior infections with hMPV and influenza A virus, respectively. A more pronounced interstitial and alveolar inflammation correlated with higher levels of inflammatory cytokines and chemokines such as interleukin-1α (IL-1α), IL-1β, IL-6, IL-12, monocyte chemotactic protein 1, macrophage inflammatory protein 1α, KC, and granulocyte colony-stimulating factor, as well as greater expression of Toll-like receptor 2 (TLR2), TLR6, TLR7, and TLR13 in the lungs of superinfected animals compared to results for single infections, with similar immunological effects seen in both coinfection models. Prior infection with either hMPV or influenza A virus predisposes mice to severe pneumococcus infection.     

Amino Acids Transitioning of 2009 H1N1pdm in Taiwan from 2009 to 2011

A swine-origin influenza A was detected in April 2009 and soon became the 2009 H1N1 pandemic strain (H1N1pdm). The current study revealed the genetic diversity of H1N1pdm, based on 77 and 70 isolates which we collected, respectively, during the 2009/2010 and 2010/2011 influenza seasons in Taiwan. We focused on tracking the amino acid transitioning of hemagglutinin (HA) and neuraminidase (NA) genes in the early diversification of the virus and compared them with H1N1pdm strains reported worldwide. We identified newly emerged mutation markers based on A/California/04/2009, described how these markers shifted from the first H1N1pdm season to the one that immediately followed, and discussed how these observations may relate to antigenicity, receptor-binding, and drug susceptibility. It was found that the amino acid mutation rates of H1N1pdm were elevated, from 9.29×10⁻³ substitutions per site in the first season to 1.46×10⁻² in the second season in HA, and from 5.23×10⁻³ to 1.10×10⁻² in NA. Many mutation markers were newly detected in the second season, including 11 in HA and 8 in NA, and some were found having statistical correlation to disease severity. There were five noticeable HA mutations made to antigenic sites. No significant titer changes, however, were detected based on hemagglutination inhibition tests. Only one isolate with H275Y mutation known to reduce susceptibility to NA inhibitors was detected. As limited Taiwanese H1N1pdm viruses were isolated after our sampling period, we gathered 8,876 HA and 6,017 NA H1N1pdm sequences up to April 2012 from NCBI to follow up the dynamics of mentioned HA mutations. While some mutations described in this study seemed to either settle in or die out in the 2011–2012 season, a number of them still showed signs of transitioning, prompting the importance of continuous monitoring of this virus for more seasons to come.     

Lactobacillus rhamnosus Strain GG Reduces Aflatoxin B1 Transport, Metabolism, and Toxicity in Caco-2 Cells

The probiotic Lactobacillus rhamnosus GG is able to bind the potent hepatocarcinogen aflatoxin B₁ (AFB₁) and thus potentially restrict its rapid absorption from the intestine. In this study we investigated the potential of GG to reduce AFB₁ availability in vitro in Caco-2 cells adapted to express cytochrome P-450 (CYP) 3A4, such that both transport and toxicity could be assessed. Caco-2 cells were grown as confluent monolayers on transmembrane filters for 21 days prior to all studies. AFB₁ levels in culture medium were measured by high-performance liquid chromatography. In CYP 3A4-induced monolayers, AFB₁ transport from the apical to the basolateral chamber was reduced from 11.1% ± 1.9% to 6.4% ± 2.5% (P = 0.019) and to 3.3% ± 1.8% (P = 0.002) within the first hour in monolayers coincubated with GG (1 × 10¹⁰ and 5 × 10¹⁰ CFU/ml, respectively). GG (1 × 10¹⁰ and 5 × 10¹⁰ CFU/ml) bound 40.1% ± 8.3% and 61.0% ± 6.0% of added AFB₁ after 1 h, respectively. AFB₁ caused significant reductions of 30.1% (P = 0.01), 49.4% (P = 0.004), and 64.4% (P < 0.001) in transepithelial resistance after 24, 48, and 72 h, respectively. Coincubation with 1 × 10¹⁰ CFU/ml GG after 24 h protected against AFB₁-induced reductions in transepithelial resistance at both 24 h (P = 0.002) and 48 h (P = 0.04). DNA fragmentation was apparent in cells treated only with AFB₁ cells but not in cells coincubated with either 1 × 10¹⁰ or 5 × 10¹⁰ CFU/ml GG. GG reduced AFB₁ uptake and protected against both membrane and DNA damage in the Caco-2 model. These data are suggestive of a beneficial role of GG against dietary exposure to aflatoxin.