Mycobacterium tuberculosis (Mtb) impairs disease resolution, and interferes with clinical test performance that relies on cell-mediated immunity. A number of mechanisms contribute to this T cell suppression, such as activation-induced death and trafficking of T cells out of the peripheral circulation and into the diseased lungs. The extent to which Mtb infection of human macrophages affects T cell viability however, is not well characterised.Depletion of T cells following infection by 9 cells/l) was prevalent among culture-positive tuberculosis patients, and lymphocyte counts significantly improved post-therapy. We previously reported that Mtb-infected human macrophages resulted in death of infected and uninfected bystander macrophages. In the current study, we sought to examine the influence of infected human alveolar macrophages on T cells. We infected primary human alveolar macrophages (the primary host cell for Mtb) or PMA-differentiated THP-1 cells with Mtb H37Ra, then prepared cell-free supernatants. The supernatants of Mtb-infected macrophages caused dose-dependent, caspase-dependent, T cell apoptosis. This toxic effect of infected macrophage secreted factors did not require TNF-α or Fas. The supernatant cytotoxic signal(s) were heat-labile and greater than 50 kDa in molecular size. Although ESAT-6 was toxic to T cells, other Mtb-secreted factors tested did not influence T cell viability; nor did macrophage-free Mtb bacilli or broth from Mtb cultures. Furthermore, supernatants from Mycobacterium bovis Bacille de Calmette et Guerin (BCG)- infected macrophages also elicited T cell death suggesting that ESAT-6 itself, although cytotoxic, was not the principal mediator of T cell death in our system.We found that lymphopenia (<1.5×10Mtb-Infected macrophages secrete heat-labile factors that are toxic to T cells, and may contribute to the immunosuppression seen in tuberculosis as well as interfere with microbial eradication in the granuloma. Mycobacterium tuberculosis (Mtb) must suppress immunity to survive within an infected host Tuberculosis (TB) in an immunosuppressive illness and lymphopenia often occurs in TB patients et al., that infected cells at the granuloma centre express reduced levels of apoptotic markers such as caspase 3 than those that are uninfected proapoptotic surface molecules such as Fas ligand are expressed at the periphery of the granuloma, where T cells accumulate et al. have described an increased susceptibility to apoptosis in T cells taken from tuberculosis patients et al. indicated that infected macrophages can signal T cell apoptosis T cells enhance the activity of phagocytes against Mtb and other intracellular microbes by delivering activating signals, including interferon-γ (IFN-γ) We found that lymphopenia was prevalent in our culture positive tuberculosis patients, and there was a statistically significant recovery of lymphocyte counts after anti-TB treatment. Because animal model work had associated low lymphocyte counts with death following mycobacterial infection For patient lymphocyte counts, all analysis was performed anonymously on preexisting clinical data, therefore consent was not required. For alveolar macrophage isolation, informed written consent was obtained as part of the project “Airway gene expression profiling and immune response to lung disease”, approved by the St. James’s Hospital/AMNCH Research Ethics Committee. Only the “Immune Response to Lung Disease” aspect of the ethics approval was used in this manuscript.Except where specified, all reagents and materials were obtained from Sigma-Aldrich. Anti-human TNF-α blocking antibody (cat# MAB210) and mouse IgG1 isotype control (cat# MAB002) were obtained from R&D systems. Anti-human Fas blocking antibody ZB-4 (cat# 05-338) was obtained from Millipore. Mtb secreted factors 16 kDa (cat# NR-14860), 45 kDa (cat# NR-14862), Antigen85 (cat# NR-14855), ESAT-6 (cat# NR-14868), GroES (cat# NR-14861), ManLAM (cat# NR-14848) and Phos1 (cat# NR-14859) were obtained from BEI resources, NIAID, NIH under the TB Vaccine Testing and Research Materials (TBVTRM) contract no. HHSN266200400091C awarded to Colorado State University.9/l. Immunosupressed patients  and cancer patients were excluded.All analysis of patient data was carried out anonymously. We performed a retrospective analysis of lymphocyte counts of twenty Mtb-culture positive patients recruited consecutively from our TB service at St James’ Hospital, starting November 2009. Pre-treatment lymphocyte counts were performed on the date of diagnosis prior to initiating anti-tuberculous treatment and post treatment lymphocyte counts were performed after at least 2 months of treatment. Lymphopenia was defined as a lymphocyte count of less than 1.5×10M.tuberculosis H37Ra and M.bovis BCG, obtained from ATCC, were grown to log phase at 37°C in 5% CO2 in Middlebrook 7H9 broth (Difco), supplemented with albumin-dextrose-catalase (Becton Dickinson) and 0.05% Tween-80 (Difco), and made up in endotoxin-free water.5 cells per ml, and incubated overnight at 37°C in 5% CO2 before use.Human alveolar macrophages were obtained at bronchoscopy, after written consent, under a protocol approved by the St. James’s Hospital/AMNCH ethics board. Cells in bronchoalveolar lavage fluid were passed through a 100 µm nylon cell strainer (BD Bioscience), then centrifuged for 15×min at 200×g and subsequently resuspended in RPMI supplemented with 10% human serum, 0.2% Fungizone, and 0.1% Cefotaxime. AMs were then seeded onto tissue culture plates at a density of 5×102 in RPMI 1640, supplemented with 10% FBS (hereafter referred to as ‘complete RPMI’), at a density of between 1×105−1×106 and 2×105−8×105 cells/ml respectively.Cells of the human T cell leukaemia line, Jurkat (ATCC #TIB-152), and the monocytic leukaemia line, THP-1 (ATCC #TIB-202) were routinely cultured at 37°C in 5% CO2 to remove monocytes. Nonadherent cells were then harvested and resuspended in complete RPMI +2 µg/ml PHA and incubated for 3 days at 37°C in 5% CO2. Subsequently, cells were washed in complete RPMI and resuspended at a density of 1.5×106 cells/ml in complete RPMI supplemented with 20 ng/ml IL-2 (Peprotech), then incubated for 5 days at 37°C in 5% CO2 to generate PBTLs.Lymphocytes were isolated from human whole blood provided by the Irish Blood Transfusion Service. Briefly, blood was diluted 1∶2 with sterile PBS, then layered onto lymphoprep and spun at 1200×g without brake for 20 min at room temperature. Buffy coats were removed, and then washed three times with PBS. Cells were then resuspended in serum-free RPMI, and incubated for 2 h at 37°C in 5% COM.tuberculosis H37Ra or M.bovis BCG bacilli growing in the log phase were centrifuged at 3000×g for 10 min, and then resuspended in complete RPMI. This suspension was then passed 8 times through a 25 G needle, before centrifugation at 100×g for 3 min to remove remaining clumped bacteria. Bacterial suspension was then transferred to a fresh tube, and subsequently used for infection of AMs prepared as described above, or THP-1 cells, previously differentiated to a macrophage phenotype by 72 h incubation with 100 nM Phorbol 12-myristate 13-acetate (PMA). Cells were infected with a range of volumes of Mtb H37Ra or BCG suspension, then following 3 h incubation, remaining extracellular bacilli were washed off with PBS, and cells were fixed with 2% paraformaldehyde in PBS for 5 min, and subsequently stained using a Rapid Modified Auramine O Stain Set (Scientific Device Laboratory Inc.) according to the manufacturer’s protocol, followed by 5 min staining with 10 µg/ml Hoechst 33258. Cells were imaged using fluorescent microscopy in order to determine the Multiplicity of Infection (MOI), where a low MOI was defined as >70% of cells infected with 1–5 bacteria, and a high MOI as >80% of cells infected with 6–10 bacteria. Subsequently, AMs at a density of 5×105 cells/ml or THP-1 macrophages growing at a density of 5×104 cells/ml in tissue culture plates, were infected in this manner, incubated for 3 h, then remaining extracellular bacilli were washed off and infected cells were incubated for a further 48 h in complete RPMI. For uninfected controls, cells were processed in parallel, but without addition of bacilli. Following this incubation, supernatants were removed from the cells, then centrifuged at 6,800×g to remove any free bacteria, and stored at −20°C.4 cells/well, in triplicate, in a volume of 150 µl/well, in cell-free supernatants from Mtb- or BCG- infected macrophages. 1 µM Staurosporine was used as a positive control for cell death. Following 24 h incubation, cells were stained simultaneously with 5 µg/ml Propidium Iodide (PI), 20 µg/ml Hoechst 33342 and 50 µg/ml Hoechst 33258, briefly resuspended by gentle pipetting, then plates were centrifuged at 400×g for 2 min to bring cells in suspension to the bottom surface of the well. Plates were then imaged using an INCell Analyzer 1000 cellular imaging system  and images were analyzed using GE INCell Analyzer 1000 Workstation software version 3.6  as we have previously published Jurkat T cells or PBTLs were incubated in 96-well plates at a density of 1×104 cells per well in triplicate, then imaged with Hoechst/PI staining as before. T cells were also incubated in triplicate in 96-well plates at 1×104 cells per well for 24 h in increasing dilutions of cleared culture supernatant taken from Mtb growing in the log phase in free culture, and then imaged. For secreted antigen experiments, T cells were incubated in the same manner, in logarithmic dilutions of each antigen in a concentration range of 10 µg/ml to 0.1 µg/ml, for 24 h before imaging.For coincubation experiments, Mtb H37Ra bacilli growing in the log phase were incubated in 96-well plates with T cells at varying ratios, with a constant 1×10For blocking antibody experiments, anti-human TNF-α (hereafter referred to as anti-hTNF-α) (5 µg/ml), anti-human Fas (hereafter referred to as anti-Fas) (2.5 µg/ml) or isotype control IgG at the same concentration were added to supernatants, and incubations carried out as described above. In positive control experiments for TNF-mediated cell killing, cells were incubated for 15 min in the presence of 0.2 µg/ml cycloheximide, then recombinant human TNF-α (R&D Systems) was added to 5 ng/ml. In positive control experiments for Fas ligand-mediated cell killing, cells were incubated in the presence of 10 ng/ml recombinant human Fas ligand (R&D systems), crosslinked with 10 µg/ml anti-6× Histidine (R&D systems).For heat treatment experiments, supernatants were heated to 95°C for 20 min, and then allowed to cool to room temperature before use. Initial experiments (data not shown) indicated that to preserve normal T cell viability, medium heated in this manner requires subsequent resupplementation with 10% FBS, therefore all supernatants were resupplemented following heat treatment.For molecular weight cutoff experiments, supernatants were spun on Amicon Ultra-15 centrifugal filter units with Ultracel-50 membrane (Millipore), then filtrate (hereafter referred to as <50 kDa fraction) and retentate (hereafter referred to as >50 kDa fraction) recovered. During optimisation experiments (data not shown), the >50 kDa fractions were found to affect the basal levels of cell death when applied to T cells, due to concentration of soluble factors from the medium itself, therefore fractions were diluted to 25% or 10% in fresh medium for subsequent experiments.t test was used in Microsoft Excel to determine significance between pre- and post-treatment lymphocyte counts. In all other cases, experiments were carried out in triplicate, and were performed a minimum of three times, except where specifically stated otherwise, with representative data from one experiment shown in each figure. Results were expressed as mean ± SEM, and statistical analysis was performed using a two-tailed, unpaired Student’s t test in Microsoft Excel. A p-value of less than 0.05 was considered to be statistically-significant.In data from patients who underwent antitubercular chemotherapy, a two-tailed, paired Student’s 9/l in 13 out of 20 cases) were prevalent at presentation, but significantly improved after treatment (p<0.01)  . This suTo investigate whether soluble mediators released during Mtb infection could alter the viability of T cells in a contact-independent manner, we first infected primary human alveolar macrophages for 48 h with Mtb H37Ra bacilli, then harvested culture supernatants and applied them to Jurkat T cells for 24 h before assessing cell death. In Jurkats incubated in cell-free supernatants from uninfected AMs, 5.85±0.13% cell death was observed, whereas in supernatants from low and high MOI Mtb-infected AMs, this increased significantly (p<0.001) to 18.12±0.24% and 21.13±0.25%, respectively. 1 µM Staurosporine used as a positive cell-killing control caused 67.33±0.85% death . In thisIn the past, we have found that PMA-treated THP-1 human macrophages serve as a good model for the human alveolar macrophage, with respect to innate responses after infection with Mtb. Specifically we have shown that the host responses of infection-induced macrophage apoptosis and phagolysosomal maturation arrest are well reproduced in this cell-line To confirm that soluble mediators contained in the medium of infected macrophages were responsible for T cell death, we carried out a dose-response assay using infected supernatants. In uninfected control THP-1 supernatants, 6.11±0.36% Jurkat T cell death was observed. This was significantly (p<0.001) elevated in undiluted, high MOI-infected THP-1 supernatant, to 17.23±0.49%, and progressively reduced to 10.88±0.45% at a 50% dilution, and 9.49±0.42% at a 20% dilution, both of which were also significantly (p<0.01) higher than the uninfected control .Mtb bacilli alone, and without macrophages, were insufficient to cause toxicity to T cells; in PBTLs coincubated for 24 h with live bacilli at 1∶1, 1∶5 and 1∶10 ratios; 12.89±0.94%, 11.72±1.55% and 12.01±0.34% cell death was observed, respectively, and none of these was significantly higher than the medium-only control, where 9.21±0.36% cell death occurred . This reM.bovis BCG, then cultured Jurkat T cells in the resulting cell-free supernatants as described previously. In uninfected AM supernatants, T cell death was 9.56±0.22%; this significantly increased to 14.39±1.38% (p<0.05) and 18.30±0.31% (p<0.001) in low and high MOI H37Ra-infected AM supernatants, respectively. In low and high MOI BCG-infected AM supernatants, T cell death also increased significantly (p<0.01) to 12.38±0.52% and 17.1±0.91%, respectively  to 24.39±0.61% and 25.07±0.81% in low MOI and high MOI supernatants respectively. Following incubation in the same supernatants after addition of 20 µM Z-VAD-FMK, cell death was 2.01±0.19%, 1.88±0.03% and 2.56±0.03% in uninfected, low MOI and high MOI supernatants, respectively . In posiSecondly, supernatants from infected macrophages were applied to T cells in the presence of TNF-α blocking antibody, at a concentration of 5 µg/ml, or isotype IgG at the same concentration. In the presence of isotype control antibody, 11.29±0.10% death occurred in cells incubated in uninfected macrophage supernatants. This increased significantly (p<0.001) to 17.79±0.38% and 20.63±0.45% in cells incubated in low and high MOI-infected macrophage supernatants. This cytotoxicity was not inhibited by coincubation with anti-hTNF-α; cell death increased significantly from 10.72±0.63% in the uninfected supernatants up to 17.13±0.13% and 19.18±0.51% (p<0.001 in both cases) in low and high MOI-infected macrophage supernatants, respectively . In posiAnti-Fas blocking antibody, or isotype control IgG were added at a concentration of 2.5 µg/ml to supernatants. Cell death increased significantly from 6.16±0.65% in uninfected macrophage supernatants up to 21.44±0.12% (p<0.001) in low MOI, and 23.03±0.38% (p<0.001) in high MOI-infected macrophage supernatants in the presence of isotype control IgG, and this pattern was recapitulated in supernatants in the presence of anti-Fas, where cell death increased from 6.55±0.42 in uninfected supernatants to 21.79±1.13% and 25.70±0.58% in low and high MOI-infected supernatants, respectively (p<0.001 in both cases) . In posiTo investigate the composition of the signalling factor(s) that induced T cell death, supernatants from uninfected and infected macrophages were heat-denatured at 95°C for 20 min, and then applied to Jurkat T cells. Following 24 h incubation, cell death was measured by Hoechst/PI staining, and expressed as fold-change in death relative to uninfected control. Unheated supernatants from low and high MOI-infected macrophages caused a significant  fold-increase in cell death to 1.52±0.07 and 2.04±0.05 of that in the uninfected control supernatants. This effect was mitigated by heat denaturing; in Jurkats incubated in heat-treated supernatants, fold-change in cell death was 0.88±0.02 and 1.01±0.08 of that in uninfected control supernatants .Subsequently, culture supernatants were passed through 50 kDa molecular weight cutoff filters, diluted in fresh medium as outlined in M.bovis BCG were used. Although T cell death in macrophage supernatants was caspase-dependent, it was not mediated by TNF-α or Fas. Further characterisation of the death-inducing factor(s) revealed them to be heat-labile and greater than 50 kDa in molecular size.In our tuberculosis patients, we found that lymphopenia was prevalent and reversible after treatment for this infectious disease. This systemic lack of T cell function may be linked to the poor performance of clinical tests for Mtb infection in tuberculosis patients that rely on lymphocyte function - such as the Tuberculin skin test and the Interferon-γ release assay. To study the effect of Mtb-infected macrophages on lymphocyte depletion, we examined T cell viability when exposed to the cell-free culture supernatant of infected phagocytes. Our results show that culture supernatants from Mtb-infected human macrophages induce contact-independent T cell apoptosis, in a dose-dependent manner. We found that T cell survival was unaffected by (macrophage-free) Mtb bacilli or their secreted products in bacterial culture, with the exception of ESAT-6, which reliably induced T cell death. However, subsequent experiments showed networked T cell death occurred in our system even when culture supernatants of AMs infected with the ESAT-6-deficient M.tuberculosis, several strategies facilitate pathogenesis and avoidance of bacillary destruction, including perturbation of phagosomal maturation During infection by et al. demonstrated that conditioned medium from infected monocytes elicited release of matrix metalloproteinase from microglia, representing a novel mechanism of pathology arising from TB disease in the central nervous system Elimination of immune effector cells in the proximity of infected phagocytes is thematically consistent with the concept of establishing the Mtb infection site as an immune-privileged location et al. have shown that activated T cells undergo increased apoptosis when exposed to Mtb-infected macrophages Since T cells arriving at the site of infection are likely to be activated, further investigation is now warranted into the importance of activation status on Mtb infection- induced cell death. However, whilst Sharma Exposure to viable Mtb bacilli did not cause an increase in T cell death during our coculture experiments, nor did incubation in their spent culture medium. However, changing physiological conditions, such as those encountered by Mtb upon phagocytosis for example, may lead to the expression of a distinct subset of genes that mediate bacillary survival and persistence et al., and Hirsch et al., had previously implicated TNF-α and Fas in deletion of T cells during Mtb infection in a contact-dependent manner TNF-α and Fas/Fas Ligand interactions have both been shown to regulate cell death during mycobacterial infections Heat treatment showed that the signal(s) present in infected macrophage supernatants responsible for T cell killing were heat-labile, and we subsequently employed a molecular size separation protocol in order to more clearly delineate these factor(s). In these experiments, the cytotoxic effect separated to the >50 kDa fraction, and suggested that a large protein or multimeric cytokines could be responsible. The lack of increased death in cells incubated in the <50 kDa fraction indicated that small molecule mediators such as nitric oxide Chlamydia trachomatisM.tuberculosis, this phenotype likely represents one of several manipulations of host immunity that enable persistence. It is possible that by protecting T cells from this toxic influence of infected macrophages in-vivo, the balance may be tipped towards preserving immune effector capacity, and improved disease resolution.Networked T cell death by soluble host- and/or pathogen-derived mediators released during Mtb infection may contribute to the T cell lymphopenia observed in patients with active TB disease; furthermore, premature deletion of T cells as they approach the infection site could inhibit the delivery of activating cytokines such as IFN-γ, thus rendering the infected phagocytes incompetent to degrade and clear the pathogen. A similar mode of infection-induced networked death signalling occurs during infection by other intracellular bacteria such as Figure S1Automated cell death image analysis. Images were acquired at 10× magnification using 360/460 nm and 535/620 nm excitation/emission filter sets to detect fluorescence from Hoechst and PI staining, respectively . Normal nuclei were distinguishable from condensed nuclei (example arrow 1), whilst mitotic cells did not show increased Hoechst staining, thus precluding false-positives (arrow 2). PI staining was also clearly distinguishable (arrow 3). Intensity measurements for Hoechst and PI in each cell were analysed on a cell-by-cell basis; cells with condensed nuclei were identified by an elevated coefficient of variance of nuclear intensity  above a user-defined threshold, and PI-positivity was also identified by intensity above a user-defined threshold. Using a decision-tree protocol, cells were categorised into four classes: Live, PI-positive, condensed nucleus, and condensed nucleus + PI. The latter three categories were then summed to give percentage cell death for each field of view (B). 12 fields of view were acquired per treatment, and each treatment was performed in triplicate.(TIF)Click here for additional data file.


Staphylococcus aureus can cause severe infections, including bacteremia and sepsis. The spread of methicillin-resistant Staphylococcus aureus (MRSA) highlights the need for novel treatment options. Sodium new houttuyfonate (SNH) is an analogue of houttuynin, the main antibacterial ingredient of Houttuynia cordata Thunb. The aim of this study was to evaluate in vitro activity of SNH and its potential for synergy with antibiotics against hospital-associated MRSA.A total of 103 MRSA clinical isolates recovered in two hospitals in Beijing were evaluated for susceptibility to SNH, oxacillin, cephalothin, meropenem, vancomycin, levofloxacin, minocycline, netilmicin, and trimethoprim/sulfamethoxazole by broth microdilution. Ten isolates were evaluated for potential for synergy between SNH and the antibiotics above by checkerboard assay. Time-kill analysis was performed in three isolates to characterize the kill kinetics of SNH alone and in combination with the antibiotics that engendered synergy in checkerboard assays. Besides, two reference strains were included in all assays.10 CFU/mL.SNH inhibited all test strains with minimum inhibitory concentrations (MICs) ranging from 16 to 64 µg/mL in susceptibility tests, and displayed inhibition to bacterial growth in concentration-dependent manner in time-kill analysis. In synergy studies, the combinations of SNH-oxacillin, SNH-cephalothin, SNH-meropenem and SNH-netilmicin showed synergistic effects against 12 MRSA strains with median fractional inhibitory concentration (FIC) indices of 0.38, 0.38, 0.25 and 0.38 in checkerboard assays. In time-kill analysis, SNH at 1/2 MIC in combination with oxacillin at 1/128 to 1/64 MIC or netilmicin at 1/8 to 1/2 MIC decreased the viable colonies by ≥2login vitro antibacterial activity against 103 hospital-associated MRSA isolates. Combinations of sub-MIC levels of SNH and oxacillin or netilmicin significantly improved the in vitro antibacterial activity against MRSA compared with either drug alone. The SNH-based combinations showed promise in combating MRSA.SNH demonstrated  Staphylococcus aureus (MRSA) has been reported worldwide and has become one of the leading causes of hospital-associated and community-acquired infections Staphylococcus aureus (MSSA) infections Over the past few decades, antimicrobial resistance has been recognized as a major public health problem. Since it was first recovered from patients in southern England in 1961 Houttuynia cordata Thunb  is the main antibacterial ingredient in volatile oil of Houttuynia cordata Thunb. As houttuynin is chemically unstable, sodium houttuyfonate  and sodium new houttuyfonate , analogue of sodium houttuyfonate, were synthesized and have been approved by China Food and Drug Administration to be used in clinical practice mainly in the formulations of tablets and injection in vitroin vitro activity of SNH and its potential for synergy when combined with antibiotics against a collection of hospital-associated MRSA (HA-MRSA) isolates recovered from various clinical samples in recent years.Historically, plants have provided us with a good source of antimicrobial agents. Drugs of plant origin including artesunate, berberine, and quinine are still effective in some cases in treating infectious diseases ta Thunb . A is a No permits were required for the described study, which complied with all relevant regulations.mecA gene identification and SCCmec typing. Relevant characteristics of the MRSA isolates used in this study are presented in mec type III is the predominant type in HA-MRSA strains in China S. aureus) were used as quality control strains.A total of 103 MRSA strains collected from a variety of clinical samples in two hospitals in Beijing between 2005 and 2010 were used in this study. All MRSA isolates were identified by automated antimicrobial susceptibility test systems (BD or bioMerieux systems) in hospital laboratories and were further confirmed by VITEK 2-COMPACT system  and the standard oxacillin agar dilution method recommended by Clinical and Laboratory Standards Institute  in our laboratory. In addition, genotypic features of these isolates were characterized by multiplex PCR for the Sodium New Houttuyfonate was provided by Beijing Standardherbs Medical Science & Technology Development Co. LTD. Eight antimicrobial agents, including oxacillin (OXA), cephalothin (CEF), meropenem (MEM), vancomycin (VAN), levofloxacin (LVX), minocycline (MIN), netilmicin (NET), and trimethoprim-sulfamethoxazole (SXT) were commercially purchased. SNH was dissolved in distilled water. Stock solutions of antibiotics were prepared in solvents and diluents recommended by CLSI CAMH broth was used for all susceptibility testing, checkerboard testing and time-kill analysis. Colony counts were determined using tryptic soy agar  plates.5 CFU/mL. The concentrations of SNH ranged from 1 µg/mL to 128 µg/mL. The concentration ranges for other antibiotics were as follows: 0.03 µg/mL to 128 µg/mL for MEM, VAN, LVX, MIN, and NET; 0.06 µg/mL to 1024 µg/mL for OXA and CEF; 0.008/0.15 µg/mL to 32/608 µg/mL for SXT. Testing of oxacillin was performed in CAMH broth supplemented with 2% NaCl. The microtiter plates were incubated at 35°C for 24 h. The MIC was defined as the lowest concentration of an antimicrobial agent that prevented turbidity. All MIC determinations were performed in duplicate.The minimum inhibitory concentrations (MICs) of the antibacterial agents for all 103 clinical isolates were determined by broth microdilution method according to CLSI guidelines 6 CFU/mL in a 250 mL flask for each strain. Distilled water or SNH was added to yield concentrations of 0×, 1/4×, 1/2×, 1×, 2×, and 4×MIC in the broth at standard inocula.Kill kinetics of SNH was determined by time-kill experiments for five MRSA strains  according to the method described by Verma et al. with slight modifications 10 reductions in bacterial counts within a specific time period. Bactericidal activity was defined as ≥3 log10CFU/mL reduction (99.9% kill) in colony count from the starting inoculum. The detection limit was 2log10CFU/mL.Viability counts were performed at 0, 2, 4, 6, 8 and 24 h of incubation at 37°C by plating 0.1 mL undiluted and 10-fold serial diluted samples onto TSA plates in duplicate. Drug carryover effect was eliminated by saline and agar dilution. The experiments were performed three times on different days and the results were presented as mean and standard deviation. The time–kill curves were recorded as log5 CFU/mL was added to wells of 96-well microtiter plates containing two-fold diluted SNH and the other antimicrobial in CAMH broth. After incubation at 35°C for 24 h, the combined effect of SNH with each antimicrobial was analyzed by calculation of the fractional inhibitory concentration index (FICI) using the following equation: FICI = +. The antimicrobial combination was defined to be synergistic when the FICI was ≤0.5; indifferent when 0.5< FICI <4; antagonistic when FICI ≥4 Eight combinations including SNH-OXA, SNH-CEF, SNH-MEM, SNH-VAN, SNH-LVX, SNH-MIN, SNH-NET, and SNH-SXT were evaluated on 10 randomly selected MRSA isolates and two quality control strains (ATCC 33591 and Mu 50) using microdilution checkerboard technique 10 decrease in CFU/mL between the combination and its most active constituent at 72 h, with the number of surviving organisms in the combination at ≥2log10 CFU/mL below the starting inoculum.Time-kill assays were performed for ATCC 33591, Mu 50, and the clinical isolates MRSA 5–20, MRSA 6–29 and MRSA 8–36 to evaluate the killing dynamics of SNH-OXA, SNH-NET and SNH-MEM, the combinations of interest. The time-kill for the combination study was similar to that described in the time kill assays of individual use of SNH. Antimicrobial-free inoculation of each strain was included as growth control. SNH was combined with oxacillin, meropenem or netilmicin and tested at concentrations below MIC of each drug. The concentrations of antimicrobials used for different strains were 1/2 MIC or MIC for SNH, 1/128 to 1/64 MIC for OXA, 1/8 to 1/2 MIC for NET and 1/16 to 1/4 MIC for MEM. Colony counts were determined at 0, 2, 4, 6, 8, 24, 48, and 72 h. Synergy was defined as ≥2 logmec types at concentrations ranging from 16 to 64 µg/mL. Both MIC50 and MIC90 values of SNH were 32 µg/mL. The MICs of the antibiotics for the quality control strains ATCC 33591 and Mu 50 were within the expected ranges. Most of the strains were resistant to three β-lactams , LVX, MIN, NET, or all of the six antibiotics. Among the eight antibiotics, only vancomycin and trimethoprim-sulfamethoxazole showed favorable antibacterial activity, except for the vancomycin-intermediate strain Mu 50.The susceptibility results of all antimicrobials tested against 103 MRSA isolates and 2 reference strains are presented in 10 CFU/mL for 4×MIC of SNH when compared with the initial inocula; the antibacterial effect of SNH on the five strains was determined to be bacteriostatic or marginal bactericidal at test concentrations.The time–kill curves of SNH for three MRSA clinical isolates and two reference strains are displayed in The results of the checkerboard analysis are summarized in 10 CFU/mL when compared with any of the most active agent used individually. The synergistic effect could still be observed at 72 h for some of the strains in certain combinations. In addition, the three combinations reduced the viable count by >3log10 when compared with the starting inoculum and bactericidal effect was demonstrated.The time-kill results of SNH-OXA, SNH-NET and SNH-MEM against the five MRSA strains are displayed in S. aureus in vitro. Although correlation between inhibition of autolysis of S. aureus and antimicrobial effect of SH needs further elucidation, the findings provided a possible explanation of response of S. aureus to SNH-like compounds SNH contains a hydrophilic sulfinyl head and a hydrophobic alkyl tail with 12 carbon atoms, which is considered to be surfactant-like structure. Although antibacterial mechanism of SNH remains unknown, it was indicated that SNH exerted its antimicrobial effect mainly through binding of non-polar tail group to bacterial hydrophobic membrane proteins or cytoplasmic enzymes Staphylococcus aureus, Staphylococcus epidermidis, and Klebsiella pneumoniae, etc. 50 and MIC90 values of 32 µg/mL for 103 MRSA strains and displayed growth inhibitory or marginal killing activity against MRSA in a concentration-dependent manner. In another set of experiments, we demonstrated that there was no significant difference between MSSA and MRSA on susceptibility to SNH ; B. MRSA 6–29, 1/2×MIC SNH-1/8×MIC MEM ; C. MRSA 8–36, 1/2×MIC SNH-1/16×MIC MEM ; D. ATCC 33591 1/2×MIC SNH-1/4×MIC MEM ; E. Mu 50, 1×MIC SNH-1/4×MIC MEM ; ▪, GC, growth control; ▴, SNH; □, MEM; ○, combination of SNH and MEM.(TIF)Click here for additional data file.Figure S2Activity of SNH at different concentrations in time-kill analysis against MSSA strains. A. MSSA 7–3 (MIC = 32 µg/mL); B. MSSA 7–4 (MIC = 32 µg/mL); C. MSSA 8–8 (MIC = 32 µg/mL); D. ATCC29213 (MIC = 32 µg/mL); ▪, GC, growth control; ▴, 1/4×MIC; ×, 1/2×MIC; ⧫, 1×MIC; ○, 2×MIC; □, 4×MIC.(TIF)Click here for additional data file.Figure S3Sub-MIC levels of SNH and oxacillin alone and in combination against MSSA strains. A. MSSA 7-3, 1/2×MIC SNH-1/4×MIC OXA; B. MSSA 7-4, 1/2×MIC SNH-1/4×MIC OXA ; C. MSSA 8-8, 1/2×MIC SNH-1/4×MIC OXA ; D. ATCC29213 1/2×MIC SNH-1/4×MIC OXA ; ▪, GC, growth control; ▴, SNH; ◊, OXA; ○, combination of SNH and OXA.(TIF)Click here for additional data file.Table S1MICs and FICI of SNH and eight antibiotics against 12 MRSA strains in checkerboard assay.(PDF)Click here for additional data file.Table S2Susceptibility of 20 MSSA strains to SNH and oxacillin.(PDF)Click here for additional data file.


Pseudomonas aeruginosa (GFP-P. aeruginosa) in the house fly alimentary canal along with antimicrobial peptide (AMP) expression. Motile, viable GFP-P. aeruginosa were found in all regions of the alimentary canal and were culturable throughout the observation period (2–24 h). A significant decrease in recoverable bacteria occurred between 2 and12 h, followed by an increase between 12 and 24 h. qRT-PCR analysis showed expression of the AMPs cecropin, diptericin, and defensin both locally (gut) and systemically. Furthermore, mRNA of all AMPs were expressed throughout gut tissues, with some tissue-specific temporal variation. Interestingly, fluctuation in recoverable P. aeruginosa was associated with AMP protein expression in the gut , but not with mRNA (qRTPCR). In regards to vector competence, flies excreted GFP-P. aeruginosa throughout the 24 h period, serving as both reservoirs and disseminators of this bacterium. Collectively, our data show flies can harbor and disseminate P. aeruginosa, and that the interactions of fly defenses with bacteria can influence vector competence.House flies associate with microbes throughout their life history. Bacteria ingested by adult flies enter the alimentary canal and face a hostile environment including antimicrobial defenses. Because the outcome of this interaction impacts bacterial survival and dissemination, our primary objective was to understand the temporospatial dynamics of fly-bacteria associations. We concurrently examined the temporospatial fate of GFP-expressing  Salmonella, Escherichia coli, Shigella, Streptococcus, Staphylococcus, and Pseudomonas, especially in developing countries where sanitation practices are poor Since larvae require bacteria for survival and development House flies transmit bacteria both mechanically, via contaminated mouthparts and legs, and biologically, via ingestion of microbes and excretion in vomit or feces. Food, including ingested bacteria, usually is first stored in the crop and predigested by salivary carbohydrases before entering the gut Drosophila melanogaster, which also resides in microbe-rich habitats, recognize gram-negative bacteria (and some gram-positive bacilli) when diaminopimelic acid-type peptidoglycan (DAP-PGN) fragments released from lysed or dividing microbes bind transmembrane peptidoglycan recognition receptors (PGRPs) on epithelial cells and other tissues Part of the dipteran antimicrobial defense is the humoral immune response, whose end products are effector molecules that have a range of functions from protecting flies from pathogen assault to bacterial population control in the gut. Pseudomonas aeruginosa is a pan-antibiotic resistant pathogen of humans and is commonly isolated from wild-caught house flies P. aeruginosa) including viability, location in the alimentary canal, population dynamics, and excretion and (2) the house fly perspective, by concurrently examining the spatial expression of selected AMPs . We determined that GFP-P. aeruginosa persisted in the entire house fly alimentary canal for up to 24 h post-ingestion (PI), and was excreted throughout the observation period. As soon as 2 h PI, AMP mRNAs were upregulated in response to bacteria both systemically (e.g. fat body) and locally in the alimentary canal. Interestingly, a decline in the amount of GFP-P. aeruginosa recovered from flies was preceded by local (gut) expression of AMP protein. This study provided a unique view of the dynamic interactions between house flies and bacteria and revealed that these associations influence bacterial persistence, survival and, ultimately, transmission potential.Pseudomonas aeruginosa strain PAKgfp (GFP-P. aeruginosa) was obtained from Dr. Marina Ulanova at Lakehead University P. aeruginosa cultures were maintained on tryptic soy agar  with 30 µg/ µl gentamicin. Before all fly feedings, described below, bacteria were grown in brain heart infusion broth  with gentamicin (30 µg/ µl) at 37°C to an OD600 near 0.1, which is ≈108 colony forming units (CFU) per ml.P. aeruginosa culture. The amount of bacteria in the droplet was determined by serial dilution and culture on TSA-gentamicin as described above.Adult house flies from the Georgia Southern University colony were maintained as previously described P. aeruginosa as described above . At 2, 6, 10, 12, and 24 h PI, flies (n = 4/replicate) were killed and aseptically dissected in sterile phosphate buffer saline (PBS) to remove the intact alimentary canal . Tissues were examined for the location, presence, motility, and viability of GFP-P. aeruginosa using epifluorescent and light microscopy. Digital photographs were captured with a Leica DFC420 digital camera on a Laborlux 12 microscope .Flies  were individually-housed and fed GFP-P. aeruginosa as described above . At 2, 6, 10, 12, and 24 h PI, flies (n = 4) were sacrificed then surface sanitized in 70% ethanol followed by 10% Clorox® bleach for 10 min each. After drying, individual flies were macerated in 500 µl sterile PBS, and homogenate was serially diluted and cultured overnight at 37°C on TSA-gentamicin to enumerate the surviving CFU in each fly. Changes in the number of recoverable GFP-P. aeruginosa were calculated as the difference between the CFU recovered at each time point and the CFU fed to each fly in that replicate. Only agar plates with 30 to 300 CFU were included in calculations  and subsequent statistical analyses. Kruskall-Wallis and Mann-Whitney U tests were used to analyze the overall change in bacterial survival over time and differences between time points, respectively .Flies  were individually housed and fed GFP-P. aeruginosa as described above . After feeding, flies were immobilized by chilling at 4°C and transferred to individual 35×10 mm sterile Petri dishes (Fisher Scientific). At 2, 6, 10, 12, and 24 h PI, flies (n = 4) were euthanized and each dish was washed with 1 ml of sterile PBS. Each wash was individually cultured on selective media as described above in order to enumerate GFP-P. aeruginosa shed in excreta during those time intervals (from 0 h to each time point listed above).Flies  were individually-housed and fed GFP-600 = 0.145; mean 3.41×105 CFU/fly; SD = 5.77×103). Flies (n = 5) were aseptically dissected at 2, 6, 10, 12, and 24 h PI to obtain intact digestive tracts  and carcasses . Calibrator state flies (n = 4) were fed BHI with gentamicin (30 µg/ µl) and dissected at 2 h PI as above. Tissues were homogenized in Trizol® reagent  and RNA was extracted and purified using an Ambion® Ribopure™ Kit (Life Technologies) following the manufacturer’s protocol. RNA (1 µg/reaction) was reverse transcribed using the Quantitect® Reverse Transcription Kit . Individual qRT-PCR reactions were performed in triplicate using the RealMasterMix SyBR ROX kit (5-PRIME) with sense and antisense primers (diptericin (dpt), cecropin (cec), and defensin (def), and the reference gene, ribosomal protein s18 (rps18), and with 1∶10 diluted cDNA template. Reactions containing all components except cDNA template served as negative controls. Amplification was performed under the following conditions in a realplex2 mastercycler : 2 min at 95°C, 32 cycles of 20 s at 61°C, 15 s at 68°C, 15 s at 95°C, and a final extension for 2 min at 68°C. Threshold cycles (CT) for each reaction were collected and absolute expression ratios were determined Flies  were housed and fed bacteria as described above . Individual alimentary canal tissues  were dissected from flies (n = 10) at 2, 6, and 12 h PI and pooled for RNA extraction. cDNA synthesis and qRT-PCR was performed as described above. Tissues from newly-emerged, unfed adult house flies (n = 3) served as calibrator state. CT values and absolute expression ratios were determined as above, and log10 transformed for use in statistical analyses. Since the dataset was normally distributed, a one-way analysis of variance (ANOVA) with Tukey-Kramer post hoc test was used to determine (1) the effect of time within AMP and tissue and (2) the effect of tissue within AMP and time.Flies  were housed and fed bacteria as described above . At 4, 6, and 8 h PI, intact house fly alimentary canals (n = 4) were removed and fixed in a 4% paraformaldehyde solution for 2 h. As a biological control, house flies (n = 10) were fed 2 µl BHI broth with gentamicin (30 µg/ µl) and dissected at 5 h PI as described above. Tissues were dehydrated using a series of alcohol washes (50%–100%) and cleared with Citrosolv™ (Fisher Scientific). Tissues from each replicate were pooled by time point and embedded into Paraplast® plus Tissue Embedding Medium (Fisher Scientific). Five micron serial sections were affixed to slides , rehydrated with a reverse alcohol series, and blocked for 1 h in StartingBlock™ T20 Blocking Buffer . Tissues were incubated overnight at room temperature with custom primary polyclonal antibodies  diluted in StartingBlock™ T20 Blocking Buffer (Thermoscientific) at the following concentrations: rabbit anti-Cecropin (20 µg/ml), chicken anti-Defensin (5 µg/ml), or mouse anti-Diptericin (6.69 µg/ml). Tissue sections were washed twice in PBS (pH = 7.4) for 5 min followed by incubation in secondary Alexa Fluor®  fluorescent antibodies (2 µg/ml) overnight at room temperature: Alexa Fluor® 568 goat anti-rabbit, Alexa Fluor® 488 goat anti-chicken, or Alexa Fluor® 488 goat anti-mouse. Two slides from each time-point were incubated with secondary antibodies only as a technical control. Tissue sections were mounted with ProLong Gold© antifade reagent (Invitrogen) containing DAPI nuclear stain. Immunofluorescence was visualized using a Laborlux12 microscope  equipped with appropriate bandpass filters and a Leica DFC420 microscope camera .Flies  were housed and fed bacteria as described above  and quantitative (culture) experiments beyond 24 h to assess the long term fate of bacteria in flies, and to determine whether the peak mean CFU we enumerated (∼105 CFU) is the maximum sustainable number that can persist in the house fly gut.After qualitatively observing the temporospatial fate of GFP- 24 h PI ; P≤0.04.P. aeruginosa persisted and proliferated in flies, we additionally assessed transmission potential by monitoring excretion of bacteria within several time intervals in the 24 h observation period. While relatively few house flies excreted bacteria in each time interval (incidence ranged from 16.7–41.7% of flies), flies that did shed GFP-P. aeruginosa had excreted from as few as 470 to as many as 2.03×105 CFU (4 CFU) between 0 and 12 h PI, which incidentally was the same time interval after which we noted a significant decrease in culturable GFP-P. aeruginosa from whole flies  and culture plates outside the countable range were not reported . Nonetheless, taken together, the excreta and culture recovery experiments suggest that factors other than excretion were important in eliminating and controlling GFP-P. aeruginosa numbers within the fly. Therefore, we explored the activity of antimicrobial peptide expression during house fly-bacteria interactions.Because GFP-×105 CFU . While tle flies . Howeverinterval , we infeinterval . NotablyP. aeruginosa, we examined systemic (carcass) and local (gut) expression of three AMPs, cec, dpt, and def, whose expression is upregulated after house flies ingest bacteria . House flies expressed all three AMPs in both the gut and carcass as early as 2 h after ingesting GFP-P. aeruginosa, and expression was sustained throughout the rest of the observation period  avoid increasing or inducing the rate of peristalsis, which may have caused bacterial elimination and (2) not provide the bacteria with additional nutrients after being ingested, which may have influenced their population dynamics. Future studies should incorporate more realistic conditions where flies ingest a bolus of bacteria, yet are able to feed ad libitum on food as they would in nature.Previous studies have demonstrated that short-term starvation of cec, dpt, and def were expressed at similar levels across all alimentary canal tissues  and bacterial peptidoglycan (PGN) In addition to local and systemic AMP expression analyses above, the temporospatial expression of AMPs in the alimentary canal was assessed via qRTPCR of mRNA from specific gut tissues  and immunofluorescent detection of AMP protein in whole gut sections. The aims were to (1) delineate tissue-specific AMP expression profiles and (2) to determine a possible correlation between AMP expression and location of bacteria in the gut. At 2 h PI,  tissues , which c tissues . However 12 h PI  and cult 12 h PI , the difP. aeruginosa using immunofluorescent microscopy . Notably, these two AMPs were consistently expressed in the midgut at 6 and 8 h PI  at 4, 6, and 8 h PI  to areas where populations could presumably recover and proliferate. The exact role that motility serves in bacterial survival in house flies deserves further study, as many non-motile bacteria or immobilized strains of motile bacteria (discussed above) consistently seem to be rapidly lysed and excreted from house flies once they enter the midgut.Although we observed a significant decrease in the number of recoverable GFP-P. aeruginosa expressed AMPs both locally in the gut and systemically in the carcass. While the activity of these AMPs against P. aeruginosa was not investigated, AMP protein expression in the midgut, where we bacteria were observed, coincided with a decrease in the number of bacteria recovered from flies. We did not determine whether AMP protein production ceases when bacteria levels drop, and it would be intriguing to measure the long term fluctuations and feedback between bacteria population counts and AMP protein-level expression. Other AMPs and antimicrobial effectors likely play a role in house fly-microbe dynamics and warrant further study. Additionally, the underlying feedback mechanisms between fly immune responses and bacteria population densities still remain unknown, and we know very little about gut-microbe interactions in higher diptera except for the model organism D. melanogaster. Since the outcome of these dynamic interactions impacts bacterial persistence, survival and vector competence, and because different bacterial species have different fates within house flies, we can conclude that house flies do not have uniform vector competence across bacteria species. Future studies assessing the molecular and microbiological interactions between house flies and other microbe species will help us understand the role these interactions play in vector competence for pathogens and may serve as a platform for designing novel interventions for fly-transmitted diseases.Flies that ingested GFP-Figure S1Viable GFP-P. aeruginosa in the crop and rectum of the house fly suggests both oral and fecal transmission of bacteria. House flies (n = 20 in each of three replicates) were fed an average of 2.12×105 CFU (SD = 8.36×104) bacteria and at 2, 6, 10, 12, and 24 h post-ingestion (PI), flies (n = 4 per replicate) were dissected to obtain intact alimentary canals for epifluorescent microscopy. GFP-expressing bacteria  were seen in the crop and rectum at all time points. A and B, representative images of the crop at 6 and 24 h PI, respectively. C, bright field image of rectum with fecal material at 10 h PI (blue box); D is an epifluorescent view of the fecal material  showing viable bacteria (arrows). Scale bars: A, B, and D = 10 µm and C = 100 µm.(TIF)Click here for additional data file.Table S1Primer sequences used for qRT-PCR analyses of antimicrobial peptide gene expression in house flies fed GFP-P. aeruginosa.(DOCX)Click here for additional data file.Table S2Systemic and local antimicrobial peptide expression analysis using REST-MCS©. House flies (n = 25 in each of three replicates) were fed an average of 3.41×105 CFU (SD = 5.77×103) of GFP-P. aeruginosa and processed as described in the text. A pairwise fixed allocation randomization test was performed using REST-MCS® to analyze AMP gene expression. P-values are for comparison to the calibrator state using the reference gene rps18. Statistically significant P-values are shown in yellow. Red and blue represent upregulation and downregulation of target genes, respectively.(XLSX)Click here for additional data file.Table S3Tissue-specific antimicrobial peptide expression analysis using REST-MCS©. House flies (n = 30 in each of two replicates) were fed an average of 6.6×104 CFU GFP-P. aeruginosa and processed as described in the text. A pairwise fixed allocation randomization test was performed using REST-MCS® to analyze AMP gene expression. P-values are for comparison to the calibrator state using the reference gene rps18. Statistically significant P-values are shown in yellow. Red and blue represent upregulation and downregulation of target genes, respectively.(XLSX)Click here for additional data file.


The endoplasmic reticulum (ER) stress response participates in many chronic inflammatory and autoimmune diseases. In the current study, we sought to examine the contribution of ER stress transducers in the pathogenesis of three principal facets of allergic asthma: inflammation, airway fibrosis, and airways hyperresponsiveness.in vitro and in vivo challenge of primary human and murine airway epithelial cells. ER stress transducers were modulated using specific small interfering RNAs (siRNAs) in vivo. Inflammation, airway remodeling, and hyperresponsiveness were measured by total bronchoalveolar lavage (BAL) cell counts, determination of collagen, and methacholine responsiveness in mice, respectively.House Dust Mite (HDM) was used as an allergen for Challenge of human bronchiolar and nasal epithelial cells with HDM extract induced the ER stress transducer, activating transcription factor 6 α (ATF6α) as well as protein disulfide isomerase, ERp57, in association with activation of caspase-3. SiRNA-mediated knockdown of ATF6α and ERp57 during HDM administration in mice resulted in a decrease in components of HDM-induced ER stress, disulfide mediated oligomerization of Bak, and activation of caspase-3. Furthermore, siRNA-mediated knockdown of ATF6α and ERp57 led to decreased inflammation, airway hyperresponsiveness and airway fibrosis.Collectively, our work indicates that HDM induces ER stress in airway epithelial cells and that ATF6α and ERp57 play a significant role in the development of cardinal features of allergic airways disease. Inhibition of ER stress responses may provide a potential therapeutic avenue in chronic asthma and sub-epithelial fibrosis associated with loss of lung function. Airway inflammation and fibrosis impact lung structure and function in allergic asthma . For insDermatophagoide (Der) family of proteins and lipopolysaccharide (LPS) [House Dust Mite (HDM) is one of the most commonly found airborne allergens , inducinde (LPS) -10. Studde (LPS) ,11,12.Physiological demand for increases in protein folding can create an imbalance in synthesis and capacity to fold. This leads to an increase in misfolded proteins in the endoplasmic reticulum (ER), initiating the ER stress response . In mammIn vivo siRNA mediated knockdown of ATF6α and ERp57 attenuated inflammation and AHR, and abrogated airway fibrosis. These results indicate a critical role of airway epithelial ER stress in allergen-induced airway inflammation and fibrosis.The goal of the present study was to evaluate the impact of HDM, an asthma-relevant allergen, on ER stress responses, apoptosis in airway epithelial cells and subsequent effects on fibrosis and lung function. Our results demonstrate enhanced expression of ER stress transducers in murine and human epithelial cells in response to HDM challenge. In mice, airway epithelial ER stress was associated with up regulation of apoptotic and fibrotic markers after HDM exposure. A human bronchial epithelial cell line (HBE) was kindly provided by Dr. Albert van der Vliet-University of Vermont, and cultured as described previously ,23 and pFor all experiments, 8 to 12 wk old WT BALB/c mice (Jackson Laboratories) were used, as approved by the Institutional Animal Care and Use Committee. Mice (n = 10/group) were anesthetized with isofluorane and exposed to 50 μg of the allergen, HDM (GREER-containing 35 endotoxin units/mg) extract, resuspended in PBS, via intranasal administration on day 0 and boosted again on day 7. Mice were then administered 50 μg of HDM consecutively on days 14–18, and euthanized 48 h post final exposure. The control group was given 50 μl of sterile PBS alone at all time points. Alternatively, mice were sensitized via oropharyngeal administration of 100 μg of low endotoxin Ovalbumin  in PBS with 0.1 μg of LPS on days 0 and 7, challenged using 6 doses of aerosolized 1% OVA in PBS for 30 min on days 14–19, and euthanized on day 21. This protocol was adapted from a previously described method of airway sensitization and challenge .Mice (n = 10/group) were anesthetized with isofluorane and administered 10 mg/kg of scrambled small interfering (si) RNA or siRNA for ERp57 (Thermo Scientific-L45187) and ATF6α (ORIGENE-SR418766) oropharyngeally on days −1, 6, and 13, and again on days 16 and 19X. Simultaneously, mice were exposed to 50 μg of HDM resuspended in PBS, or PBS alone via intranasal administration on days 1 and 7. Mice were then administered 50 μg of HDM consecutively, on days 14–18 and euthanized 72 h following the final HDM exposure. On day 16, when siRNA administration coincided with HDM exposure, mice received siRNA 6 h prior to intranasal administration of HDM.Mice (n = 10/group) were anesthetized with an intraperitoneal injection of pentobarbital sodium (90 mg/kg), tracheotomized using an 18 gauge cannula, then mechanically ventilated at 200 breaths/min using a FlexiVent™ computer controlled small animal ventilator (SCIREQ). While on the ventilator mice also received the paralytic, pancuronium bromide. The parameters Newtonian resistance (Rn), tissue damping (G), and elastance (H) were calculated as previously described ,29. AirwBronchoalveolar lavage (BAL) from mice (n = 10/group) was collected. Total and differential cell counts were performed as previously described . Briefly50 and CHOP (Abcam), ERp57, GRP94 (Stressgen), Poly (ADP-ribose) polymerase (PARP) (BD Pharmingen) and β-actin (Sigma).Following dissection, right lung lobes were flash frozen for protein analysis. Lungs were pulverized, and lysed in buffer containing 137 mM Tris∙HCl (pH 8.0), 130 mM NaCl, and 1% NP-40. Proteins from cell lysates were prepared in the same buffer. Insoluble proteins were pelleted via centrifugation, and following protein quantitation of the supernatant, samples were resuspended in loading buffer with dithiothrietol (DTT), and resolved by SDS-PAGE. Proteins were transferred to PVDF and membranes were probed using a standard immunoblotting protocol using the following primary antibodies: P-IRE, IRE, GRP78, ATF6Lung homogenates were resuspended in loading buffer without the reducing agent dithiothrietol (DTT). A separate set of samples were resuspended in loading buffer with DTT to reduce the disulfide bonds. The samples were resolved by SDS-PAGE and subjected to western blot analysis.Following euthanization, left lobes were fixed with 4% paraformaldehyde, stored at 4°C overnight for fixation of the tissue, mounted in paraffin, and 5 μm sections were affixed to glass microscope slides for histopathology as previously described . SectionCollagen content was measured via the Sircol assay (n = 10/group) . Briefly, lung lobes were diced and placed in 500 μl of 10 mg/mL pepsin in 0.5 M acetic acid for 3 h at 37°C, or until lungs were completely digested. The digest was spun at 10,000 g for 10 min at room temperature. Fifty microliters of the supernatant was mixed vigorously with 500 μL of sircol dye solution for 30 min and then spun again at 10,000 g for 10 min. Excess dye was decanted off, and the resulting pellet was dissolved in 500 μL of an alkaline solution, 200 μL of which was pipetted in duplicates into a 96 well plate and measured at 540 nm. To evaluate regional changes in alpha-smooth muscle actin (αSMA), fixed sections were prepared for immunostaining by deparaffinizing with xylene and rehydrating through a series of ethanols. For antigen retrieval, slides were heated for 20 min in 95°C citrate buffer (pH 6.0), then rinsed in distilled water. Sections were then blocked for 1 h in blocking serum as per manufacturer’s instructions . Slides were then washed in TBS with 0.1% TWEEN-20 3×5 min, followed by incubation with primary antibody for αSMA (Sigma) overnight at 4°C. Sections were washed again and incubated with a biotinylated universal secondary antibody  for 30 min at room temperature. Slides were washed and incubated with the Vectastain ABC-AP reagent (prepared as per manufacturer’s instructions) for 30 min at room temperature. Sections were then incubated with Vector Red Alkaline Phosphatase Substrate Kit I (Vector) for 10 min at room temperature, rinsed with tap water, and counterstained with Mayer’s Hemotoxylin.All assays were performed in triplicates. Data were analyzed by one-way analysis of variance (ANOVA) using the Tukey’s test to adjust for multiple comparisons or student’s t test where appropriate. Histopathological scores were analyzed using the Kruskal-Wallis test and Dunn's multiple comparison post hoc tests. Data from multiple experiments were averaged and expressed as mean values ± SEM.HDM is a complex allergen known to activate multiple receptors and their consequent downstream pathways . In the During ER stress, activation of ATF6α is known to specifically up regulate PDIs, chaperones, as well as CEBP homologous protein CHOP, and consequently, these events are known to lead to apoptosis ,31. To aTo elicit allergic airways disease we challenged mice with a ubiquitous allergen-HDM, or Ovalbumin and compared the responses with mice treated with LPS (a model of acute lung injury and inflammation). Mice were initially sensitized and challenged via intranasal administration of HDM, LPS or low endotoxin OVA with 0.1 μg of LPS (as an adjuvant) and were euthanized on day 21   or siRNA targeting ERp57 and ATF6α. Subsequently, the mice were challenged via the intranasal administration with 50 μg of HDM consecutively on days 15–19 and euthanized 72 h after final challenge  at either the lowest or highest dose of methacholine in HDM-challenged siRNA-treated groups of mice, but we did observe significant increases in Rn in mice that received Si-ERp57 + ATF6α as compared to Si-scr, after HDM challenge , ATF6aspase-3 ,33,34. Iaspase-3 , this upaspase-3 ,35-37. Aaspase-3 .in vitro results in human epithelial cells provide a clue that ATF6α as a transcription factor is responsible for ERp57 expression. As shown by others, ATF6α is also known to regulate inflammatory responses in models of other diseases [Our diseases ,39 alludAs reported in neuronal diseases, a severe ER stress response can lead to neuronal cell death ,40. ReceER stress transducers, such as ATF6 and CHOP, are known to play a prominent role in apoptosis of alveolar type II epithelial cells in fibrotic lung diseases, such as Idiopathic Pulmonary Fibrosis (IPF) ,20 and Hin vivo analyses.Complex allergens, such as HDM , may indn in these models are yet to be determined.Eosinophilic inflammation is one of the hallmarks of allergic asthma . In our Collectively, our work illuminates a previously unexplored mechanism for HDM-induced airway inflammation and fibrosis via ER stress and apoptosis of epithelial cells. We also show that knocking down ER stress transducers ATF6α and ERp57 decreases HDM-induced apoptosis of airway epithelial cells, bronchiolar fibrosis, inflammation, and airway hyperresponsiveness.HDM: House Dust Mite; OVA: Ovalbumin; LPS: Lipopolysaccharide; UPR: Unfolded protein response; IRE: Inositol requiring enzyme; GRP78: Glucose regulated protein 78; ATF6: Activating transcription factor 6; ERp57: Endoplasmic reticulum protein 57; CHOP: C/EBP homologous protein; PDI: Protein disulfide isomerase.The authors declare that they have no competing interests.SMH, MEP and VA designed research. SMH, JET, JDN, KGL, DHG, ND, MA and VA conducted experiments. AED provided human epithelial cells and necessary reagents to grow human cells. CGI, MEP and AED provided help with data interpretation. SMH and VA wrote the paper. All authors read and approved the final manuscript.Endoplasmic Reticulum Stress Mediates House Dust Mite-induced Airway Epithelial Apoptosis and Fibrosis.Click here for file


ARF, and p16INK4A), in astrocytoma samples and cell lines. RASSF1A was the most frequently hypermethylated gene in all grades of astrocytoma samples, in cell lines, and in adult secondary GBM. It was followed by MGMT. PTEN showed a slight methylation signal in only one GBM and one pilocytic astrocytoma, and in two cell lines; while p14ARF and p16INK4A did not show any evidence of methylation in primary tumors or cell lines. In pediatric GBM, RASSF1A was again the most frequently altered gene, followed by MGMT; PTEN, p14 and p16 showed no alterations. Lack or reduced expression of RASSF1A in cell lines was correlated with the presence of methylation. RASSF1A promoter hypermethylation might be used as a diagnostic marker for secondary GBM and pediatric GBM. Promoter hypermethylation might not be an important inactivation mechanism in other genes like PTEN, p14ARF and p16INK4A, in which other alterations  are prevalent.While allelic losses and mutations of tumor suppressor genes implicated in the etiology of astrocytoma have been widely assessed, the role of epigenetics is still a matter of study. We analyzed the frequency of promoter hypermethylation by methylation-specific PCR (MSP) in five tumor suppressor genes (PTEN, MGMT, RASSF1A, p14 The role of epigenetics in the control of gene expression is having an increasing interest in cancer research. Aberrant cytosine methylation can promote tumor initiation or progression, both by excess (promoter hypermethylation of tumor suppressor genes) and defect .ARF, APC, MLH1, and MGMT in colon cancer [ARF), DNA damage repairing , cell adhesion (APC), apoptotic response , and microtubule stability (RASSF1A).In the last decade, several authors have reported the importance of epigenetic silencing of tumor suppressor genes in a wide range of human tumors. Some of these tumor suppressor genes inactivated by cytosine methylation on their promoters are p14n cancer , CDKN2B n cancer , Caspasen cancer . Some ofTo date, chromosomal gains (+7) and losses (−10), allelic losses , oncogene amplifications , and homozygous deletions and mutations of tumor suppressor genes  have been the most frequently found alterations in astrocytoma. However, methylation has been studied in a much lesser extent in astrocytic tumors. Recently, MGMT promoter hypermethylation has been associated to secondary glioblastoma phenotype and to an increase in GC→AT mutations in TP53 gene . But proPTEN, in 10q23.3, which has been demonstrated as a critical tumor suppressor gene frequently mutated in high-grade astrocytomas, has also been reported to be inactivated by methylation-dependent mechanisms in NSCLC  and carcloci map 9p21: CDKN2B (p15INK4B) and CDKN2A, which code for two alternative protein products, p14ARF and p16INK4A. These three protein products negatively regulate cell cycle progression: p15 and p16INK4A act in the CDK/cyclin/Rb pathway, while p14ARF participates in the p14/MDM2/p53/p21 route. So, homozygous deletion of CDKN2A locus alters cell cycle regulation in two different but synergistic variants: p53 and Rb pathways. CDKN2A has a high frequency of homozygous deletions in high-grade astrocytomas, while mutations and promoter methylation of p14ARF or p16INK4A have a lesser importance in these tumors [Two important tumor suppressor e tumors , 14.Finally, recent reports have pointed at the RASSF1A gene, on 3p21.3 , as an iARF, p16INK4A, MGMT, PTEN, and RASSF1A), in a subset of astrocytomas and cell lines. While the methylation frequency of some of them  has been widely assessed in high-grade astrocytomas, it has not been much studied in low-grade astrocytic tumors. Moreover, promoter hypermethylation as an inactivation mechanism of PTEN and RASSF1A genes has rarely been studied in astrocytomas.Our aim was to study the promoter methylation status of these five important tumor suppressor genes , and MOG-G-CCM (grade III) were purchased from the European Collection of Cell Cultures ; cell lines U251, U118 (grade IV astrocytomas), SW1783, and SW1088 (grade III astrocytomas) were purchased from the American Type Culture Collection ; finally, cell lines LN-405 (grade IV astrocytoma) and GOS3 (grade II/III astrocytoma/oligodendroglioma) were obtained from the Deustche Sammlung von Mikroorganismen und Zellkulturen GmbH . Cell lines were grown in RPMI supplemented with 10% FBS, 1% penicillin/streptomycin, and 5% MEM nonessential aminoacids. Medium was restored three times a week, and subcultures were done before cells reached confluence.μL proteinase K (20 mg/mL) at 56°C, overnight. After that, 0.4 mL saturated NaCl were added to the mixture, shaked, and spun at 13.000 rpm, 10 min. Supernatant was carried to a new tube, and DNA was precipitated by adding 2 volumes of absolute ethanol at −80°C. The DNA was washed twice in 70% ethanol, dried, and resuspended in 200–500 μL Tris-EDTA Buffer .DNA was obtained with phenol-chloroform purification from the frozen tissues. Paraffin embedded samples were taken into a series of alcohol in decreasing grade and, then, left until they were totally dried. Tissues were resuspended in 1 mL lysis solution buffer  and were incubated with 20 RNA from cell lines was extracted with TRIzol Reagent , following manufacturers' instructions.μg DNA was mixed with 10 μg Herry Sperm's DNA and 1,7 μL 6 M NaOH to a final volume of 50 μL and denatured at 37–42°C, 15 min. Then, 30 μL 100 mM hydroquinone and 520 μL 3 M sodium bisulfite pH 5.0 were added, and the reactions were incubated at 56°C for 16 h. To avoid liquid evaporation, eight drops of mineral oil were added to the mixture. Modified DNAs were recovered and purified by using the Wizard DNA Clean-Up System , according to the manufacturer and eluted in 30–50 μL Tris-EDTA Buffer . These elutions were stored at −20°C until their use.Sodium bisulfite DNA modification was done as previously described . One μg μL modified DNA were used as template for each methylation-specific PCR. DNAs were added to mixes which contained 0.8 mM dNTPs, 2-3 mM MgCl2, 2.5 μL 10X reaction buffer, 5–15 pmol primers, 5%  μL DMSO, and 1 U AmpliTaq Gold DNA polymerase . For p14ARF and p16INK4A, amplification BioTaq DNA polymerase  was used, in a final volume reaction of 50 μL. Five μL 0.1 M β-mercaptoethanol were added to the PCR mix for p14ARF amplification. Primers for MGMT and RASSF1A amplification were designed by using MethPrimer software [ARF, and p16INK4A  were added. The mixture was heated at 42°C for 2 min and incubated with 1 U Superscript II RNA Retrotranscriptase . Samples were heated at 42°C for 50 min, and a final step at 72°C for 10 min was used. cDNAs were diluted 1 : 5 and stored at −20°C until their use.Five ARF, p16INK4A, MGMT, and RASSF1A expression, 1.5–2.0 μL 1 : 5 diluted cDNA were used as template for each RT-PCR. cDNAs were added to a mixture that contained 0.8 mM dNTPs, 2.5 μL 10X Reaction Buffer, 1.5 mM MgCl2, 5 pmol primers, and 1 U AmpliTaq Gold DNA polymerase , in a final volume of 25 μL. Primers were designed with Oligo 4.0 software  and are depicted in RT-PCR conditions for studying PTEN expression had been previously described . For p14ARF and p16INK4A promoter hypermethylation, respectively. We could not find any evidence of methylation in all samples tested , without finding any case of methylation  without detecting any alteration. Cell lines U87MG, U118 MG, SW1088, and U251 MG did not show any interpretable band either for p14ARF or for p16INK4A methylation-specific PCR.p14hylation . p16INK4PTEN promoter status was analyzed in a total number of 43 adult astrocytomas . We coulWe could study all cell lines and found two cases with methylation in one PTEN allele (cell lines GOS3 and MOG-G-CCM) , Table 2MGMT could be studied in 48 astrocytoma cases, and alterations were found in all grades but in pilocytic astrocytomas . Six gliP = 0,028)  . All cel= 0,028) .ARF, or p16INK4A showed methylation in any sample.Thirteen high-grade pediatric astrocytomas were studied for methylation in the five genes . RASSF1AWe studied expression of both five genes in all astrocytoma and neuroblastoma cell lines by an RT-PCR approach . ExpressRASSF1A was the most frequently altered gene and lacked expression in all cell lines .Astrocytoma cell line SW1088 lacked PTEN expression (not shown); however, this lack was not due to promoter hypermethylation, but to a homozygous deletion encompassing this gene (data not shown). Cell lines GOS3 and MOG-G-CCM, both presenting methylation in one PTEN allele, did not show a significant loss of expression when compared with other cell lines and a control with normal human astrocytes .Cell lines LN405, SW1088, and U87MG, which suffered MGMT promoter methylation, also showed lack of expression of this gene; on the contrary, SW1783 cells did not have MGMT hypermethylation and expressed the gene . We did ARF, while T98G, U87MG, GOS3, and SW1088 did not . Recently, RASSF1A promoter methylation has been revealed as a frequent epigenetic alteration in high-grade astrocytomas [RASSF1A showed the highest methylation frequency in all grades of astrocytoma studied (33% of high-grade (8 GBM and 2 AIII) and 40% of low-grade adult astrocytomas were methylated). Moreover, RASSF1A hypermethylation significantly associated to secondary glioblastoma phenotype, as 4 of 5, secondary glioblastomas, suffered hypermethylation on its promoter, while only 4 of 19 primary glioblastomas did so . On the contrary, in the case of PTEN, we could not see a reduction of mRNA expression in GOS3 and MOG-G-CCM cells, which had previously shown to be hypermethylated for this gene; however, they both had just one methylated allele, the other one remaining free of methylation, which might be enough to maintain normal PTEN expression levels.ARF and p16INK4A genes, while U87MG, GOS3, SW1088, and T98G did not. All cell lines that expressed these genes did not show any detectable methylation signal. U87MG and SW1088 cells did not produce a band for p14ARF and p16INK4A MSP; this might be due to a big homozygous deletion that affected their promoter. Other mechanisms different from methylation, like homozygous deletion, can also be responsible of p14ARF silencing in U87 MG and SW1088 cells [INK4A in MOG-G-CCM cells, when the MSP approach did not give any interpretable result: maybe MSP could not work as well as in other experiments, or a mutation in the priming site, that did not affect gene expression, was the cause of data misinterpretation after MSP.SW1783, MOG-G-CCM, and LN405 cells expressed the p1488 cells ; howeverIn the case of MGMT, although 7 cell lines showed methylated or hemimethylated alleles, only three of them lacked expression, while MOG-G-CCM and T98G retained the MGMT transcript .ARF and p16INK4A genes, while offer doubts about the direct cause-effect relationship between promoter methylation and lack of expression of PTEN and MGMT in astrocytoma cell lines. This lack of correlation between PTEN hypermethylation and lack of expression has partially been described by Baeza et al. [Our results suggest the existence of mechanisms of inactivation different from methylation for p14a et al.  by immun


Botulinum neurotoxin serotype A (BoNT/A), a potent therapeutic used to treat various disorders, inhibits vesicular neurotransmitter exocytosis by cleaving SNAP25. Development of cell-based potency assays (CBPAs) to assess the biological function of BoNT/A have been challenging because of its potency. CBPAs can evaluate the key steps of BoNT action: receptor binding, internalization-translocation, and catalytic activity; and therefore could replace the current mouse bioassay. Primary neurons possess appropriate sensitivity to develop potential replacement assays but those potency assays are difficult to perform and validate. This report describes a CBPA utilizing differentiated human neuroblastoma SiMa cells and a sandwich ELISA that measures BoNT/A-dependent intracellular increase of cleaved SNAP25. Assay sensitivity is similar to the mouse bioassay and measures neurotoxin biological activity in bulk drug substance and BOTOX® product (onabotulinumtoxinA). Validation of a version of this CBPA in a Quality Control laboratory has led to FDA, Health Canada, and European Union approval for potency testing of BOTOX®, BOTOX® Cosmetic, and Vistabel®. Moreover, we also developed and optimized a BoNT/A CBPA screening assay that can be used for the discovery of novel BoNT/A inhibitors to treat human disease. The mouse bioassay presents several challenges including assay time required, inability to differentiate between serotypes, sample capacity, and need for highly trained personnel and special animal facilities. Alternatives  include the localized muscle paralysis  Detection of BoNTs in drug products, contaminated foods, and clinical and environmental samples is challenging because of their potency . The currently approved method for measuring BoNT biological activity is the mouse LD50∼1-0.4 U/well) is equivalent or better than the mLD50, and improving the mLD50, it is specific for BoNT/A by measuring SNAP25197. It is based on a neuronal cell line and a sandwich ELISA read-out, it is accurate, reproducible, and amenable to QC validation. Moreover, it measures BoNT/A biological activity in BOTOX® (onabotulinumtoxinA) vials.A replacement to the mouse bioassay poses challenging limit of detection (LOD) requirements, in the low pM, because of the minute quantity of BoNT in drug products, and the required sensitivity, accuracy, precision, and reproducibility for Quality Control (QC) standards 197 by cleaving 9 amino acids at the C-terminus of SNAP25206197. Monoclonal 2E2A6 is highly specific for SNAP25197 with no detectable cross-reactivity to SNAP25206 in Western blot (405 = 0.892 for SNAP25134–197 vs. OD405 = 0.036 for SNAP25134–206 peptides). 2E2A6 has been characterized using Surface Plasmon Resonance (SPR) analysis and compared with a commercial antibody, MC-6053 (Research & Diagnostic Antibodies), claiming to be specific for SNAP25197, but that was found to bind SNAP25206 with a KD of 240 nM in the SPR analysis demonstrating some cross-reactivity. SPR analysis demonstrated that the 2E2A6 antibody has excellent affinity and specificity for SNAP25197 (KD = 0.075 nM for SNAP25197 and no binding to SNAP25206 at doses up to 10 µM) with a very low dissociation constant , European Collection of Cell Cultures , and German Collection of Microorganisms and Cell Cultures  and screened for their sensitivity to BoNT/A. Differentiated Neuro-2a cells were previously identified as BoNT/A sensitive and served as comparison for screening additional cell lines. The primary screen was performed using BoNT/A complex at 0 and 1 nM with 6 h treatment followed by overnight incubation in toxin free medium to allow for cleavage of SNAP25. Samples were analyzed in Western blots (WB) using anti-SNAP25 antibodies (mAb SMI-81 or pAb S9684) that detect both SNAP25and SiMa . Undiffeand SiMa  with 22.197 WB-assay  electrochemiluminescence (ECL) detection platform. The combination producing the best results consisted of 2E2A6 monoclonal for capture and anti-SNAP25 polyclonal S9684 (N-terminus) for detection  exposed to 0.4 pM BoNT/A for two days rendering 50% SNAP25 cleavage 197. To replace the bioassay, the CBPA must distinguish a fully active neurotoxin from altered or damaged neurotoxins. In the specificity studies, differentiated SiMa cells were treated with recombinant LHN/A, lacking the binding domain but containing the Light Chain and Translocation domains, and a recombinant iBoNT/A containing an inactivating mutation in the LC 197 was only detected at the higher doses of LHN/A tested, suggesting a non-specific internalization of LHN/A  and there was no SNAP25197 detected after iBoNT/A treatments. LHN/A uptake was at least 60,000 fold lower than 150 kDa BoNT/A (EC50 = 1.6 pM). To determine the effects of higher concentrations of LHN/A in the SiMa CBA, differentiated SiMa cells were treated with BoNT/A complex (at pM concentrations) or LHN/A with a highest dose of 50 µM. The data in N/A in the assay at concentrations ∼106 higher than those of active BoNT/A. The EC50 for the LHN/A molecule was 2.1 µM versus 0.85 pM for the fully active BoNT/A. Moreover, the assay can measure the potency of pure neurotoxin (150 kDa) as well as BoNT/A complex. These results demonstrate that the CBPA mirrors BoNT/A mechanism of action in vivo: binding, internalization-translocation, and catalytic activity The specificity of a method defines its ability to measure the analyte of interest and differentiate it from similar compounds. This CBPA is specific to BoNT/A by design since the 2E2A6 monoclonal antibody only recognizes SNAP25197, and are summarized in 197 accumulation  of rows A and H and columns 1 and 12 were compared to the inner wells in the plate. Edge effects were detected (p<0.001) and the assay was designed avoiding the use of the plate edges .System suitability studies (linearity), to ensure that small differences in potency between samples can be detected in the assay, were performed by two operators. To simulate samples with different potencies, samples from 2-fold more concentrated to 0.1 (10-fold) diluted compared to the BoNT/A reference preparation were tested in the assay. For these assays 150 kDa BoNT/A was used. Accuracy was evaluated by calculating the relative potency (RP) and 95% confidence intervals (CI) between the 1× and the concentrated or diluted samples, and by obtaining a percent recovery from the expected and the experimental relative potencies. The data obtained with dilutions from 0.5 (2-fold) to 0.1 (10-fold)  demonstrmax+SD0)/(Meanmax−Mean0)) values for 49 assays conducted by three operators were calculated. The Z′ parameter compares the assay dynamic range to data variation and measures how statistically different the experimental values are from the negative control To measure the quality and power of the CBPA, the z-prime . For both assays hands-on time is approximately 4 h. The screening CBPA is sensitive, EC50∼120 pM, and possesses excellent S/B>200 at 120 pM and ∼40 at 10 pM . Reconstitution of BOTOX®  with isotonic culture medium (BoNT/A concentration 500 U/mL or ∼25 pM) results in a hypertonic medium with detrimental effects on cells in culture. Testing each excipient separately in the assay demonstrated that only NaCl at the 500 U/mL dose negatively affected SiMa cells and reduced toxin uptake and/or cell viability. To circumvent hypertonicity, a custom EMEM medium was designed and used to reconstitute 100 U BOTOX® vials (500 U/mL or ∼25 pM). The matrix was kept constant for all concentrations along the dose-response curve by adding NaCl and HSA to the dilution medium to match the amount of excipients present at 500 U/mL. The assay became more sensitive with an EC50 of 9 U/mL (0.9 U/well) and an S/B ratio of 50 at 2 U/mL (0.2 U/well) (50 was 3.96±0.16 U/mL for operator 1 (n = 8) and 4.44±0.16 U/mL (n = 9) for operator 2. To compare the performance of the CBPA when different forms of BoNT/A are tested, the ECL-ELISA assay with differentiated SiMa cells was used to test the biological activity of BoNT/A complex, 150 kDa neurotoxin, and BOTOX® in different plates  contains 0.9 mg NaCl and 0.5 mg human serum albumin (HSA) in each 100 U vial  . The dat U/well)  generati U/well) . The avet plates . The EC550∼1-0.4 U/well) and is specific for BoNT/A, by the use of monoclonal antibodies specific for SNAP25197. The assay utilizes a stable cell line of neuronal origin The sensitive BoNT/A CBPA reported here utilizing differentiated human neuroblastoma SiMa cells and an ECL sandwich ELISA satisfies all the requirements for a fully in vitro replacement of the mouse bioassay for BoNT/A 50 values in the mid and low pM (50 = 6.5 pM in WB and 3 pM in ELISA) to BoNT/A and produced excellent S/B at all doses tested, especially at sub-pM concentrations, comparable to the primary and embryonic cell derived neurons For over 25 years there has been a strong desire to replace the mouse bioassay with a fully in vitro assay that enables sensitive evaluation of all key steps in BoNT/A action d low pM  that wer197 monoclonal antibody. The 2E2A6 antibody, displaying high affinity and a very low dissociation constant  that can produce reliable measurement of BoNT/A activity in only 2½ days. Additionally, we rigorously optimized and characterized both CBPAs  to the mouse bioassay and being specific for BoNT/A by design. Further development, validation, and cross-validation of a version of this CBPA has resulted in FDA, Health Canada, and European Union approval for use in the potency testing of BOTOX® (onabotulinumtoxinA), BOTOX® Cosmetic, and Vistabel®.A very sensitive CBPA is needed for measuring BoNT/A biological activity in pharmaceutical products and in other situations in which the toxin is present in very low concentrations. But during outbreaks when patients are waiting for a diagnosis or for research to identify BoNT/A inhibitors, the speed of the assay becomes more important. Our team was able to develop a shorter, but yet sensitive assay  were cultured in collagen IV plates in ATCC's recommended medium. Differentiation medium: RPMI medium with 2 mM GlutaMAX™, 1× B27 supplement, 1× N2 supplement, 10 mM HEPES, 1 mM Sodium Pyruvate, 50 ng/mL NGF, 100 U/mL Penicillin, and 100 µg/mL Streptomycin. Neuro-2a- Murine neuroblastoma  were cultured in Costar flasks in ATCC's recommended medium. Differentiation medium: EMEM with 2 mM GlutaMAX™, 0.1 mM NEAA, 10 mM HEPES, 1× N2 supplement, and 1× B27 supplement. SH-SY5Y- Human neuroblastoma  were cultured in Costar tissue culture flasks in ECACC's recommended medium. Differentiation medium: EMEM with 2 mM GlutaMAX™, 0.1 mM NEAA, 10 mM HEPES, 1× N2 supplement, and 1× B27 supplement. N18- Mouse neuroblastoma×Rat glioma hybrid  were cultured in Costar tissue culture flasks, 162 cm2 . Growth medium: DMEM with 2 mM glucose, 2 mM glutamine, and 10% heat-inactivated FBS. LA1-55n- human neuroblastoma  were cultured in Costar flasks in ECACC's recommended medium. SiMa- human neuroblastoma  were cultured in collagen IV flasks in DSMZ's recommended medium. Differentiation medium: Minimum Essential Medium with 2 mM GlutaMAX™ I with Earle's salts, 0.1 mM Non-Essential Amino-Acids, 10 mM HEPES, 1× N2 supplement, and 1× B27 supplement. For optimal differentiation, PC-12, Neuro-2a, LA1-55n, and SiMa cells were plated in 96-well plates at 5×104 cells/well in 100 µL differentiation medium for three days.197 were generated with the (C)DSNKTRIDEANQ peptide utilizing standard immunization protocols. Antibodies to SNAP25197 were screened in WB and ELISA. Antibodies were affinity purified from ascites before use in the SPR and ELISA assays.Murine monoclonal antibodies specific for SNAP25134–197 and SNAP25134–206 peptides, were immobilized on a CM5 chip using amine coupling. Analytes, Anti-SNAP25197 2E2A6  or MC-6053  antibodies, were injected at 0–125 nM. Sensorgram curves were fitted to a 1∶1 Langmuir binding model . The association, ka (1/Ms), the dissociation, kd (1/s) and the equilibrium, KD (KD = kd/ka) (M) constants were determined.Experiments were performed on a BIAcore 3000 instrument . Ligands, SNAP25N/A or iBoNT/A, or with BOTOX®  for different amounts of time as specified in each assay. The medium containing BoNT/A was then replaced with fresh medium without toxin and cells were incubated for additional periods of time as specified in each assay. Each concentration of BoNT/A was tested in triplicate.Cells were treated with BoNT/A complex, 150 kDa BoNT/A , recombinant LH2, 1 mM EGTA, 1% Triton X-100, and one tablet of EDTA-free protease inhibitors) on ice for 20 min. Lysed cells were centrifuged in the plate at 4000 rpm for 20 min at 4°C. Western blots (WB) for detection of SNAP25206 and SNAP25197 or only SNAP25197 were described previously 197 polyclonal antibody 206 and SNAP25197 were detected). Antibodies were diluted in 2% blocking agent/TBS-T buffer and incubated overnight at 4°C with gentle shaking. Secondary antibodies were anti-rabbit or anti-mouse IgG H+L HRP conjugated (Invitrogen) diluted 1∶5,000 or 1∶10,000 in 2% blocking agent/TBS-T buffer for 1 h at room temperature. The membranes were washed and exposed for 5 min to ECL Plus Western Blotting System Detection Reagents . Chemifluorescence was captured by scanning the blots in the Typhoon 9410 Imager  at λex 452/λem 520. The intensity of the gel bands were calculated using ImageQuant TL software . The data was analyzed using SigmaPlot v 10.0 (Systat Software Inc.) or PLA2.0 (Stegmann Systems). Intensity values were plotted against concentration of BoNT/A in log scale and fitted to a 4-parameter logistics function (Y = Y0+a/[1+(X/X0)b]) without constraints. Based on the fitted curves the EC50 values, corresponding to “X0”, were determined.Cells were washed once with PBS and lysed in freshly prepared Triton X-100 Lysis Buffer  in diluent buffer was added. Plates were sealed and shaken at room temperature for 1 h, washed with PBS-T, and 150 µL of 1× Read Buffer was added per well. Plates were immediately read on the SI6000 Image plate reader. For the chemiluminescence sandwich ELISA, white plates (Greiner) were coated with 100 µL/well of anti-SNAP25197 2E2A6 MAb at 4°C overnight. Plates were blocked with 2% ECL blocking with 10% goat serum for 1 h at RT. Fifty microliters of cell lysate were added to each well and the plates were incubated at 4°C. S9684 anti-SNAP25 pAb conjugated with HRP was used for detection. The plates were developed with SuperSignal ELISA Pico 1∶1 mixture (Pierce) and read at 395 nm on a Luminometer (Molecular Devices). Data was fitted to a 4PL model as detailed above.For the ECL sandwich ELISA, MSD High Bind plates  pre-spotted with anti-SNAP25Figure S1The ECL-ELISA CBPA with SiMa cells can be used to test the biological activity of BoNT/A complex, 150 kDa neurotoxin, and BOTOX®. Comparison of CBPAs performed with A. BoNT/A complex, B. 150 kDa BoNT/A, and C. BOTOX®  utilizing the optimized assay conditions. The EC50 values obtained in the assays are very similar demonstrating that the assay is robust, versatile, and can detect BoNT/A biological activity at very low concentrations in the presence of formulation excipients.(TIF)Click here for additional data file.Table S1(TIF)Click here for additional data file.


During injury, HMWHA breaks down to low molecular weight hyaluronic acid .Following tissue injury, monocytes can enter the tissue and differentiate into fibroblast-like cells called fibrocytes, but little is known about what regulates this differentiation. Extracellular matrix contains high molecular weight hyaluronic acid  inhibits HMWHA-induced fibrocyte differentiation and potentiates LMWHA-induced inhibition. Conversely, LMWHA inhibits the ability of HMWHA, interleukin-4 (IL-4), or interleukin-13 (IL-13) to promote fibrocyte differentiation.We hypothesize that hyaluronic acid signals at least in part through CD44 to regulate fibrocyte differentiation, with a dominance hierarchy of SAP>LMWHA≥HMWHA>IL-4 or IL-13. Cells appear to be able to sense the difference between HMWHA, LMWHA, and oligo-HA 3 Da HA, but not HMWHA or LMWHA During tissue injury, the extracellular matrix component hyaluronic acid (HA) breaks down into smaller fragments One of the major receptors that monocytes and lymphocytes express to detect HA is CD44 There are several known factors that either promote or inhibit fibrocyte differentiation Blood was collected from healthy adult volunteers with specific approval from the Institutional Review Boards of Rice University and Texas A&M University. Written consent was received and all samples were de-identified before analysis. Peripheral blood mononuclear cells (PBMC) were isolated and incubated in RPMI-based serum free media (SFM) as described previously Streptomyces hyalurolyticus (Sigma-Aldrich) for 1 or 4 hours at 37°C, and the digestion was stopped by boiling the solution for 5 minutes at 100°C. Electrophoresis of HA on agarose gels was done following 3 mg/ml of HMWHA or 3 mg/ml of LMWHA in PBS were treated with 10 U/ml of hyaluronidase from 1 µg/ml 1 Kb DNA ladder, 10 µg/ml, 1 µg/ml, or 0.1 µg/ml of HMWHA, LMWHA, or digested HMWHA were electrophoresed on 1% agarose gels containing 2 µg/ml ethidium bromide for 2 hours at 40 V. The gels were visualized by UV and photographed. We also examined 10 ng/ml, 100 ng/ml, or 1000 ng/ml of HMWHA, or digested HMWHA at 260/280 nm on a Synergy MX  microplate reader using a Take3 UV plate to examine the presence of DNA, RNA, or protein contamination, using BSA as a protein control. The detection limit of the Synergy MX is 2 ng/µl dsDNA, 2 ng/µl RNA, or 6 ng/µl protein. We tested for the presence of endotoxin in digested HMWHA using THP-1 blue cells  and QuantiBlue (Invivogen) following the manufacturer's instructions.7 PBMC using an EasySep Monocyte Depletion Kit  following the manufacturer's instructions 5 cells/ml in SFM was mixed with 100 µl of 600 µg/ml HMWHA in SFM, 600 µg/ml LMWHA in SFM, or a water dilution control. On day 5, the cells were fixed, stained, and the number of fibrocytes was counted.CD16-negative monocytes were purified from 5×10PBMC were cultured on eight-well glass slides  in the presence or absence of 300 µg/ml HMWHA or SFM for 5 days, as described previously PBMC were cultured on eight-well glass slides  for 5 days, as described previously To stain for hyaluronic acid, purified human monocytes were cultured in the presence or absence of 300 µg/ml HMWHA or LMWHA in eight-well glass slides as above for 30 minutes. The media was gently pipetted out and cells were fixed, permeabilized, and non-specific binding reduced as above. The monocytes were then incubated with biotinylated hyaluronic acid binding protein (bio-HABP)  diluted 1∶500 in TBS-5% BSA at room temperature for 60 minutes. Wells were washed with 400 µl of TBS, and then incubated with 1.0 µg/ml streptavidin-FITC (BD Biosciences) at room temperature for 30 minutes. After washing the slides with TBS, the slides were mounted as above.Statistical analysis was performed using Prism . Statistical significance was defined as p<0.05 as determined by the statistical methods indicated in the figure legends.6 Da and LMWHA has a range of molecular masses with a peak at 7.5×105 Da; as described below, these sizes were verified by gel electrophoresis. In our serum-free culture media, when PBMC were incubated with HMWHA or LMWHA, we found that 300 µg/ml HMWHA potentiated fibrocyte differentiation, whereas 300 µg/ml LMWHA inhibited fibrocyte differentiation . We also stained the day 5 PBMC cultured in 300 µg/ml HMWHA or SFM for the monocyte marker CD14 and the macrophage marker PM2K. Fibrocytes from both conditions did not stain for CD14 or PM2K (data not shown). There were few non-fibrocyte cells that stained positively for CD14 and PM2K (data not shown). Together, these observations indicate that the long spindle-shaped cells are fibrocytes.6 Da–2×106 Da (5 Da–8×105 Da) as analyzed by gel electrophoresis, while hyaluronidase treatment of LMWHA slightly decreased the upper end of the size range of the LMWHA , there was no difference in the number of fibrocytes compared to SFM . This inHyaluronic acid binds to the cell-surface molecule CD44, and this interaction plays an important role in development, inflammation, tumor growth, and the recruitment and activation of many immune cells into injured tissues To determine whether the hyaluronic acid binding receptor CD44 could directly affect fibrocyte differentiation, we cultured PBMC with two different anti-human CD44 antibodies, anti-human CD43, or mouse IgG1 isotype control for 5 days. We used CD43 as a second hematopoietic marker as it plays an important role in adhesion, cell-signaling, and cytoskeleton interactions, but does not bind HA + cells HA can also bind to other surface receptors such as RHAMM As HMWHA and LMWHA directly affect the differentiation of monocytes to fibrocytes, we examined if the internalization of HMWHA and LMWHA was different in purified monocytes. Purified monocytes were incubated with or without 300 µg/ml of HMWHA or 300 µg/ml of LMWHA, and after 30 minutes the cells were fixed. The fixed cells were then stained for hyaluronic acid. We detected a low intensity fluorescence signal on untreated monocytes, which could have been due to autofluorescence, non-specific binding of the biotinylated hyaluronic acid binding protein (bio-HABP), or the presence of HA bound to the cells ex vivo, as these were freshly prepared cells, and HA is present in plasma at 0.01–0.03 µg/ml A variety of pro-fibrocyte and anti-fibrocyte factors are present during tissue injury IL-4 and IL-13 are cytokines that potentiate fibrocyte differentiation We found that 300 µg/ml HMWHA potentiates fibrocyte differentiation and 300 µg/ml LMWHA inhibits fibrocyte differentiation. These HA concentrations are similar to what has been observed in tissues HMWHA and LMHWA bind to CD44, TLR2, TLR4, LYVE, and RHAMM (CD168) receptors to accomplish their biological effects 6 Da) did not affect the secretion of pro-inflammatory chemokines or cytokines 5 Da) stimulated these cells to produce inflammatory chemokines such as macrophage inflammatory protein-1α (MIP-1α), macrophage inflammatory protein-1β (MIP-1β), and monocyte chemoattractant protein-1 (MCP-1) 5–2.8×106 Da) inhibited phagocytosis, while LMWHA (9.0×104 Da) enhanced phagocytosis 5 Da) actively stimulate many types of cells at sites of injury to facilitate the clearance of debris and infectious agents, while HMWHA (>106 Da), which is the dominant HA in resolving wounds, acts as a signal for repair.Several studies have described differential effects of HMWHA and LMWHA on different types of cells such as macrophages, dendritic cells, osteoclasts, and T cells 5 Da 6–3.1×106 Da, 6 Da). Combined with our results, this suggests that the presence of LMWHA at an injured site would inhibit fibrocyte differentiation and collagen deposition to allow macrophages to freely move about the injured site to phagocytose debris and clear any infectious agents. As wound healing progresses and the ratio of HMWHA to LMWHA increases During tissue injury, cirrhosis, and fibrosis, the concentration of HA increases in serum and tissue fluids, and this HA appears to be mainly LMWHA