In the hippocampus of the control APP-tg mice, there were many Iba-1+

and CD11b− microglia cells surrounding the senile plaques (Fig. 4a), while nasally vaccinated mice with rSeV-Aβ showed the uniform distribution of Iba-1+ CD11b+ microglia (Fig. 4b). GFAP positive cells were less frequent in mice nasally vaccinated with rSeV-Aβ. Synaptophysin immunoreactivity was shrunken and disrupted in control mice with rSeV-LacZ. The nasally vaccinated mice with rSeV-Aβ showed the amelioration of abnormal change in synaptic densities and distribution patterns (Fig. 4c and d). We examined the changes of body weight in Tg2576 mice treated with SeV-Aβ nasally at the age of 12 months. The body weight measured at the age of 15 months was 28.2 ± 1.4 g for rSeV-LacZ-vaccinated non-tg mice, 26.3 ± 1.1 g for rSeV-Aβ-vaccinated non-tg mice, 23.8 ± 0.9 g for rSeV-LacZ-vaccinated Tg2576 OSI-744 mice, and 22.6 ± 0.7 g for rSeV-Aβ-vaccinated Tg2576 mice. Results with the two-way ANOVA were significantly different in genotype (F(1,38) = 17.08, p < 0.01) but not vaccination (F(1,38) = 2.24, p = 0.14)

nor interaction of genotype with vaccination (F(1,38) = 0.10, p = 0.74). During the training session, there were no significant differences in exploratory preference between the two objects and total exploratory time among the groups (data not shown), suggesting that all groups of mice have the same levels of motivation, curiosity, and interest in exploring TGF-beta inhibitor novel objects. For the retention session at age 12 months, the level of exploratory preference for the novel object in Tg2576 mice was significantly Thalidomide decreased compared to that in non-tg mice (supplemental Fig. 1). At age 15 months, the rSeV-LacZ-vaccinated Tg2576 mice also showed a significant reduction in the exploratory preference for the novel

object compared with rSeV-LacZ-vaccinated non-tg mice, however rSeV-Aβ vaccination improved the impairment of recognition memory in Tg2576 mice significantly (supplemental Fig. 1). There was no significant difference in the number of arm entries among the groups (data not shown), suggesting that all mice have the same levels of motivation, curiosity, and motor function. At age 12 months, Tg2576 mice showed significantly reduced spontaneous alternation behavior in a Y-maze test compared with non-tg mice (Fig. 5a). At age 15 months, the rSeV-LacZ-vaccinated Tg2576 mice also showed a significant reduction in spontaneous alternation behavior compared with rSeV-LacZ-vaccinated non-tg mice, however rSeV-Aβ vaccination improved alternation behavior in Tg2576 mice significantly (Fig. 5b). In the preconditioning phase, the mice hardly showed any freezing response. There were no differences in basal levels of freezing response between the groups (data not shown).

15 and 16 The phytochemicals Angiogenesis inhibitor induce toxicity in tumor cells either by scavenging constitutive reactive oxygen species or by generating paradoxically additional amount of free radicals resulting in the imbalance of cellular oxidative status, leading to inhibition of cell proliferation and eventually cell death.17, 18 and 19 In a recent study,20 the bark extract of S. oleosa was examined for its cytotoxic potential against different cell lines such as 502713 (colon), SW-520 (colon), HCT-13 (colon), A-549 (lungs), HEP-2 (liver), SK-NS-H (central nervous system), and IMR-32 (neuroblastoma). SRB dye assay following the method of Skehan et al 21 is used to evaluate the cytotoxic potential. The

ethyl acetate, methanol, and water extract showed a significant cytotoxicity against all ATM Kinase Inhibitor cell lines, except the IMR-32 cell line whereas hexane and chloroform extract did not show any significant inhibition against any of the cell lines. The cytotoxic potential was correlated with their hydroxyl radical scavenging potential. Hexane and chloroform extracts were found to have least hydroxyl radical scavenging ability, hence least cytotoxicity against the different cell lines. Oxygen is used for generating

metabolic energy in our body but it also produces reactive oxygen species as by product during its various reactions in the body. Reactive oxygen species are usually atoms or a group of atoms having odd (unpaired) electrons, in aerobic cells these are produced during mitochondrial electron transport and several Calpain oxidation reactions.22

These reactive species can, react with DNA and several other biomolecules causing what is called ‘oxidative damage to DNA’ This damage causes changes in DNA such as strand breaks; changes at cross links between DNA and protein; changes at base free sites among other changes.23 Several medicinal plants, fruits, vegetables can decrease the risk of oxidative damage as they comprise of vitamins, carotenes, phenolic compounds, flavanoids, alkanoids, tannins etc. which act as chemopreventive agents.24, 25 and 26 These phytochemicals can prevent damage by their radical scavenging ability. Thind et al evaluated the hydroxyl radical scavenging potential of S. oleosa. Extracts of roots of S. oleosa with different solvents were tested for their antiproliferative activity. Methanol extract was effective against a colon cell line (SW-620), ethyl acetate against SK-NS-H (CNS cell line) and water extract against 502713 and SW-620 (colon) cell lines. Hydroxyl radical which was used to determine radical scavenging potential of extracts, was generated by Fenton’s reaction, in site-specific and non-site-specific deoxyribose degradation assays. The extracts showed radical scavenging potential following the order of inhibition at 100 μg/mL as ethyl acetate extract (67.72%) > water extract (65.68%) > methanol extract (64.32%) in site-specific assay and as methanol extract (83.38%) > ethyl acetate extract (81.

Individuals were identified through the literature search and personal contacts using snowball sampling. The contact list was reviewed by country experts to identify the most relevant contacts and facilitate interviews in some cases. All interviews were carried out face-to-face by two interviewers, where one individual took detailed notes. see more Interviews were held in the capital cities, lasted one hour, and not digitally recorded. Questions were asked mostly in English with professional

translators used in Taiwan and Russia. In Chile and Mexico, some respondents explained some answers in Spanish in response to questions in English. An interview guide was developed and pretested where questions focused on perceptions of disease burden and the evidence supporting hepatitis A vaccination as well as the decision-making processes for adoption of a ZD6474 manufacturer hepatitis A vaccine into

national immunization programs. Interviews also assessed respondent beliefs about general policymaker agreement with a series of statements about hepatitis A severity and its vaccine. Detailed interview notes were analyzed by line-by-line coding using ATLAS.ti software. A codebook including a priori research questions was developed and applied. We present numbers of responses among those who answered specific questions. Results are presented in aggregate across respondents to protect the confidentiality of individuals. Analyses were conducted at the country level and by themes across countries. Data from the literature review, internet search and key informant

interviews were analyzed together to identify gaps between the two sources around epidemiological data, economic data and policies around hepatitis A vaccine adoption. For each topic, we compared what was said or reported in the literature with what stakeholders reported. The literature and internet search yielded 797 articles. The initial screening removed 343 articles based on titles and abstracts. Another 114 articles were excluded upon reading of full-length articles. Ketanserin This resulted in 340 articles, or 352 by country, as some articles covered multiple countries (see Fig. 1 for a flow diagram). The majority of included articles were identified through PubMed. India, South Korea and Taiwan (88, 77 and 72 articles) had twice as many publications as Russia, Chile and Mexico (43, 40 and 32 articles). 312 articles discussed the epidemiology of hepatitis A, 36 articles were on policy and 4 articles on economic analyses. While all the articles on India were in English, many of the articles in the other countries were in local languages (Russia 83%, Chile 75%, Mexico 63%, South Korea 47% and Taiwan 13%).

For dexamethasone, the cell monolayer used for the permeability assay was of low resistance Smad inhibitor (TEER ∼ 140 Ω cm2) and high log Ppara (−4.85) ( Fig. 3c). Fig. 4 illustrates carrier-mediated effects in the case of naloxone (Fig. 4a), vinblastine (Fig. 4b), colchicine (Fig. 4c), and digoxin (Fig. 4d). For naloxone and vinblastine, Ppara was estimated from the simultaneously determined sucrose Papp, while for colchicine and digoxin, Ppara was estimated using the relationships in Eqs. (A.8) and (A.11) in Appendix A (cf., Table 3). Since naloxone was measured

without stirring, the propranolol ABL marker could not be used. Since PC filter inserts were used in the cases of naloxone and vinblastine, Pfilter did not contribute to the determined log P0 in any significant way. However, PE filter inserts were used in the cases of colchicine and digoxin, which increased the contribution to the ABL effect. Nevertheless, this did not have a deleterious effect on the refinement of

log P0 values (cf., Fig. 4c and d). The big difference between the log Papp–pH (solid curve) and log PC–pH (dashed curve) curves at low pH in Fig. 4a for naloxone showed evidence for uptake via transporters. The permeability assay was repeated to include unlabelled naloxone (300 and 3000 μM) to confirm transporter saturation. The tracer (0.02 μM) naloxone set could not be refined for log P0 since the ABL was nearly entirely limiting the permeation. TSA HDAC clinical trial Consequently, the two partly-saturated sets (300 and 3000 μM cold naloxone added to the tracer level) were combined in refinement to obtain log P0 = −3.28 ± 0.02, log PABL = −5.13 ± 0.03, and log Puptake = −4.81 ± 0.06. These values were then used in the tracer set to refine just log Puptake, which produced

−4.23 ± 0.26, a value that was nearly masked by the swamping ABL effect. The three sets were then combined in a overall calculation to produce the final set of refined constants log P0 = −3.34 ± 0.12, log PABL = −5.13 (fixed), and three values of log Puptake (−4.29 ± 0.26, −4.78 ± 0.09, −4.77 ± 0.05), corresponding to the 0.02, 300, and 3000 μM sets, respectively. This Thymidine kinase analysis clearly indicated that the positively charged form of naloxone crosses the cell membranes via a saturable uptake mechanisms, apparently involving a high capacity transporter, since 3000 μM cold naloxone was not enough to saturate the transporter entirely. The efflux substrate vinblastine showed higher P0 when P-gp efflux transporter was inhibited by 50 μM PSC833 ( Fig. 4b, checkered circles). The curves were shifted both in the region of the cation and the neutral species, suggesting that vinblastine in both forms may be subject to efflux. Hence, it appeared that vinblastine was simultaneously subject to uptake and efflux carrier-mediated processes. Sucrose Papp was used to estimate Ppara in the vinblastine assay.

, 2009, Maier and Watkins, 2005, Risbrough et al., 2009 and Risbrough et al., 2004). For the purpose of this review, the CRF effects discussed will be those mediated by CRF1 unless otherwise noted. The LC-NE system is a target of CRF neurotransmission. CRF-immunoreactive

axon terminals synaptically contact LC dendrites, particularly those that extend into the peri-LC (Tjoumakaris et al., 2003 and Van Bockstaele et al., 1996). The majority of these synapses are asymmetric or excitatory-type and approximately one third co-localize glutamate, IDO inhibitor whereas few co-localize GABA (Valentino et al., 2001). Additionally, CRF axon terminals are apposed to non-labeled axon terminals that synapse with LC dendrites

suggesting that CRF can affect LC neuronal activity through both direct and indirect effects. CRF afferents to LC this website dendrites in the peri-LC derive from the central amygdalar nucleus (CeA) and the paraventricular hypothalamic nucleus (Reyes et al., 2005, Valentino et al., 1992, Van Bockstaele et al., 1998 and Van Bockstaele et al., 1999), whereas those to the nuclear LC include the nucleus paragigantocellularis, Barrington’s nucleus and the paraventricular hypothalamic nucleus (Reyes et al., 2005, Valentino et al., 1992 and Valentino et al., 1996). Hypothalamic CRF neurons that project to the LC are a distinct population from those that project to the median eminence to regulate adrenocorticotropin release (Reyes et al., 2005). In slice preparations in vitro, CRF increases LC discharge rates in the presence of tetrodotoxin or cadmium, suggesting that these are direct effects on LC neurons (Jedema and Grace, 2004). These actions are mediated by CRF1 Gs-protein

coupled receptors, are cyclic AMP dependent and are mediated by a decreased potassium conductance (Jedema and Grace, 2004 and Schulz et al., 1996). In vivo, CRF mimics the effects of stressors on LC neuronal activity when administered intracerebroventricularly or directly almost into the LC. Thus, CRF increases LC spontaneous discharge rate and attenuates sensory-evoked phasic discharge, thereby shifting discharge to a high tonic mode that would promote increased arousal, going off-task, scanning the environment and behavioral flexibility (Curtis et al., 1997, Valentino and Foote, 1987 and Valentino et al., 1983). Consistent with this, bilateral intra-LC CRF injections activate forebrain EEG activity (Curtis et al., 1997), behavioral arousal (Butler et al., 1990) and enhance behavioral flexibility in a rat attention set shifting task (Snyder et al., 2012). The increased CRF-elicited LC neuronal activation also translates to elevated forebrain NE release (Page and Abercrombie, 1999).

At 14 days post-boosting, MenB-TCM frequencies (mean of 65%) were higher (P < 0.05) than MenB-TEM frequencies (mean of 35%). By 28 days after boosting MenB-TCM frequency (mean of 59%) decreased to levels not significantly different from the ones detected before booster (mean of 57% from Selleck ABT737 days 0 to 14) but remained higher (P < 0.05) than MenB-TEM frequency (mean of 41%). Similar changes were observed for MenB-TEM frequencies at day 28 (mean of 41%) which returned to levels statistically similar to pre-boosting (mean of 51%) ( Fig. 4B). Therefore, these data indicated that in contrast to the early primary T-cell response, the 14 day-recall response to

vaccination was marked by a predominance of TCM. This difference may be attributed to the fact that the analysis of T-cell frequency after the primary series was restricted to a period of 3 days. By day 28, post-boosting T memory-cells returned to homeostatic levels. In agreement with the significant increase of Galunisertib datasheet MenB-TCM frequency at 14 days after booster immunisation, these cells reached a maximal (P < 0.05) frequency of activation by day 14 after booster (mean of 26%) as determined by the expression of CD69 ( Fig. 5C). From days 3 to 14 after boosting frequencies of activated MenB-TCM (13–26%) were significantly higher than activated MenB-TEM frequencies (5.8–9.2%) ( Fig. 5C and D). MenB-TEM reached its maximal expression of CD69 at day 28 (mean of

14.6%, P < 0.05 compared to day 14 but not to day 0) after boosting but were still lower in Dipeptidyl peptidase frequency than the TCM/CD69+ (mean of 22.8%) at the same time point. No significant differences were seen in activation status of specific TCM and TEM after primary immunisation (Fig. 5A and B), although a discrete increase of TCM/CD69+

was detected after the third dose (mean of 4.1%) of vaccine when compared with 1 dose (mean of 2.3%) or before vaccination (mean of 1.3%) (Fig. 5A). Fig. 5B shows that about 1.7% of TEM cells were activated before or after immunisation. In conclusion, vaccination with the Cuban MenB vaccine induced a significant memory CD4+ T-cell population that was activated by the booster immunisation. As expected for an efficient recall response, TCM was readily activated after stimulation with specific antigen. The design of optimal strategies to improve MenB vaccine efficiency is an ongoing challenge [4] and [17]. We reported here that the porin PorA, the serosubtype protein of meningococci, had a prominent role in inducing bactericidal as well as opsonic antibodies after immunisation of volunteers with the VA-MENGOC-BC® vaccine. Similarly, previous studies have demonstrated the potential of PorA, especially loops 1 and 4, for evoke bactericidal antibodies [18] and [19]. In contrast, opsonic antibodies have been shown to be directed mainly to PorB proteins [20] and [21]. Maintenance of long-term antibody responses is critical for protective immunity against N. meningitidis.

Haematoxylin eosin staining was applied for optical microscope observation. Controls were performed by replacing the primary antibody with buffer solution. The density of positive staining cells was calculated by MetaMorph®

Imaging System (Downingtown, PA, U.S.A.) After fixation in 4% paraformaldehyde, the specimen was kept in 30% sucrose at 4 °C overnight, and sectioned at 5 μm in thickness by freezing microtome. Then, it was incubated in 3% H2O2 at 37 °C for 30 min, rinsed in PBS Epacadostat price for 3 times, and blocked with 5%BSA at 37 °C for 30 min. Specimen was treated by chicken anti-Ag85A IgY (1:400) at 4 °C overnight, followed by rinsing in PBS for 3 times, and incubated with FITC-goat-anti-chicken GSK2656157 mw IgY (1:200, Gene Corporation) at 37 °C for 30 min. It was provided for fluorescence microscopic observation after sealing samples with 10% glycerol. The density of positive staining cells was calculated by MetaMorph® Imaging System, as shown in total grey value average. The procedure is in the similar manner as described in Section 2.4, except replacement of the primary antibody with chicken anti-Ag85A IgY (1:400) at 4 °C

overnight. After intensively washing, they were incubated with TRITC conjugated UEA-1 (1:40, Vector Laboratories) and FITC-goat-anti-chicken IgY (1:200, Gene Corporation) simultaneously at 37 °C for 30 min, and provided for fluorescence microscope observation as described. The procedure is in the

similar manner as described in Section 2.4, except replacement of the primary antibody with chicken anti-Ag85A IgY (1:400) and purified Armenian Hamster-anti-mouse CD11c (1:20, BD Pharmingen Corporation) and secondly replacement of the antibody with Texas Red conjugated Goat Anti-Armenian Hamster IgG (1:75, Jackson ImmunoResearch Laboratories) and FITC-goat-anti-chicken IgY (1:200). Total RNA from 2 × 106 IELs was extracted by Rneasy Mini Kit (QIAGEN, China) according to the manufacturer’s instructions. DNA of FasL and β-actin (as internal parameter) was respectively amplified by PCR with sense primer5′-AAT TAC CCA TGT CCC CAG ATC-3’and that of antisense primer was 5′-GCT GCT GTG GGC CCA TAT CTG-3′ for FasL gene. For β-actin gene, sense primer was 5′-TCA GAA GGA CTC CTA TGT GG-3′ and that of antisense primer however was 5′-TCT CTT TGA TGT CAC GCA CG-3′. The total volume of PCR system was 50 μl. PCR cycling conditions were; pre-denaturation at 95 °C for 2 min; denaturation at 95  °C for1 min, annealing at 55 °C for 1 min, extension at 72 °C for 2 min, in total 35 cycles; and extension at 72 °C for 10 min. Size of FsaL product amplified was 709 bp and that of β-actin was 500 bp. The products were scanned and analyzed by ChemiImager 5500 gel imaging analyzer (UVP, USA) after electrophoresis and staining by Ethidium Bromide. FasL amount expression was measured as the density of FasL and β-actin. IELs were isolated as described [15].

Based on PFGE profile analyses, no capsular switch events were detected and thus no evidence was found in our study of vaccine escape recombinant isolates as reported by Bruegemann et al. in 2007 [40]. However, Hydroxychloroquine it should be noted that the failure to detect capsular switch events could be linked to the relatively small sample size of 174 PFGE profiles. In the present

study, besides the pneumococcal prevalence comparisons that allowed detection of the known serotype replacement phenomenon between VT and NVT isolates (Table 2 and Table 3), we actually identified the mechanism of the vaccine’s effect in our setting. We show that within a month, in children aged between 12 and 24 months, a single dose of PCV7 decreases VT colonization as it prevents de novo acquisition, and conversely increases NVT colonization, namely by enhancing NVT unmasking ( Table 4). Our data is in accordance with previous studies, which suggest that conjugate

vaccines reduce VT carriage by preventing de novo acquisition rather than clearance [19], [41], [42] and [43]. Besides this major mechanism of the vaccine’s effect we propose that an additional one is the enhancement of NVT unmasking ( Table 4). Assessment of this last mechanism was only possible due to the study of multiple colonization. As a result of the paucity of multiple carriers, we were unable to conclude about a specific Histone demethylase tendency Gefitinib supplier of serotype associations before and after a single vaccine dose. Nevertheless, we found that 13 serotypes (6A, 6B, 7F, 11A, 14, 16F, 17F, 19A, 19F, 23B, 23F, 33F, and 38) and non-typeable isolates were able to co-colonize, associating with other serotypes in the children’s nasopharynx. In the vaccinated group, serotype 6A was the most common serotype observed among multiple carriers. Worthy of note is the fact that in the PCV7 era, the nasopharynx of multiple carriers can constitute

a reservoir for VT isolates. Some VTs (e.g. 6B, 14 and 19F) prevailed as minor serotypes “masked” by the dominant NVT isolates, in opposition to what occurred in the control. Whether or not the preferred co-existence of some serotypes reflects similarity of their chemical structures, similar nutritional requirements and/or bacteriocin compatibility [44] of the particular isolates remains to be determined. In summary, the present study demonstrates that, as early as 1 month after vaccination with a single dose, PCV7 causes serotype replacement of VT by NVT isolates in single and multiple carriers, with the mechanisms of the vaccine’s effect being the prevention of VT de novo acquisition and enhancement of NVT unmasking.

Analysis of the VP8* subunit of VP4 of the outbreak samples revealed two conserved amino acid substitutions at positions 237 (Ser-Leu) and 242 (Thr-Ser) when compared to the previously circulating strains. NSP4, the rotavirus enterotoxin, was also analysed. Conserved amino acid changes were observed in the 2007 outbreak G9P[8] strains. All changes were located in the cytoplasmic

domain that has numerous overlapping functional domains. In particular, the amino acid changes at positions 137 and 168 resulted in changes of the polarity, these alteration may have a functional impact on the maturation process of the virus [32]. There are DAPT supplier six described G9 VP7 lineages, Lineage I contains strains isolated in the 1980s in the USA and Japan and Lineage II contains asymptomatic neonatal strains from India [33]. Lineage III contains strains currently circulating globally including the G9 VP7 gene of the 2007 Alice Springs outbreak strains which clustered GABA cancer into sub-lineage D [33]. Four lineages of P[8] VP4 genes have been described [34]. The 2007 Alice Springs outbreak strain clustered within P[8] Lineage 3 which contains

G9P[8] and G1P[8] human strain in current global circulation. Nine enterotoxin genogroups have been described for NSP4, the 2007 Alice Springs outbreak strains clustered within enterotoxin genogroup 1 with the other characterised Australia isolates. All three genes analysed clustered closely with a 2008 G9P[8] isolate from the USA, and the VP7 gene clustered with a 2005 G9P[8] Brazil isolate. Thus sequence analysis demonstrates that

the Alice Springs 2007 outbreak strain was caused by a single G9P[8] strain, more similar to strains isolated in the USA and Brazil than Thymidine kinase to previously detected Australian isolates. The gastroenteritis outbreak occurred between March and July 2007, and during this period 173 children were admitted to Alice Springs Hospital. Seventy-eight patients had confirmed rotavirus infection. Ninety-two percent of hospitalisations involved Indigenous children and 74% involved children from remote communities [35]. A good vaccine efficacy of Rotarix against G9P[8] strains was observed. Vaccine efficacy for two doses against all hospitalisations for gastroenteritis was 77.7% and for confirmed cases of rotavirus gastroenteritis was 84.5% [35]. These results were similar to Rotarix™ vaccine efficacy against G9P[8] strains in a European trial, 85% and 83.76% from the pooled data of the phase II and III clinical trials [12] and [36]. In Brazil where 63% of disease caused by G9 strains, 80% protective efficacy has been demonstrated [37]. This outbreak occurred just 6 months after vaccine introduction, and this is highly unlikely to have influenced virus or genotype selection. However, vaccine introduction is expected to influence the genetic evolution of rotavirus strains over time.

Cohorts of 6–8 week old female BALB/c mice were obtained from Charles River Laboratories (St. Constant, QC). All experiments were conducted in accordance with the ethical guidelines by the University of Saskatchewan and the Canadian Council for Animal Care. The mice (n = 12 per group) were given a single immunization by subcutaneous injection on the back with formulations containing 10 μg of PCEP, 20 μg of IDR 1002, 10 μg CpG ODN 10101 as SOL, MP or AQ formulations, with Quadracel®

(Sanofi-Pasteur) diluted to 1 μg of PTd per animal and one group received only phosphate buffered saline pH 7.4 (PBS). The mice were immunized on day 1 and serum was separated from blood collected by tail vein puncture on days 14 and 28 after immunization. Z-VAD-FMK price find more B. pertussis Tohoma-1 strain were streaked onto charcoal agar plates supplemented with 10% sheep blood (CBA) and incubated at 37 °C for 48 h to obtain single colonies. A few single colonies were subsequently spread onto fresh CBA plates and incubated as above. After 48 h, plates were overlaid with 300 μl of 1% casamino acids, bacteria were scraped off into the casamino acid solution and 200 μl of the suspension was used to inoculate fresh CBA plates. These were incubated and harvested as described above and transferred into 2 ml of

SS medium and quantified using a spectrophotometer. Bacterial concentration was adjusted to 5 × 106/20 μl and administered intranasally. After 2 h, 2 animals from each group were humanely euthanized and their lungs were collected and homogenized in 1 ml of SS

medium and 10-fold dilutions were plated on CBA agar plates to determine the number of viable bacteria. Lungs from 5 mice per group were collected at days 3 and 7 after challenge and processed as described above. The lung homogenates were stored in 0.1 mg/ml of PMSF at −20 °C and used to examine MCP-1, TNF-α, IL-12p40, and IFN-γ cytokine production and to evaluate total IgG and IgA antigen-specific antibody responses. Antigen specific total IgG, IgG1, IgG2a and IgA immune responses were determined by end-point ELISA using methods previously described [14]. Briefly, 100 μl of pertussis toxin (PT, Sigma–Aldrich Inc., CA, USA; 0.25 μg/ml) science in carbonate coating buffer (15 mM Na2CO3, 35 mM NaHCO3, pH 9.6) was added to each well. Wells were washed 6 times with Tris-buffered saline pH 7.3 (TBS) containing 0.05% TWEEN™ 20 (TBS-T). Diluted mouse serum samples (for IgG1 and IgG2a) or lung homogenates (IgG and IgA) were added to the wells at 100 μl/well and incubated for 1 h at room temperature. Wells were washed again with TBS-T and biotinylated goat-anti mouse IgG, IgG1, IgG2a, and IgA antibodies (Caltag Laboratories, CA, USA) were added to wells (1/5000) at 100 μl/well and incubated for 1 h at room temperature.