Within 2 months after the diagnosis Panobinostat order of BL1 and 4 months after BL2, rejection appeared, thus the patients in BL1 tended to experience rejection earlier. Statistically, graft survival did not significantly differ between the BL1 and BL2 groups (P = 0.44), and events of acute rejection in patients with BL had no detrimental effect on graft survival up to the late examination (P = 0.69) (Fig. 2). Results of the second biopsy for all BL patients who

underwent that procedure showed 13 categorized as BL1 (32.5%), 3 as BL2 (7.5%), 8 with ATMR Ia (20.0%), 2 with ATMR Ib (5.0%), 1 with chronic T-cell mediated rejection (CTMR) (2.5%), 1 chronic antibody mediated rejection (CAMR) (2.5%), 4 with normal findings (NF) (10.0%), and other findings in 8 (20.0%). Furthermore, PLX4032 nmr when divided into BL1 and BL2, the 21 BL1 patients led to 6 as BL1 (28.5%), 1 as BL2 (4.8%), 6 with ATMR Ia (28.5%), 1 ATMR Ib (4.8%), 1 with CAMR (4.8%), 2 with NF (9.5%), and 8 others (19.0%), while the 19 with BL2 led to 7 as BL1 (36.8%), 2 as BL2 (10.5%), 2 with ATMR Ia (10.5%), 1 with ATMR Ib (5.3%), 1 with CTMR (5.3%), 2 NF (10.5%), and 4 others (21.0%). We also analysed predictive factors associated with rejection onset by using univariate logistic

regression. No significant difference was observed between B1 and B2 in regard to rejection development (odds ratio (OR) = 1.16, confidence index (CI): 0.31–4.28, P = 0.816). There were also no significant factors relevant Thalidomide to rejection among the other factors (human leukocyte antigen mismatch (OR = 0.99, CI: 0.59–1.64, P = 0.97); spousal transplantation (OR = 0.90, CI: 0.20–3.66, P = 0.89);

ABO incompatible (OR = 0.99, CI: 0.01–1.75, P = 0.18); use of tacrolimus (OR = 0.56, CI: 0.14–2.07, P = 0.38); donor age (OR = 1.01, CI: 0.93–1.11, P = 0.75); recipient age (OR = 1.02, CI: 0.97–1.07, P = 0.41); male (OR = 1.62, CI: 0.38–8.65, P = 0.52). There is no clear consensus regarding clinical outcome after development of BL or the treatment strategy for it, while appropriate clinical management for patients showing such changes in biopsy findings also remains controversial. Moreso et al. reported a significantly higher incidence of clinical acute rejection in patients with BL and the same for graft survival rate in patients with BL as compared with those with normal findings.[3] The incidence rate of acute rejection after BL was 48% in that report, while we found a rate of 35% in the present. BL cases have a high probability of rejection onset and should be treated, however, it does not have an influence on rate of survival. With such a background in mind, it is not surprising that contradicting reports recommend and do not recommend treatment. Since Saad et al.

7A–C). In addition, whereas stressed mice demonstrated a significant increase in the frequency of splenic CD4+CD25+ T cells as compared with nonstressed mice (17.3 and 14.7%, respectively, p < 0.05; Fig. 7D and E), the fraction of CD127− cells among CD4+CD25+ T cells was significantly lower in stressed than in nonstressed mice in the spleen (76 and 82%, respectively, p < 0.05; Fig. 7D and E) and in the blood (65.6 and 77%, respectively, p < Midostaurin order 0.01; Supporting Information Fig. 5A and B). Comparing the frequency of cells expressing CD127+ and CD127+ within splenic (Fig. 7D and F) and blood-derived (Supporting Information Fig.

5A and C) CD4+ T cells revealed a significant decrease in the CD127+/CD127+ ratio in stressed mice compared with nonstressed mice. This was evident primarily within the CD4+CD25high subpopulation EPZ-6438 purchase and to a lesser extent within the CD25low population, but was not evident in the CD25+ subpopulation. Notably, the frequency of CD25+CD127+, but not CD25+CD127+, within splenic (Fig. 7G) and blood-derived (Supporting Information Fig. 5D) CD4+ T cells was significantly higher in stressed than in nonstressed mice. This indicates that the increased Teff/Treg ratio in stressed mice resulted from an increase in the effector T-cell population with no change in the Treg-cell population. The frequency of Foxp3+ cells and the CD127−/CD127+ ratio among CD4+CD25+

T cells were then examined following EAE induction. As shown in Figure 7H, whereas the frequency of splenic Foxp3 Treg cells among CD4+ T cells was generally reduced in stressed mice prior to EAE induction, no difference was observed between stressed and nonstressed mice following EAE. Similarly, no difference was observed in the CD127+/CD127+ ratio among blood-derived CD4+CD25+ T cells between stressed and nonstressed mice

following EAE induction or remission (Supporting Information Fig. 5E). Notably, both the frequency of Foxp3+ cells (Fig. 7H) and the CD127+/CD127+ ratio among CD4+CD25+ T cells (Supporting Information Fig. 5E) were reduced at EAE onset and gradually recovered toward disease remission. The present study aimed to test the effects of chronic variable stress on immunoregulatory processes involved in autoimmune diseases. Although stress has been traditionally considered to suppress the immune system and shift it toward an antiinflammatory Bay 11-7085 response through the secretion of CORT [3, 13], our results show that prolonged stress exposure exacerbates, rather than ameliorates, EAE in female C57BL/6 mice; this phenomenon, however, could be prevented by blocking CORT signaling throughout the stress exposure period. We also show that CORT levels under basal conditions are significantly lower in male than in female mice, which is associated with exacerbated EAE symptoms. Finally, we show that stress decreases the Treg/Teff ratio, and increases the Th1-Th17/Th2 ratio, within the Teff-cell subsets.

,

Hercules, CA, USA). The primer pairs utilized for qPCR are shown in Table 1. The data are presented as the mean + SD and are representative of at least two independent experiments that employed at least four mice in each group, unless otherwise indicated. Data were analyzed using the Student’s t-test. A value of P < 0·05 was considered significant. The administration of ES proteins to the airways induced immune cell infiltration, particularly neutrophil and lymphocyte infiltration, into the lung (Figure 1a,b). The level of IL-17 cytokines in bronchial alveolar lavage (BAL) was increased profoundly after six repetitions of ES protein airway treatment, as compared with what was noted in the OVA-only treatment group (Figure 1c). In addition, the cells from the ES protein-treated selleck chemical mouse lung could generate more IL-17 cytokines than those of the OVA-only treatment group (Figure 1d). The cells of the lung draining lymph node could secrete more IL-17 cytokine than those of the mesenteric lymph node cell in response to OVA re-stimulation. This finding demonstrated that the ES protein contained some molecule that could activate Th17 cells. However, we were unable to detect any difference in the spleen cells between the ES proteins and the mice treated only with OVA. In

addition, the levels of Th2 cytokines (IL-4, -5 and click here -13) were not increased after ES protein treatment (data not shown). To determine the mechanism underlying immune cell recruitment by ES proteins, we measured IL-6, CXCL1, MDC (CCL22), TARC (CCL17) and GM-CSF gene expression levels from lung epithelial cells using ELISA, real-time PCR and RT-PCR. It is well known that CXCL1 and IL-8 (CXCL8) perform a key role in the recruitment of neutrophils during lung inflammation (25). In addition, IL-17 levels are very closely related to IL-6 levels (25,26). The lung epithelial cell line (MLE12) cells could generate IL-6 and CXCL1 as a response to ES protein treatment; we also observed the same result in a study of

primary lung epithelial cells (Figure 2a). The ES proteins induced lung inflammation via the production of IL-6 and CXCL1. Thymidine kinase In addition, The GM-CSF, TARC and MDC gene expressions in the MLE12 cells were increased by parasite ES proteins (Figure 2b). These chemokines are also related to neutrophil and T-cell and B-cell recruitment. To determine whether or not the ES protein can activate TLR, we analyzed TRIF KO and MyD88/TIRAP KO mouse embryonic fibroblast (MEF) cells after ES treatment. The ES proteins were shown to enhance the expression of IL-6 and CXCL1 in wild-type (WT) MEF, similar to what was observed in lung epithelial cells. However, we did not find that the ES protein could not enhance IL-6 and CXCL1 levels in TRIF KO MEF cells (Figure 3a,b, Supplementary Figure S1). We assessed this again with ES proteins after the administration of RNase A and C treatment to MEF cells. The results we observed, however, did not differ between the RNase-treated and nontreated samples.

These circulating AGE can deposit in the kidney and cause cellular dysfunction and renal damage. Elevated serum and urine levels of the AGE pentosidine can be detected

by HPLC or ELISA and help to predict the development of diabetic nephropathy.17 In addition, plasma levels of pentosidine have been shown to increase with loss of residual renal function in patients on peritoneal dialysis and to decrease with patients recovering renal function after transplantation.19,20 The excretion rate of albumin is the most commonly used biomarker of renal injury. Albumin is the most abundant protein in the circulation and during normal kidney function very little intact albumin is excreted by the kidney (<30 mg/day in humans). However, following renal injury, glomerular filtration of albumin is increased and the find more reabsorption and degradation of albumin by tubules are decreased, resulting Ibrutinib chemical structure in increased levels of intact albumin in the urine (i.e. albuminuria). Patient albuminuria is usually classified by ranges of severity, which are: microalbuminuria (30–300 mg/day), macroalbuminuria (300 mg–3 g/day) and nephritic range albuminuria (>3 g/day). Albuminuria is commonly used as

an early marker of renal injury because it often precedes a decline in renal function. However, it cannot distinguish different types of proteinuric kidney disease and has a limited ability to predict disease progression and determine therapeutic efficacy. Albuminuria is commonly measured by immunological

techniques, which include: immunonephelometry, immunoturbidimetry, radioimmunoassay and ELISA.21 These techniques are good for assessing albumin excretion, which is distinctly higher than normal. However, newer HPLC-based methods (e.g. the Accumin Test) can identify both immunoreactive and non-immunoreactive albumin providing greater sensitivity than conventional immunological methods for distinguishing microalbuminuria from normal Glycogen branching enzyme albumin excretion.22,23 Podocyte injury is a feature of many kidney diseases that is postulated to increase glomerular filtration of albumin. Severely damaged podocytes can detach from the glomerular basement membrane and be collected in the urine sediment. Analysis of the urine sediment by quantitative PCR or ELISA can determine mRNA or protein levels of podocyte-specific molecules (e.g. nephrin, podocin, podocalyxin) as markers of podocyte injury. Increased urine sediment levels of nephrin and podocin have been detected in patients with diabetic nephropathy and active lupus nephritis.24,25 Similarly, increased levels of podocalyxin have been found in the urine sediment of patients with IgA nephropathy, lupus nephritis and post-streptococcal glomerulonephritis.26 Sensitive markers of tubular injury have been identified in acute and CKD. N-acetyl-beta-D-glucosaminidase is a proximal tubular lysosomal enzyme, which is released during damage to proximal tubules.

The patients were grouped into the following categories: International Federation of Gynecology and Obstetrics (FIGO) stage IB (n = 16) and stage IIA–IIIB (n = 24). All tissues were subjected to immunohistochemical staining for IL-32 as described previously27

and clinically correlated with FIGO stage and survival, and the following results were obtained. In the serial section, immunohistochemical staining for COX-2 was also conducted to determine whether IL-32 and COX-2 are co-localized in cervical cancer cells. This study was approved by the Chungnam National University Hospital. The IL-32γ and COX-2 were amplified from the genomic DNA of human CaSki cells via PCR, using the following primers, respectively: IL-32γ: 5′-CTGGAATTCATGTGCTTCCCGAAG-3′ (forward), 5′-GAAGGTCCTCTCTGATGACA-3′ (reverse); COX-2: 5′-CCCAAGCTTGGGCTCAGACAGCAAAGC CTA-3′ (forward), 5′-CTAGTCTAGACTAGCTACAGTTCAGTCGAACGTTCTTT-3′ (reverse). Interleukin-32γ Y-27632 cell line find more was cloned into the EcoRI and XhoI sites of pCDNA3.1 using EcoRI and SalI, and COX-2 was ligated with pCDNA3.1 vector using the HindIII and XbaI sites. The promoters of IL-32 and COX-2 were amplified via PCR from human genomic DNA. The IL-32 promoter (−746/+25) was constructed as previously reported.21 The COX-2 promoter (−880/+9) used the following primers: 5′-CGGGATCCAAATTCTGGCCATCGCCGCTT-3′ (forward), 5′-CCAAGCTTTGACAATTGGTCGCTAA

CCGAG-3′

(reverse) cloned into the MluI and HindIII sites of the pGL3-basic vector, and the inserted sequences were confirmed via DNA sequencing. Both pTarget/E7 and pTarget/E7 antisense (E7AS) were described in a previous report.25,28 C33A/pOPI3, C33A/E7, SiHa and CaSki cells were seeded on six-well plates at a density of 3 × 105 cells per well, then grown to confluence, reaching approximately 80% at the time of transfection. For each well, plasmid DNA (1 μg) was introduced into the cells using an identical volume of Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA) in accordance with the manufacturer’s instructions. The pTarget Baricitinib and pTarget/E7AS plasmid were transfected into C33A/E7, SiHa and CaSki cells to confirm the E7 oncogene-specific effect on IL-32 and COX-2 expression in HPV-expressing cervical cancer cells. The pGL3 basic, pGL3b/IL-32 promoter, and pGL3b/COX-2 promoter were respectively co-transfected with pTarget, pTarget/E7 and pTarget/E7AS into C33A/pOPI3, C33A/E7, SiHa and CaSki cells to determine the specific effects of E7 on the transcriptional activities of IL-32 and COX-2. Additionally, pCDNA3.1, pCDNA3.1/COX-2, pCDNA3.1/IL-32γ, siCONTROL and siIL-32 (Dharmacon, Lafayette, CO) were respectively transfected into SiHa and CaSki cells to evaluate expression between COX-2 and IL-32 by the HPV E7 oncogene. Interleukin-32γ is the most active form of IL-32 isoforms.

Melt-curve analysis was included to identify nonspecific products. All RNA samples were tested for DNA contamination using a one-step RT-PCR kit with SYBR Green (Bio-Rad Laboratories) lacking reverse transcriptase. For RNA analysis, the program of the iCycler was Roscovitine cost as follows: RT reaction for 10 min at 50 °C, followed by 5 min at 95 °C. The PCR was carried out in 45 cycles consisting of denaturation for 10 s at 95 °C and elongation for 30 s at 60 °C. A final denaturation step of 1 min at 95 °C and a

final elongation step for 1 min at 55 °C were also conducted. For DNA analysis, the program was as indicated but excluded the initial cDNA synthesis step (10 min at 50 °C). To determine the half-lives of different types of RNA, we assumed that the amount y of a specific RNA at time t was given by an exponential function, where y0 and T represent the initial amount of RNA and the half-life, respectively. For each of the graphs, we determined which values of the constants y0 and T minimized the least square error. The values of T obtained by this procedure are given in Table 2. Statistical analysis was performed using Student’s t-test (two-tailed distribution, two-sample

equal variance) when indicated in the figure legends. Mean relative amounts of each target mRNA, normalized LEE011 clinical trial to individual control RNA, were added together and divided with the corresponding number of control RNAs (four control RNAs). During a C. pneumoniae infection, the amount of DNA and the number of bacteria increase between 14 and 26 h p.i., but not before that time (Ouellette et al., 2006, Fig. 1). Also, the microorganisms differentiate from metabolically inactive EBs to metabolically active RBs before 14 h p.i. (Wolf et al., 2000). click here On the contrary, addition of the growth inhibitor INP0010 abolished C. pneumoniae proliferation and the amount of DNA increased only slightly between 2 and 26 h p.i. (Fig. 1, Bailey et al., 2007). To further analyze the mechanism of INP0010, it was of interest to measure gene expression in INP0010-treated and untreated C. pneumoniae during the transition phase (14 h p.i.).

We chose to investigate several genes coding for components of the virulence-associated type 3 secretion system (T3SS), as well as the gene groEL_1, which encodes the housekeeping chaperone GroEL (Table 3). Expression of these mRNAs was correlated with different control RNAs [16S rRNA, rpoA, rpoD, and gyrA (Goellner et al., 2006; Bailey et al., 2007; Fink et al., 2007)]. Data obtained in previous experiments had indicated that treatment with INP0010 reduced the transcription of some T3SS genes when 16S rRNA was used as an internal control (Bailey et al., 2007). Therefore, to examine the effect of INP0010 on T3SS gene expression when using different internal expression controls, we allowed C. pneumoniae to infect HEp-2 cells in the presence or the absence of INP0010 for 14 h.

Preassembly of these components is believed to facilitate the rapid and efficient activation of ERK. Consistent with this idea, all studies to date show that the absence of KSR1 leads to an attenuation of ERK activity in a wide variety of different cells 18–22. Because the intensity and duration of ERK activation has been implicated in the development of thymocytes 8, 9, 32, 33, we were interested to test whether the absence

of KSR1 would have an effect on the positive and negative selection of thymocytes. Surprisingly, LY2835219 nmr our analysis using several different models showed that KSR1 was basically dispensable for both positive and negative thymocyte selection. Our findings are in contrast to a previous study on the role of KSR1 in thymocyte development that suggested it was important for positive selection 34. In that study, overexpression of KSR1

was delivered to thymocytes by retroviruses 34 and resulted in a partial block in positive selection. Although our study used a variety of in vivo models of positive and negative selection, the previous study relied on in vitro reaggregate Poziotinib clinical trial cultures 34. Differences between the studies could be due to the different approaches used. In addition, overexpression of scaffold proteins is problematic as it can act to titer down concentrations of binding partners, possibly resulting in off-target effects on pathways in addition to the ERK-MAPK pathway 35. No data were presented regarding the effect of KSR1 overexpression on negative selection 34. Numerous studies have directly implicated ERK in thymocyte development 7–11.

Although initial studies in the ERK1−/− mouse indicated that there was a slight defect Farnesyltransferase in thymocyte maturation 10, subsequent studies failed to find any defect 7. Mice lacking both isoforms of ERK, ERK1 and ERK2, have a partial block in thymocyte development at the DN3 stage 7 and a complete block in positive selection. Surprisingly, when the ERK1/2 double KO was bred to two different TCR transgenic mice, OT-I and AND, a small percentage of thymocytes could still be positively selected, suggesting that ERK is important but not absolutely required for positive selection 7. This defect in positive selection is consistent with the studies using transgenic mice expressing dominant-negative forms of Ras and MEK under the control of the Lck promoter 5, 36, 37. Our studies showing decreased, but clearly detectable, ERK activity in KSR1-deficient thymocytes is consistent with the idea that only a threshold amount of ERK activity is required to mediate positive selection. The role of ERK in negative selection is more controversial. Experiments performed using transgenic mice expressing a DN form of Ras or MEK reported normal negative selection using a superantigen-mediated deletion model or the HY TCR transgenic model 5, 36, 37.

Bioluminescence images were acquired with a 7-cm FOV, medium binning factor and exposure time of 10–30 s. Quantitative analysis was performed by measuring the luminescence signal intensity per well using the ROI settings of the living image 3.0 software. ROI measurements are expressed selleck inhibitor in total flux of photons. Per cent inhibition was calculated by the following formula; 1 – (average bioluminescence in immune plasma sample/average bioluminescence in naive plasma sample)* 100%. In all experiments and assays, comparisons between two groups were performed by a Mann–Whitney U-test

using prism software version 5.0 (Graphpad, San Diego, CA, USA). P < 0.05 is considered statistically significant. Overall comparisons over three groups or more was performed by Kruskal–Wallis test. Calculations of sample sizes were performed (power 0.85; α = 0.05) by estimation of differences between IV and ID groups. To compare protective efficacy conferred by ID or IV immunization, mice immunized by either RAS or CPS protocols were challenged by infectious mosquito bites. Irrespective of the immunization protocol, ID immunization induced lower protection in BALB/cByJ (50%) and C57BL/6J (7–13%) mice as compared to 90–100% protection after IV immunization

(Table 1). Development of blood-stage parasites in unprotected ID immunized mice showed no significant delay compared to control mice. To evaluate whether infection by IV or ID routes resulted in different magnitude of Cediranib (AZD2171) liver infection, we measured in vivo parasite liver see more loads in C57BL/6 mice by real-time imaging after IV or ID injection of identical doses of fresh PbGFP-Luccon sporozoites. Mice that received IV injection showed a clear bioluminescent signal originating from the site of the liver as from 30 h post-infection

onwards. This signal subsequently further increased covering the whole liver area at 44 h post-infection (Figure 1a). In contrast, ID injection did not result in a bioluminescent signal distinct from background at 30 and 35 h post–infection, while a weak signal was visible at 44 h. After ID injection, mice showed approximately a 30-fold lower parasite liver load (P < 0.0001) compared to IV injected mice (Figure 1b). These data show a strong association [P < 0.001 (χ2 = 49.08, (d.f. = 1)] between the number of parasites reaching the liver in this experiment and the level of protection conferred by different routes of sporozoite administration as shown in preceding immunization experiments. We next assessed cellular immune responses after IV or ID immunization of C57BL/6j mice. Following RAS or CPS IV immunization, proportions of CD8+ T cells with effector memory phenotype (Tem) were significantly increased in both liver (P = 0.008) and spleen (P = 0.008). With the exception of one CPS mouse, this expansion of CD8+ Tem cells was not observed in any of the ID immunized mice, remaining at baseline levels similar to naïve mice (Figure 2a).

4C). Antibodies recognizing pS73 c-Jun were not sensitive enough to detect binding to the TNF proximal promoter/TSS in quiescent polarized T cells (Fig. 4C). No binding of NFATc2 or c-Jun was detected at the proximal promoter of the LTα gene (−148 −44); therefore, we considered

the corresponding amplicon PI3K inhibitor as a negative control (Fig. 4B and C). Overall, the level of c-Jun binding better correlated with the open conformation of TNF TSS than the level of NFATc2 binding. To investigate further the possible role of the TCR-activated transcription factors in the regulation of chromatin conformation at the TNF TSS, we performed Western blot analysis of the nuclear fractions from quiescent and activated T cells. In accordance with earlier reports [25-27, 49, 51], we detected an increase in NFATc2 concentration, including its active dephosphorylated form (lower band of approximately 130 kDa), in the nucleus already 15 min after activation of cells with anti-CD3 and anti-CD28 antibodies, while phosphorylation

of c-Jun (pSer63 and pSer73) became prominent only 1 h after stimulation and increased further at 3 h (Fig. 5). Such kinetics correlated with binding of NFATc2 and c-Jun with the TNF proximal promoter/TSS (Fig. 4B and C). Extended analysis of nuclear concentrations buy RAD001 of AP-1, NFAT, and NF-κB family members (Supporting Information, Results and Fig. 5) demonstrated that both NFATc2 and c-Jun transcription factors are required for chromatin remodeling at the TNF

TSS in T cells upon activation. We next compared chromatin status of the TNF TSS and the nuclear concentrations of NFATc2 and c-Jun transcription factors in mouse CD4+ T-cell subsets (Fig. 6A). In quiescent polarized T cells, we observed higher levels of expression and phosphoryl-ation of transcription factor c-Jun in Th1 and Adenosine Th17 cells regardless of the polarization method (either with soluble or immobilized anti-CD3 antibodies), while NFATc2 in quiescent polarized T cells remained at comparable levels except Th17 cells, where it was higher (Fig. 6A). We also detected similar or comparable levels of RelA/p65 and c-Rel transcription factors in the nuclei of quiescent polarized T cells (Fig. 6A), while c-Fos member of AP-1 family was not detected (data not shown). The level of JunB transcription factor was higher in Th2 and Th17 cells polarized in the presence of soluble anti-CD3 antibodies (Fig. 6A). Importantly, c-Jun appeared to be critical for the maintenance of open chromatin conformation at the TNF TSS in quiescent T cells polarized under Th1 and Th17 conditions. Incubation of these cells with c-Jun N-terminal kinase (JNK) inhibitor SP600125, blocking c-Jun phosphorylation (Supporting Information Fig. 5C), but not with cyclosporine A (CsA), blocking NFATc2 migration to the nucleus (Supporting Information Fig. 5C), facilitated the restoration of closed chromatin configuration at the TNF TSS (Fig. 6B and Supporting Information Fig. 6).

The data showing induction of sustained and predominantly polyfunctional T-cell responses agree with results from two studies of MVA85A-induced immunity in adults from the site in South Africa 25, and from the UK 32. Although BCG vaccination alone induces polyfunctional T cells, specific T cells expressing only IFN-γ are the most common T-cell subset, both in infants 33 and in adults 20. The reason for the more polyfunctional response after in vitro Ag85A peptide pool stimulation, compared with viable BCG,

is most likely related to differential signalling between Ag presenting cells and www.selleckchem.com/products/r428.html T cells. BCG is taken up by innate cells, such as monocytes and dendritic cells, which are known to become activated and secrete inflammatory cytokines, whereas no innate response to peptides is expected. This is supported by our previous observation that more polyfunctional T-cell responses are detected after PPD stimulation of whole blood from healthy, mycobacteria-exposed persons, LY294002 price compared with BCG stimulation 20. We hypothesize that the polyfunctional T-cell population may be the best predictor of vaccine efficacy, because polyfunctional T cells, and not T cells expressing IFN-γ alone, have been associated with protection against another intracellular infection, Leishmania major, in mice 13. As mentioned above, recent animal data from novel TB vaccination

studies also suggest that polyfunctional T-cell responses may correlate with protection against TB 14. Whether this is also true for humans is not known. PPD-specific T-cell responses in TB patients were recently shown to be more polyfunctional than responses from healthy, household TB contacts 34. Until the efficacy of novel TB vaccines are assessed in large phase III clinical trials we have to rely on surrogates,

such as vaccine take or immunogenicity, to assess these vaccines 35. Ag85A-specific CD8+ T cells were not detected after MVA85A vaccination. This contrasts with results from a Gambian 24 and a UK 23 MVA85A trial, in which the Ag85A-specific CD8+ T-cell response was boosted. In the latter trial, a dose of 1×108 plaque forming units (pfu) of MVA85A was administered to BCG-vaccinated participants, which is double the standard dose (5×107 pfu) used in DNA ligase this and in other trials until recently 25, 36. Further, in another study, low frequencies of Ag85A-specific CD8+ T cells were only detected after in vitro expansion of specific T cells in persons vaccinated with 5×107 pfu of MVA85A 37. These results suggest that a higher dose of MVA85A may lead to more readily detectable CD8+ T-cell boosting. Increased CD4+ and CD8+ T-cell responses have also been described with increasing doses of MVA using Ag other than Ag85A 38–40. Vaccination with non-recombinant MVA of humans elicited detectable virus-specific CD8+ T-cell responses, even when a low dose of 1×106 pfu was used 41.