Samples were run at 37°C on the BD™ LSR cytometer (BD Biosciences), and changes in FL5-H/FL4-H ratio were recorded for a total of 512 s (the basal line was recorded for 30 s before the cross-linking Ab was added). Isotype-matched mAb MOPC-21 was used in the assay as a negative control. Data analysis was done using the FlowJo software (Three Star). To analyze the respiratory burst kinetics, production of O was assayed by detection of reduced cytochrome c by freshly isolated monocytes as previously described 40. Briefly, cells were resuspended

in HBSS buffer supplemented with 10% FBS, 0.5 mM Ca2+ and 1 mg/mL glucose and plated over the coated mAb BMS-777607 in vivo at 1.5×105/100 μL in 96-wells plate. After 15 min of incubation at 37°C in 5% CO2 atmosphere, 80 μM cytochrome c (Sigma Aldrich) was added and the plate was kept at 37°C in VersaMax™ microplate reader (Molecular Devices,

Sunnyvale, CA, USA). Absorbance was measured at 550 and 468 nm during 3 h in 10-min intervals. Supernatants of cells (1×106/mL) stimulated either with plate-coated mAb, AZD1208 research buy ultra pure E. coli LPS or recombinant human M-CSF (rhM-CSF, ImmunoTools GmbH) for 24 h were collected and frozen at −20°C until required. Supernatants were analyzed by ELISA for IL-6, IL-8/CXCL8, IL-10, TNF-α (all from ImmunoTools GmbH) and IL-12p70 (eBioscience, San Diego, CA, USA) according to the manufacturer’s instructions. Freshly isolated cells were stimulated Liothyronine Sodium with plate-coated mAb or medium alone in a 24- or 48-well plate (Corning, Corning, NY, USA). Ultra pure E. coli LPS at 100 ng/mL or rhM-CSF (ImmunoTools GmbH) at 10 ng/mL were used as positive controls. After 24 (mDC) or 48 h (monocytes) of incubation, cells were harvested and apoptotic cells were detected by labeling with Annexin-V-FLUOS (Roche Applied Sciences, Penzberg, Germany) followed by flow cytometry analysis. mDC were visualized using an inverted Leica SP2 Confocal

microscope (Leica Microsystems, Wetzlar, Germany) under the 63×/1.32 oil Ph3 CS objective; final total magnification ×200. CbT were obtained from umbilical cord blood samples supplied by Cord Bank of Barcelona, according to guidelines approved by Ethical Committee with donor consent. Cord blood mononuclear cells were separated by Ficoll-Paque PLUS centrifugation (GE Healthcare Bio-Sciences AB) and CbT cells were purified by negative selection using the RosetteSep™ human T-cell enrichment cocktail (StemCell Technologies) that contained anti-CD16, anti-CD19, anti-CD36, anti-CD56, anti-CD66b and anti-glycophorin A mAb. Purity of the cell preparation was assessed by FACS using CD3 and CD45RA markers. In each experiment, >80% of the cells were CD3+CD45RA+. CFSE labeling of CbT cells was performed as previously described 41.

Transfer experiments using OT-II transgenic T cells, which are specific for an ovalbumin peptide, revealed that T cells that had undergone multiple rounds of cell division up-regulated S1P1 and down-regulated CCR7, and cells that had undergone a high number of divisions were more frequently found in the circulation.[24] Presumably, this would allow effector cells to exit the lymph node and scan the periphery

for antigen. Similarly, transgenic mice over-expressing S1P1 in T cells had increased T cells in blood, had elevated IgE before and after immunization, and selleck compound exhibited aberrant activation profiles in delayed-type hypersensitivity responses, including decreased cell recruitment to the site of inflammation and lower surface CD69 expression by lymph node T cells.[29] These studies suggest that proper cell activation is a function of cell localization, and a model constructed from balancing lymph node retention Autophagy inhibitor cell line versus escape mechanisms demonstrates that these signals dictate lymphocyte dwell time within the lymph node, potentially

affecting the generation of the adaptive immune response.[30, 31] Sphingosine-1-phosphate receptor 1 is coupled to Gαi, and is therefore pertussis-toxin-sensitive. Signals from S1P1 are transduced via multiple downstream pathways, including mitogen-activated protein kinase, phospholipase C, phosphoinositide 3 kinase/Akt and adenylyl cyclase.[32] Activation of these different signalling cascades

is known to result in diverse biological outcomes; however, their applicability to T-cell biology is, in some cases, unknown. For instance, Akt-mediated phosphorylation of S1P1 RG7420 solubility dmso is required for Rac activation and chemotaxis in endothelial cells, yet it is unclear if this same mechanism is active within T cells.[33] Phosphoinositide 3 kinase and mammalian target for rapamycin are known to affect T-cell trafficking by regulating Kruppel-like factor 2 (KLF2) expression.[34] KLF2 is a transcription factor that can modulate expression of CD62L (l-selectin), CCR7 and S1P1[35, 36] and may maintain T-cell quiescence, as its loss results in unrestrained expression of inflammatory chemokine receptors.[37] Phosphoinositide 3 kinase and/or mammalian target for rapamycin inhibition resulted in higher expression of KLF2, CD62L, CCR7 and S1P1. Lymph node homing chemokine receptors such as CCR7 and CD62L are expressed on naive T cells and are lost on T effector cells, which home to tissues to fight infection.[30] It is unclear how CCR7 is lost while S1P1 surface expression increases when expression of both factors are controlled by KLF2, although post-translational modifiers and protein–receptor interactions may be involved. It is also possible that transcription of S1P1 or CCR7 can be initiated by other transcription factors, since expression of both receptors is dependent on the T-cell developmental stage as well as phenotype and location.

Therefore, together with other recent studies [31, 32], these observations may help to understand why rapamycin monotherapy is not very effective in preventing graft rejection, and is sometimes even Venetoclax mouse accompanied by inflammatory side effects, including

pneumonitis and glomerulonephritis [36]. The authors would like to thank Dr Gwenny M. Fuhler for advice on immunoblotting. The authors declare no financial or commercial conflicts of interest. “
“In mice, the plasma cell (PC) niche in the bone marrow is close to the haematopoietic stem cell (HSC) niche. We investigated whether PCs can be mobilized into the peripheral blood (PB) in healthy donors receiving granulocyte colony-stimulating factor (G-CSF) for the induction of HSC mobilization into the PB.

G-CSF increased the count of circulating PCs 6-fold, that of circulating B lymphocytes 4-fold and that of circulating HSCs 44-fold. Mobilized circulating PCs comprised CD138− (62·2%) and CD138+ (37·8%) PCs, the latter being more mature based on increased CD27, CD38 and cytoplasmic immunoglobulin selleck inhibitor expression. Mobilized PCs had a phenotype close to that of steady-state PB PCs or in vitro generated PCs, but they expressed L-selectin only weakly. Finally, a median value of 0·4 × 106/kg donor PCs – one-thirtieth of the overall PC count in a healthy adult – was grafted into patients, which could contribute to immune memory recovery. After they have been generated in the lymph nodes, plasmablasts exit into the lymphatic system. They flow out into the peripheral blood (PB) via the thoracic duct and have to find a niche in the bone marrow (BM), spleen, Farnesyltransferase mucosa-associated lymphoid tissues (MALTs) or lymph nodes.1 In these niches, plasmablasts further differentiate into mature plasma cells (PCs) and may survive for decades.2 Long-term surviving PCs are responsible for the long-term humoral immune memory. Consistent with this, treatment with anti-CD20 monoclonal

antibodies (mAbs), which completely delete B cells, did not affect the levels of circulating immunoglobulins.3 The rarity of the niche supporting the long-term survival of PCs is a key factor of the regulation of humoral responses. In fact, newly generated plasmablasts have to compete with already established long-lived PCs to gain access to these rare niches.4 In mice, the PC niche has been shown to be similar to the haematopoietic stem cell (HSC) and pre-pro B-cell niche. Insertion of the green fluorescent protein (GFP) gene into the stromal cell-derived factor-1 [SDF-1 or chemokine (C-X-C motif) ligand 12 (CXCL 12)] gene made it possible to show that all murine BM PCs as well as HSCs and pre-pro B cells adhere to SDF-1+ vascular cell adhesion molecule (VCAM1)+ cells, which represent 1% of BM cells.

Pharmacological inhibition of both NK1R and NK3R significantly affected the downstream SP signaling. We further examined whether there was any crosstalk between the two pathways upon SP stimulation. Alpelisib Inhibition of ERK1/2 decreased levels of p-MLC20 after SP activation, in a PKC dependent manner, indicating a potential crosstalk between these two pathways. Conclusions:  These data provide the first evidence that SP-mediated

crosstalk between pro-inflammatory and contractile signaling mechanisms exists in the lymphatic system and may be an important bridge between lymphatic function modulation and inflammation. “
“Microvascular and macrovascular endothelial function maintains vascular reactivity. Several diseases alter endothelial function, including hypertension, obesity, and diabetes mellitus. In addition, micro- and macrovascular endothelial Erlotinib datasheet dysfunction is documented in GDM with serious consequences for the

growing fetus. Increased l-arginine uptake via hCAT-1 and NO synthesis by eNOS is associated with GDM. These alterations are paralleled by activation of purinergic receptors and increased umbilical vein, but not arteries blood adenosine accumulation. GDM associates with NO-reduced adenosine uptake in placental endothelium, suggested to maintain and/or facilitate insulin vasodilation likely increasing hCAT-1 and eNOS expression and activity. It is proposed that increased umbilical vein blood adenosine concentration in GDM reflects a defective metabolic state of human

placenta. In addition, insulin recovers GDM-alterations in hCAT-1 and eNOS in human micro- and macrovascular endothelium, and its biological actions depend on preferential activation of insulin receptors A and B restoring a normal-like from a GDM-like phenotype. We summarized existing evidence for a potential role of insulin/adenosine/micro- and macrovascular endothelial dysfunction in GDM. These mechanisms could be crucial for a better management of the mother, fetus and newborn in GDM pregnancies. Vascular reactivity depends on several mechanisms including locally released vasoactive molecules from the endothelium [9, 16, 29]. A number of diseases are associated with dipyridamole reduced ability of this cell type to synthesize the potent local vasodilator NO [53]. In addition, a potential link between the bioavailability of the cationic amino acid l-arginine, the substrate for NO synthesis and the eNOS has been reported in human endothelium [66]. Expression and activity of l-arginine membrane transporters are phenomena playing crucial roles in the synthesis of NO in the micro- and macrovascular endothelium in diseases associated with vascular dysfunction [39, 48, 81]. Thus, unveiling the mechanisms behind abnormal regulation of endothelial l-arginine transport and NO synthesis (i.e., the endothelial l-arginine/NO signaling pathway) in the micro- and macrovasculature in adulthood diseases (e.g.

Future studies to investigate LPS-induced CGRP synthesis in monocytes/macrophages of RAMP1 over-expressing

transgenic mice20 and knockout mice37 should verify this hypothesis. In the present study, we have used exogenous CGRP, peptide CGRP receptor antagonist CGRP8-37 and non-peptide CGRP receptor antagonist BIBN4096BS, Talazoparib in vitro to establish the possible role of CGRP receptor signalling in basal and LPS-induced pro-inflammatory and anti-inflammatory chemokines and cytokines in the RAW 264.7 macrophage cell line. The affinities of αCGRP, CGRP8-37 and BIBN4096BS to bind human CGRP receptors have been well established, with the affinities BIBN4096BS (Ki = 14·4 ± 6·3 pm) > αCGRP (Ki = 31·7 ± 1·6 pm) > CGRP8-37 (Ki = 3·6 ± 0·7 nm), respectively.25 Hence, the physiological concentrations for Tamoxifen molecular weight both CGRP and BIBN4096BS are within nm range25 whereas for CGRP8-37, it is within the μm range.38 We used the physiological range of concentrations of the antagonists in the current study. The mechanisms underlying the blocking activities of both antagonists on CGRP receptors are rather different. Since CGRP8-37 peptide includes all but the first seven amino acids at the C-terminal

of CGRP, it works as a competitive antagonist to block the binding of full-length CGRP to its receptor. In contrast, the specific affinities of BIBN4096BS depend on its interaction with the RAMP1 subunit of CGRP receptor.39 From the literature, the role of CGRP in the induction of pro-inflammatory and anti-inflammatory chemokines and cytokines is controversial.21–23 In these studies, depending on the cell type and concentration, CGRP exhibits either stimulating or suppressing effect on the production of MCP-1, IL-1β, TNFα, IL-6 and IL-10. Consistently, CGRP receptor signalling in the current study also demonstrates positive or negative effects on basal and LPS-induced release of these inflammatory mediators depending on the concentration of CGRP and CGRP receptor antagonists. Generally speaking, a lower concentration of CGRP seems to facilitate the basal Axenfeld syndrome release of MCP-1, TNFα and IL-6 but had no effect on the basal release of IL-1β and IL-10. The facilitating effects were

blocked by a lower concentration of CGRP8-37 (10 nm), suggesting that CGRP receptor mediates the effect. In contrast, a higher concentration of CGRP suppressed basal TNFα release but had no effect on others. Contrary to the effect of CGRP, a higher concentration of the peptide antagonist CGRP8-37 significantly increased the basal release of all chemokines and cytokines examined, but the lower concentration had no effect at all. Non-peptide antagonist BIBN4096BS also manifested the same tendency. However, at higher concentration, it only significantly increased the basal release of MCP-1, IL-6 and IL-10 but had no effect on IL-1β and TNFα. Similar to CGRP8-37, a lower concentration of BIBN4096BS had no effect on the basal release of chemokines and cytokines.

2A and B). IL-1β levels were actually downregulated from 8.5±1.4 to 3.2±1.2 pg/mL (Fig. 2A, p<0.005). A slight increase in IL-6 levels was seen after 6 h, with a return to baseline levels by 24 h (Fig. 2B). We were further interested to know whether complement-dependent interaction between MAPK inhibitor apoptotic cells and macrophages leads to secretion

of TGF-β or IL-10. However, although we used a sensitive kit capable of detecting levels as low as 0–3.4 pg/mL, no increase was detected. TGF-β levels never exceeded baseline levels (six experiments, Fig. 2C). On the other hand, modest IL-10 secretion was clearly documented following 1 h of interaction with apoptotic cells (p<0.001, Fig. 2C), reaching an average of 30 pg/mL. Taken together, these findings suggest that apoptotic

cells interacting with monocyte-derived macrophages in a complement-dependent mechanism do not trigger a proinflammatory response, downregulate the basal level of IL-1β secretion, and induce IL-10 but not TGF-β secretion. As a model for proinflammatory activation of monocyte-derived macrophages, we used non-opsonic phagocytosis of zymosan CP-673451 and LPS. TLR and the downstream signaling pathway play a key role in innate immune recognition and activation in this model 16, but other receptors such as CD11b/CD18 17, Dectin-1 18, and mannose receptor 19, 20 have also been suggested to be involved in non-opsonic zymosan recognition and signaling. As shown in Fig. 3A and B, we documented a proinflammatory response following 1 h exposure of monocyte-derived human macrophages

to non-opsonized zymosan. IL-1β (Fig. 3A) was detected already at 6 h, and reached 40–300 pg/mL at 24 h (15 experiments, p<0.001). Variability was mainly dependent on the number of macrophages, ranging between 100 and 180 pg/mL in most experiments, indicating an average 15-fold increase in 24 h (p<0.001). There was an even more dramatic increase in IL-6 secretion following exposure to zymosan find more (Fig. 3B), reaching a 100–200 fold increase. IL-10 secretion followed, with a lag in IL-1β and IL-6 increases to 1000–5000 pg/mL, always in proportion to proinflammatory cytokine secretion (p<0.001). When we documented higher levels of IL-1β, higher levels of IL-10 followed (Fig. 3C). Taken together, these findings suggest that non-opsonic zymosan induced a proinflammatory macrophage response, manifested by IL-1β and IL-6 secretion followed by IL-10 secretion. Similar results were obtained upon exposure to LPS (see below). When monocyte-derived human macrophages were exposed for only 1 h to apoptotic thymocytes, and then washed and exposed for 24 h to zymosan, a marked inhibition of the proinflammatory response to zymosan was seen. As shown in Fig.

26 Let-7g was slightly, but not significantly, increased after LIF stimulation, which is in contrast to previous descriptions on let-7g in cancer. In hepatocellular carcinoma, ectopic expression of let-7g inhibits cell migration and growth.27 In gastric cancer, low let-7g is associated with unfavorable outcome in overall survival independent of clinical covariates, including depth of invasion, lymph-node metastasis, and stage.28 LIF-stimulated buy SCH772984 JEG-3 cells expressed significantly higher levels of miR-93, which

is in line with previous observations on tumors. In human glioblastoma, miR-93 suppresses integrin-β8 expression, which promotes tumor growth and angiogenesis.29 In human T-cell leukemia virus 1, miR-93 targets the mRNA for tumor protein 53–induced nuclear protein 1 (TP53INP1), which is a tumor suppressor protein.30 In our experiments, miR-9 did not change considerably. In human embryonic stem cell-derived neural progenitors, loss of miRNA-9 reduces proliferation and increases migration.31 On the

other hand, miR-9 targets E-cadherin, RXDX-106 chemical structure which is a suppressor of metastasization and angiogenesis. Its high expression in breast cancer is correlated with the malign properties.32 In JEG-3 cells, LIF significantly downregulated miR-141. Repression of miR-141 induces invasiveness of breast cancer cells by targeting the endothelial mesenchymal transition activators ZEB1 and ZEB2, which downregulate E-cadherin expression.18 Also in colorectal cancer, miR-141 negatively correlates with migration and invasion.9 A different function has been observed for miR-141 in gastric cancer cells, where its over-expression by the application of its precursors Thiamet G inhibited the proliferation.33 In contrast, it is upregulated in nasopharyngeal carcinoma, where it positively correlates with proliferation, migration, and invasion.34 In our hands, silencing of miR-141 inhibits proliferation of JEG-3 choriocarcinoma cells, which goes in line

with these results. The observed strong impact of LIF on various miRNA in JEG-3 choriocarcinoma cells underlines the expected involvement of miRNAs in the regulation of essential functions in trophoblastic cells and thus in tuning placentation and other crucial processes in reproduction and pregnancy. The project has been supported by the German Research Foundation (DFG, project Ma1550/7-1). DMMP has a Ph.D. grant from the regional graduate academy of the Friedrich-Schiller-University Jena, Germany. “
“Type 1 diabetes is an autoimmune disease whose clinical onset signifies a lifelong requirement for insulin therapy and increased risk of medical complications.

05; Fig. 5). Collectively, there were fewer Th2-promoting cytokine cells (IL-4) than Th1-promoting cytokine cells (IFN-γ). In our previous

study, we developed surface-displayed ApxIIA#5 expressed on S. cerevisiae and full ApxIIA-expressing S. cerevisiae and demonstrated that oral immunization of mice induced antigen-specific immune responses and protection against A. pleuropneumoniae [3, 9]. However, to develop an efficient oral vaccine, further study of the mucosal immune responses induced by transgenic S. cerevisiae was needed. We selected surface-displayed ApxIIA#5 expressed on S. cerevisiae as an oral vaccine for porcine pleuropneumonia. In mice, it has greater specific antibody activities X-396 molecular weight than other yeasts, including ApxIIA#5-secreting S. cerevisiae and full-ApxIIA expressing S. cerevisiae [20]. As APCs, DCs induce primary immune responses and have a key role in both innate and adaptive immunity [21]. In adaptive immune responses, the phenotype and function of DCs determine the initiation of tolerance, memory and polarized Th1 and Th2 differentiation [21]. Stimulation of bone marrow-derived DCs with surface-displayed ApxIIA#5

expressed on S. cerevisiae in vitro indicated that this could generally induce secretion Nivolumab mouse of the proinflammatory cytokines TNF-α and IL-1β, the Th1-inducing cytokine IL-12p70 and the Th2-inducing cytokine IL-10. Moreover, maturation of the APCs was confirmed by showing upregulation of CD40 and CD86 costimulatory molecules and surface MHC class II, all of which are required

for efficient stimulation of T cells [22]. Mucosal protection requires generation of antigen-specific T cells and antibodies [23]. In addition, following ablation of immune responses after oral and nasal immunization of mice depleted of cDCs in vivo, cDCs are reportedly essential for activation of CD4+ T cells and generation of specific antibodies [23]. In the present study, we demonstrated that surface-displayed ApxIIA#5 expressed on S. cerevisiae helped to improve both systemic and mucosal immune responses in mice by generating antigen-specific antibodies and encouraging proliferation of CD4+ T cells, which were stimulated by DCs activated by oral vaccination. Presentation of ApxIIA on activated DCs to CD4+ T cells from mice in the Cediranib (AZD2171) vaccinated group elicited specific T-cell proliferation. The induction of ApxIIA-specific T-cell proliferation demonstrated that ApxIIA was indeed presented on DCs and that the orally administered surface-displayed ApxIIA#5 expressed on S. cerevisiae induced cellular immune responses in mice. Both serum Ag-specific IgG and Ag-specific IgA antibody activities increased in the vaccinated group. Furthermore, both Apx-specific IgG and IgA antibody-producing cells in the PP, LP and SP were significantly more numerous in the vaccinated group than in the control group.

Conclusion:  This registry analysis suggests that IL-2Ra induction may be associated with

a reduction HIF inhibitor in rejection risk in cyclosporine-treated intermediate immunological risk recipients, but not in low-risk renal transplant recipients. Renal allograft outcomes have been improving over the last 10 years, perhaps related to improved immunosuppression and reduced acute rejection rates.1 Acute rejection, an important determinant of graft survival, occurs commonly in the early post-transplant period, but the incidence has decreased significantly over recent years.2 Antibodies designed to inactivate interleukin-2 receptor antibody (IL-2Ra) on T cells such as basiliximab are often used as induction therapy in immunosuppressive protocols to reduce the risk of acute rejection or to delay the introduction of calcineurin inhibitor (CNI) in those at high risk of delayed graft function.3,4 The effectiveness of IL-2Ra in reducing the risk of acute rejection is well established in deceased- and live-donor kidney transplantation.5,6 Unlike T-cell depletive therapies, IL-2Ra

is not associated with increased infection- or cancer-related morbidity and mortality.7–9 The use of IL-2Ra has been steadily increasing in Australia such that IL-2Ra induction therapy was used for >50% of new renal transplant recipients in Australia by 2005.10,11 Although the efficacy of IL-2Ra in reducing the risk of rejection is well established in renal transplant recipients, the effectiveness of this agent in renal transplant Hydroxychloroquine manufacturer recipients with differing immunological risk remains unclear.10,12,13 The aim of the present

study is to evaluate the efficacy of IL-2Ra induction on allograft outcomes including acute rejection, glomerular filtration rate (GFR), graft and patient Immune system survival in renal transplant recipients of low and intermediate immunological risk, and when stratified by initial immunosuppression. Using the Australia and New Zealand Dialysis and Transplant (ANZDATA) Registry, all live- and deceased-donor renal transplant recipients in Australia from 1995 to 2005 were included in this study. Follow up was censored at 31 December 2006. Recipients were arbitrarily divided into low immunological risk (primary grafts with ≤2 human leucocyte antigen (HLA)-mismatches and panel-reactive antibody (PRA) < 10%) or intermediate immunological risk recipients (i.e. subsequent grafts or >2 HLA-mismatches or PRA > 25%). Multiple-organ graft recipients, recipients’ age less than 16 at time of transplant and recipients initiated on corticosteroids or CNI-free immunosuppressive regimens were excluded from the study. In addition, recipients who had received induction monoclonal or polyclonal T-cell depletive agents were also excluded.

An enhanced skin test response to PPD after TNF-α treatment was associated with a reduction

3-deazaneplanocin A cost in the BCG bacillary loads in the lymph nodes when compared to the BSA-injected guinea pigs (Fig. 1b). In the present study, no viable M. bovis BCG were detected in the spleen of either TNF-α- and BSA-injected guinea pigs 6 weeks after M. bovis BCG infection. This can be explained on the basis of studies by others that a maximum level of viable BCG organisms in spleen was seen 20 days post-vaccination, after which there was a significant decrease in the bacilli in spleen [39]. It is known that in vivo injection of TNF-α increases the resistance of mice to virulent M. tuberculosis or M. avium complex, as it resulted in decreased bacteria in the tissues [16,31]. Conversely, treatment with anti-TNF-α antibody enhanced the susceptibility of mice to tuberculosis [2,13]. In M. marinum-infected zebra fish, loss of TNF-α signalling accelerated bacterial growth and caused increased

mortality, although TNF-α was not required for tuberculous granuloma formation [40]. In vitro studies from our laboratory also support our findings, as rgpTNF-α and rgpIFN-γ, alone or in combination, inhibited the intracellular growth of M. tuberculosis in guinea pig macrophages in vitro[25]. Conversely, alveolar and peritoneal macrophages from Navitoclax cost BCG-vaccinated guinea pigs treated with anti-gpTNF-α antibody in vitro showed increased mycobacterial growth [20]. Furthermore, we reported that injection of anti-TNF antibody into BCG-vaccinated and non-vaccinated guinea pigs

following aerosol challenge with virulent M. tuberculosis resulted in splenomegaly Bay 11-7085 and presence of plasma cells in the granulomas in the BCG-vaccinated guinea pigs, while splenic granulomas were more organized in the non-vaccinated guinea pigs [24]. Thus, anti-TNF-α seems to have a differential effect after M. tuberculosis infection, as large amounts of TNF-α and greater number of bacillary loads occur in non-vaccinated guinea pigs versus lower levels of TNF-α and reduced numbers of bacilli in the vaccinated animals [26,41,42]. In the tuberculous pleurisy model, no necrosis was evident after the anti-TNF-α treatment, while the treatment altered the cellular composition of the pleural effusion, as well as increasing the cell-associated mycobacterial loads in the granulomas [23]. In order to determine whether TNF-α treatment also altered the cytokine mRNA expression after BCG vaccination, lymph node and spleen cells were stimulated in vitro with PPD. TNF-α treatment enhanced the IL-12p40 mRNA expression in both lymph node and spleen cells upon antigen restimulation (Fig. 4a). These results are in agreement with previous reports as well as our in vitro experiments in which rgpTNF-α enhanced both IL-12p40 and IFN-γ mRNA expression [20,21].