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There have been unprecedented advances in the identification of new treatment targets for chronic hepatitis B that are being developed with the goal of achieving functional cure in patients who would otherwise require lifelong nucleoside analogue treatment. Many of the new investigational therapies either directly target the immune system or are anticipated to impact immunity indirectly through modulation of the viral lifecycle and antigen production. While new viral biomarkers (HBV RNA, HBcAg, small, middle, large HBs isoforms) are proceeding through validation steps in clinical studies, immunological biomarkers are non-existent outside of clinical assays for antibodies to HBs, HBc and HBe. To develop clinically applicable immunological biomarkers to measure mechanisms of action, inform logical combination strategies, and guide clinical management for use and discontinuation of immune-targeting drugs, immune assays must be incorporated into phase I/II clinical trials. This paper will discuss the importance of sample collection, the assays available for immunological analyses, their advantages/disadvantages and suggestions for their implementation in clinical trials. Careful consideration must be given to ensure appropriate immunological studies are included as a primary component of the trial with deeper immunological analysis provided by ancillary studies. Standardising immunological assays and data obtained from clinical trials will identify biomarkers that can be deployed in the clinic, independently of specialised immunology laboratories.

Supplemental Material for A single, oral dose of the TLR7 agonist JNJ-64794964 induces transcriptomic and phenotypic changes in peripheral immune cells in healthy adults by Wim Pierson, Marianne Tuefferd, Florence Herschke, Leen Slaets, Marjolein Crabbe, Dorien Verstappen, Steffi de Pelsmaeker, Ian Strickland, Edward J Gane, Christian Schwabe, Yingjie Zhang, Peter Meerts, Joris Vandenbossche, Pieter Van Remoortere, Inge Verbrugge and An de Creus in Antiviral Therapy

Background This Phase I, two-part, first-in-human study assessed safety/tolerability and pharmacokinetics/pharmacodynamics of single-ascending doses (SAD) and multiple doses (MD) of the oral toll-like receptor-7 agonist, JNJ-64794964 (JNJ-4964) in healthy adults. Methods In the SAD phase, participants received JNJ-4964 0.2 ( N = 6), 0.6 ( N = 6), 1.25 ( N = 8) or 1.8 mg ( N = 6) or placebo ( N = 2/dose cohort) in a fasted state. Food effect was evaluated for the 1.25 mg cohort following ≥6 weeks washout. In the MD phase, participants received JNJ-4964 1.25 mg ( N = 6) or placebo ( N = 2) weekly (fasted) for 4 weeks. Participants were followed-up for 4 weeks. Results No serious adverse events (AEs) occurred. 10/34 (SAD) and 5/8 (MD) participants reported mild-to-moderate (≤Grade 2), transient, reversible AEs possibly related to JNJ-4964. Five (SAD) participants had fever/flu-like AEs, coinciding with interferon-α serum levels ≥100 pg/mL and lymphopenia (9/L), between 24–48 h after dosing and resolving approximately 96 h after dosing. One participant (MD) had an asymptomatic Grade 1 AE of retinal exudates (cotton wool spots) during follow-up, resolving 6 weeks after observation. JNJ-4964 exhibited dose-proportional pharmacokinetics, with rapid absorption (tmax 0.5–0.75 h) and distribution, and a long terminal half-life (150–591 h). Overall, no significant differences in JNJ-4964 pharmacokinetic parameters were observed in the fed versus fasted state. JNJ-4964 dose-dependently and transiently induced cytokines with potential anti-HBV activity, including interferon-α, IP-10, IL-1 RA, and/or MCP-1, and interferon-stimulated genes (ISG15, MX1, and OAS1) in serum. Conclusions In healthy adults, JNJ-4964 was generally well-tolerated, exhibited dose-proportional pharmacokinetics and induced cytokines/ISGs, with possible anti-HBV activity.

Background Chronic hepatitis B (CHB) is responsible for major disease burden worldwide. However, the number of available therapies is limited; cure remains an elusive goal. JNJ-64794964 (JNJ-4964) is an oral toll-like receptor-7 (TLR7) agonist being evaluated for the treatment of CHB. Here, we investigated the capacity of JNJ-4964 to induce transcriptomic and immune cell changes in peripheral blood in healthy volunteers. Methods Peripheral blood was collected in the JNJ-4964 first-in-human phase 1 trial at multiple time points to assess transcriptomics and changes in frequency and phenotype of peripheral-blood mononuclear cells. Correlation of changes to JNJ-4964 exposure (Cmax) and changes in cytokine levels (C-X-C motif chemokine ligand 10 [CXCL10] and interferon alpha [IFN-α]) were evaluated. Results Fifty-nine genes, mainly interferon-stimulated genes, were up-regulated between 6 hours and 5 days after JNJ-4964 administration. JNJ-4964 increased frequencies of CD69, CD134, CD137, and/or CD253-expressing natural killer (NK) cells, indicative of NK cell activation. These changes correlated with Cmax, increase of CXCL10, and induction of IFN-α and were observed at IFN-α levels that are associated with no/acceptable flu-like adverse events. JNJ-4964 administration resulted in increased frequencies of CD86-expressing B cells, indicative of B-cell activation. These changes were predominantly observed at high IFN-α levels, which are associated with flu-like adverse events. Conclusions JNJ-4964 administration led to changes in transcriptional profiles and immune cell activation phenotype, particularly for NK cells and B cells. Together, these changes could represent a set of biomarkers for the characterization of the immune response in CHB patients receiving TLR7 agonists.

Background JNJ-4964 is a TLR7 agonist, which, via a type I interferon (IFN)–dependent mechanism, may enhance host immunity suppressed by persistent exposure to hepatitis B antigens in chronic hepatitis B. Methods PK and PD data were pooled from 2 studies involving 90 participants ( n = 74 JNJ-4964, dose range 0.2–1.8 mg; n = 16 placebo) in a fasted state. Food effects on PK were studied in 24 participants (1.2 or 1.25 mg). A population PK model and PK/PD models were developed to characterize the effect of JNJ-4964 plasma levels on the time course of IFN-α, IFN-γ–inducible protein 10 (IP-10 or CXCL10), IFN-stimulated gene 15 ( ISG15), neopterin and lymphocytes following single and weekly dosing in healthy adults. Covariate effects, circadian rhythms and negative feedback were incorporated in the models. Results A 3-compartment linear PK model with transit absorption adequately described JNJ-4964 PK. Bioavailability was 44.2% in fed state relative to fasted conditions. Indirect response models with maximum effect (Emax) stimulation on production rate constant (kin) described IFN-α, IP-10, ISG15 and neopterin, while a precursor-dependent indirect response model with inhibitory effect described the transient lymphocyte reduction. Emax, EC50 and γ (steepness) estimates varied according to PD markers, with EC50 displaying substantial between-subject variability. Female and Asian race exhibited lower EC50, suggesting higher responsiveness. Conclusions PK/PD models well characterized the time course of immune system markers in healthy adults. Our results supported sex and race as covariates on JNJ-4964 responsiveness, as well as circadian rhythms and negative feedback as homeostatic mechanisms that are relevant in TLR7-induced type I IFN responses.

JNJ-64794964 (JNJ-4964/AL-034/TQ-A3334), an oral toll-like receptor 7 agonist, is being investigated for the treatment of chronic hepatitis B (CHB), a condition with a high unmet medical need. The anti–hepatitis B (HBV) activity of JNJ-4964 was assessed preclinically in an adeno-associated virus vector expressing HBV (AAV/HBV) mouse model. Mice were treated orally with 2, 6 or 20 mg/kg of JNJ-4964 once-per-week for 12 weeks and then followed up for 4 weeks. At 6 mg/kg, a partial decrease in plasma HBV-DNA and plasma hepatitis B surface antigen (HBsAg) was observed, and anti-HBs antibodies and HBsAg-specific T cells were observed in 1/8 animals. At 20 mg/kg, plasma HBV-DNA and HBsAg levels were undetectable for all animals 3 weeks after start of treatment, with no rebound observed 4 weeks after JNJ-4964 treatment was stopped. High anti-HBs antibody levels were observed until 4 weeks after JNJ-4964 treatment was stopped. In parallel, HBsAg-specific immunoglobulin G–producing B cells and interferon-γ–producing CD4+ T cells were detected in the spleen. In 2/4 animals, liver HBV-DNA and HBV-RNA levels and liver hepatitis B core antigen expression dropped 4 weeks after JNJ-4964 treatment-stop. In these animals, HBsAg-specific CD8+ T cells were detectable. Throughout the study, normal levels of alanine aminotransferase were observed, with no hepatocyte cell death (end of treatment and 4 weeks later) and minimal infiltrations of B and T cells into the liver, suggesting induction of cytokine-mediated, non-cytolytic mechanisms.

Supplemental Material, sj-pdf-1-avt-10.1177_13596535211056581 for Safety, tolerability, pharmacokinetics, and pharmacodynamics of oral JNJ-64794964, a TLR-7 agonist, in healthy adults by Edward Gane, Mina Pastagia, Ullrich Schwertschlag, An De Creus, Christian Schwabe, Joris Vandenbossche, Leen Slaets, Bart Fevery, Ilham Smyej, Liviawati S Wu, Rui Li, Samia Siddiqui, Abbie Oey, Clark Musto and Pieter Van Remoortere in Antiviral Therapy

Aim To determine the frequency of expression of the tumor-associated antigens (TAAs) melanoma-associated antigen A3 (MAGE-A3) and preferentially expressed antigen of melanoma (PRAME) and the rate of EGFR mutations in a Taiwanese non–small cell lung cancer (NSCLC) population including only adenocarcinomas and squamous cell carcinomas. Furthermore, to investigate associations between TAA expression and EGFR mutations and to evaluate these TAAs as prognostic markers for overall survival. The occurrence of single nucleotide polymorphisms in MAGEA3 and PRAME was also assessed. Methods Archival fresh-frozen tumor tissue specimens were tested by quantitative reverse transcription polymerase chain reaction assays to detect MAGE-A3 and PRAME expression. EGFR mutations were detected by mass spectroscopy and single nucleotide polymorphisms by gene sequencing. Results Of the 156 adenocarcinomas examined, 3.3% expressed MAGE-A3, 32.2% expressed PRAME and 62.8% had EGFR mutations. Of the 128 squamous cell carcinomas, 29.8% expressed MAGE-A3, 59.2% expressed PRAME and 20.5% harbored EGFR mutations. TAA expression was similar across subgroups determined by patient or tumor characteristics. There was no association between TAA expression and EGFR mutation status and TAA expression was found not to be a prognostic marker for survival. Single nucleotide polymorphisms were identified, one of which with a possible impact on MAGE-A3 expression. Conclusions In this NSCLC population, expression of MAGE-A3 and PRAME was more frequent in squamous cell carcinomas than in adenocarcinomas tumors. EGFR mutations were not associated with TAA expression for either histology and were three times more frequent in adenocarcinomas than in squamous cell carcinomas tumors.

This case deals with the decision of Sanac Inc., a Belgian wholesale company, on whether to proceed with the implementation of an activity-based costing (ABC) system or switch to time-driven activity-based costing (TDABC). As a business consultant, you are hired to decide about the appropriate costing method. Your task is to decide which system the company should implement, given the desire of the president of the company to calculate profitability at the order and the customer level.

Peptides of the trefoil factor family (TFF) are expressed along the gastro-intestinal tract. They protect mucous epithelia from damage and contribute to mucosal repair, which is essential for preventing inflammation. Moreover, it has been suggested that TFF2 and TFF3, in particular, play a role in regulating immune responses. Depending on their activation status, dendritic cells (DC) can initiate either tolerance or immunity. This study, by comparing LPS-induced maturation of mTFF3-treated DC and non-treated DC, investigated whether murine TFF3 directly regulated DC function. mTFF3-treated DC and non-treated DC did not differ phenotypically or functionally. Both populations expressed, both before and after LPS-stimulation, similar levels of co-stimulatory molecules and cytokines, and were both efficient stimulators of T-cells. Our results suggest that mTFF3 does not govern immune responses on the level of DC function.

In this study, we propagated myeloid dendritic cells (DC) from BALB/c (H2d) mouse bone marrow progenitors in IL-10 and TGF-β, then stimulated the cells with LPS. These “alternatively activated” (AA) DC expressed lower TLR4 transcripts than LPS-stimulated control DC and were resistant to maturation. They expressed comparatively low levels of surface MHC class II, CD40, CD80, CD86, and programmed death-ligand 2 (B7-DC; CD273), whereas programmed death-ligand 1 (B7-H1; CD274) and inducible costimulatory ligand expression were unaffected. AADC secreted much higher levels of IL-10, but lower levels of IL-12p70 compared with activated control DC. Their poor allogeneic (C57BL/10; B10) T cell stimulatory activity and ability to induce alloantigen-specific, hyporesponsive T cell proliferation was not associated with enhanced T cell apoptosis. Increased IL-10 production was induced in the alloreactive T cell population, wherein CD4+Foxp3+ cells were expanded. The AADC-expanded allogeneic CD4+CD25+ T cells showed enhanced suppressive activity for T cell proliferative responses compared with freshly isolated T regulatory cells. In vivo migration of AADC to secondary lymphoid tissue was not impaired. A single infusion of BALB/c AADC to quiescent B10 recipients induced alloantigen-specific hyporesponsive T cell proliferation and prolonged subsequent heart graft survival. This effect was potentiated markedly by CTLA4-Ig, administered 1 day after the AADC. Transfer of CD4+ T cells from recipients of long-surviving grafts (>100 days) that were infiltrated with CD4+Foxp3+ cells, prolonged the survival of donor-strain hearts in naive recipients. These data enhance insight into the regulatory properties of AADC and demonstrate their therapeutic potential in vascularized organ transplantation.

The pro-drug FTY720 is undergoing phase III clinical trials for prevention of allograft rejection. After phosphorylation, FTY720 targets the G protein-coupled-sphingosine-1-phosphate receptor 1 (S1PR1) on lymphocytes, thereby inhibiting their egress from lymphoid organs and their recirculation to inflammatory sites. Potential effects on dendritic cell (DC) trafficking have not been evaluated. Here, we demonstrate the expression of all five S1PR subtypes (S1PR1-5) by murine DCs. Administration of FTY720 to C57BL/10 mice markedly reduced circulating T and B lymphocytes within 24 h, but not blood-borne DCs, which were enhanced significantly for up to 96 h, while DCs in lymph nodes and spleen were reduced. Numbers of adoptively transferred, fluorochrome-labeled syngeneic or allogeneic DCs in blood were increased significantly in FTY720-treated animals, while donor-derived DCs and allostimulatory activity for host naïve T cells within the spleen were reduced. Administration of the selective S1PR1 agonist SEW2871 significantly enhanced circulating DC numbers. Flow analysis revealed that CD11b, CD31/PECAM-1, CD54/ICAM-1 and CCR7 expression on blood-borne DCs was downregulated following FTY720 administration. Transendothelial migration of FTY720-P-treated immature DCs to the CCR7 ligand CCL19 was reduced. These novel data suggest that modulation of DC trafficking by FTY720 may contribute to its immunosuppressive effects.

Dendritic cell (DC) precursors were propagated from C57BL/10 (B10; H2b) mouse bone marrow in fms-like tyrosine kinase 3 ligand. Cosignaling molecule (B7-1/B7-2 and B7-H1) expression and stimulatory capacity of precursor (pre)-plasmacytoid (p)DC (CD11c+B220+CD11b-CD19-) and classic myeloid DC (MDC) for allogeneic (C3H; H2k) T cells were compared. Unstimulated pre-pDC exhibited very low levels of surface MHC class II and classic costimulatory molecules (B7-1/B7-2), whereas a minor population expressed B7-H1 at levels higher than on MDC. The pre-pDC were ineffective T-cell stimulators and induced nonspecific hyporesponsiveness to rechallenge with donor alloantigens in vitro and in vivo. Following stimulation with CpG-oligonucleotide (CpG-ODN), B7 molecule expression was upregulated on pre-pDC, however the ratio between coinhibitory (B7-H1) and costimulatory (B7-1/B7-2) signals was much higher (five- to six-fold) on pre-pDC than MDC. Blockade of B7-H1 expression on pDC increased their T-cell allostimulatory capacity significantly. A single preoperative infusion of C3H hosts with pre-pDC prolonged B10 heart graft survival significantly but nonspecifically compared with untreated mice (median survival times 22 vs. 9 days, respectively). Thus, pre-pDC of donor origin have potential to regulate T-cell responses to alloantigens and can prolong organ graft survival.

Fms-like tyrosine kinase 3 ligand (Flt3L) administration leads to dramatic increases in dendritic cells (DC) in lymphoid and nonlymphoid tissues. Conversely, mice lacking Flt3L (Flt3L−/−) show severe reductions in both myeloid (CD11c+CD8α−) and lymphoid-related DC (CD11c+CD8α+) in the thymus and secondary lymphoid organs. In this study marked reductions in CD11c+ interstitial cardiac DC and in dermal, but not epidermal, DC (Langerhans cells) were also observed. CD11c+ cells that migrated from Flt3L−/− skin explants expressed lower surface MHC class II and costimulatory molecules and naive T cell allostimulatory activity than migratory wild-type (wt) C57BL/6 (B6) CD11c+ cells. We examined the survival of Flt3L−/− heart or tail skin grafts (H2b) in allogeneic wt (BALB/c; H2d) recipients. The outcome of transplantation of BALB/c organs into Flt3L−/− recipients was also determined. Flt3L−/− mice rejected BALB/c heart or skin grafts with similar kinetics as B6 wt recipients. Trafficking of donor DC into host spleens or draining lymph nodes was markedly reduced after transplantation of Flt3L−/− heart, but not skin grafts, respectively. Compared with wt hearts, survival of Flt3L−/− hearts was markedly prolonged in BALB/c recipients (median survival time, 37 and 15 days, respectively; p < 0.001). Skin graft survival was unaffected. Rejection of Flt3L−/− hearts was precipitated by infusion of wt donor DC at the time of transplant. Thus, severe depletion of interstitial heart DC resulting from targeted gene disruption prolongs, but does not indefinitely extend, heart survival. Acute rejection of wt grafts in Flt3L−/− recipients reflects presumably an intact role of the direct pathway of allorecognition.

Possible mechanisms by which liver antigen presenting cells (APC) may facilitate tolerance induction are discussed. Tolerance may be facilitated by a distinctive combination of factors, linked to the unique anatomical and microenvironmental features of the liver.

In this study, the role of IL-15 and its regulation by the transcription factor IFN regulatory factor-1 (IRF-1) in murine V gamma 3 T cell development and activity is assessed. Compared with wild-type (WT) mice, reduced numbers of mature V gamma 3 cells were found in the fetal thymus of IL-15(-/-) mice, while IRF-1(-/-) mice displayed normal frequencies. V gamma 3(+) dendritic epidermal T cells (DETCs) were absent in IL-15(-/-) mice but present in IRF-1(-/-) mice. DETCs from IRF-1(-/-) mice displayed morphologically a less mature phenotype and showed different emergence kinetics during ontogeny. This corresponded with lower IL-15 mRNA levels in the skin epidermis. Comparable levels of IL-7 were found in the skin of WT and IL-15(-/-) mice. Adoptive transfer experiments of WT fetal thymocytes into IL-15(-/-) mice did not result in the development of V gamma 3(+) DETCs, confirming the nonredundant role of IL-15 in the skin during DETC development. In vitro, cytolytic activity of IL-15(-/-) V gamma 3 cells was normal after stimulation with IL-15 and was further enhanced by addition of IL-12. In contrast, cytolytic activity of IRF-1(-/-) V gamma 3 cells remained defective after stimulation with IL-15 in combination with IL-12. These data suggest that IL-15 is redundant for the development and/or survival of mature V gamma 3 cells in the fetal thymus, whereas it is essential for the localization of V gamma 3 cells in the skin. Furthermore, a possible role for IRF-1 in inducing morphological maturation of DETCs and cytolytic capacity of V gamma 3 cells is suggested.

Ly49 and CD94/NKG2 inhibitory receptors are predominantly expressed on murine NK cells, but they are also expressed on a subpopulation of peripheral CD8 memory TCR αβ lymphocytes. In this study we demonstrate that Ly49E and CD94/NKG2 receptors are expressed on mature TCR Vγ3+ cells in the fetal thymus. Expression correlated with a memory phenotype, such as expression of CD44, 2B4, and IL-2Rβ (CD122), and absence of IL-2Rα (CD25) expression. No expression of Ly49A, C, D, G2, or I receptors was observed. This phenotype is similar to that of fetal thymic NK cells. Skin-located Vγ3 T cells, the progeny of fetal thymic Vγ3 cells, also expressed CD94/NKG2 and Ly49E but not the other members of the Ly49 family. The development and survival of Ly49E+ or CD94/NKG2+ Vγ3 T lymphocytes was not dependent upon expression of MHC class I molecules. The cytotoxicity of TCR Vγ3 cells was inhibited when Qdm, the ligand for CD94/NKG2, was presented by Qa1b-transfected target cells. Also, upon cross-linking of CD94/NKG2 with mAb 3S9, TCR Vγ3 thymocytes were prevented from killing FcγR+ P815 target cells. These effects were most pronounced in the CD94/NKG2high subpopulation as compared with the CD94/NKG2low subpopulation of Vγ3 cells. Our data demonstrate that Vγ3 T cells expressing inhibitory Ly49E and CD94/NKG2 receptors are mature and display a memory phenotype, and that CD94/NKG2 functions as an inhibitory receptor on these T lymphocytes.

Murine NK cells express inhibitory receptors belonging to the Ly49 and CD94/NKG2 family. Ly49E and CD94 are the only NK cell receptor transcripts detectable in fetal NK cells. Still unproved is the surface expression of Ly49E on NK cells. Here we generated two novel mAbs, a mAb recognizing Ly49E with cross-reactivity to Ly49C, and a mAb against NKG2A/C/E. Ly49E was immunoprecipitated as a disulfide-linked homodimer with 46-kDa subunits. Removal of N-linked carbohydrates revealed a 31-kDa protein backbone. NKG2A was immunoprecipitated as a 38-kDa protein. Although the frequency of fetal NK cells expressing Ly49E was higher than 25%, it decreased drastically from 2 wk after birth. Phenotypic analysis showed that ∼90% of fetal NK cells and ∼50% of adult NK cells express high levels of CD94/NKG2. The remaining 50% of adult NK cells expressed low surface levels of CD94/NKG2. Expression of Ly49E and CD94/NKG2 was not restricted to NK cells, but was also observed on NK T and memory T cells. Functional analysis showed that sorted Ly49E+ and CD94/NKG2+ fetal NK cells could discriminate between MHC class I-positive and MHC class I-negative tumor cells. We also demonstrated that Ly49E becomes phosphorylated following pervanadate stimulation of fetal NK cells. The expression levels of Ly49E and CD94/NKG2 were similar in wild-type compared with β2-microglobulin−/− mice. In conclusion, generation of mAbs against Ly49E and NKG2 extended the phenotypic and functional characterization of NK cells.

Natural killer (NK) cell phenotype and activity was studied by analyzing uncultured and short-time-cultured murine NK cells from fetal day 17 spleen and thymus. In contrast to NK cells from adult mice, freshly sorted fetal NK cells did not contain NK receptor transcripts for Ly-49A, B, C/I, D, F, G2, or H. The only NK receptor transcripts that could be detected were Ly-49E and CD94. It is important that Ly-49E was present at a 10- to 30-fold higher level compared with uncultured NK cells from adult mice. After short-time interleukin-2 culture, the level of Ly-49E mRNA was comparable between fetal and adult NK cells. Functionally, fetal NK cells only killed MHC class I-negative tumor cells when activating NK receptors were cross-linked with antibody. We show that fetal NK cells are mature but are different from NK cells in adult mice regarding their NK cell receptor repertoire and function. J. Leukoc. Biol. 66: 625–633 1999.

Oral delivery of IL-10 by genetically modified Lactococcus lactis (LL-pTmIL10) has been shown to efficiently reduce intestinal inflammation in mice with chronic colitis, but the mechanisms involved have not been elucidated. It has been suggested that IL-10 controls intestinal inflammation by inhibiting microbe-induced activation of dendritic cells. We therefore investigated whether LL-pTmIL10 can modulate the functions of bone marrow-derived dendritic cells (BM-DC) responding to LPS. Incubation of these cells with LL-pTmIL10 or with the control strain LL-pTREX reduced their ability to activate allogeneic T-cell proliferation. However, in contrast to LL-pTREX, LL-pTmIL10 inhibited the LPS-stimulated secretion of MCP-1 by BM-DC and reduced the synergistic up-regulation of IL-12/IL-23p40. In addition, LL-pTmIL10 treatment of LPS-stimulated BM-DC significantly inhibited their capacity to induce strong secretion of IL-17 by CD4+ T cells. Our data suggest that the beneficial effects of LL-pTmIL10 treatment during chronic colitis might involve inhibition of CD4+ Th17 cells and a reduced accumulation of these cells as well as other immune cells at the site of inflammation.

The case deals with the decision of Sanac Inc., a Belgian wholesale company, on whether to proceed with the implementation of an Activity-Based Costing (ABC) system or switch to Time-Driven Activity-Based Costing (TDABC). As a business consultant, you are hired to decide about the appropriate costing method. Your task is to decide which system the company should implement, given the desire of the president of the company to calculate profitability at the order and the customer level.

Peptides of the trefoil factor family (TFF) are expressed along the gastro-intestinal tract. They protect mucous epithelia from damage and contribute to mucosal repair, which is essential for preventing inflammation. Moreover, it has been suggested that TFF2 and TFF3, in particular, play a role in regulating immune responses. Depending on their activation status, dendritic cells (DC) can initiate either tolerance or immunity. This study, by comparing LPS-induced maturation of mTFF3-treated DC and non-treated DC, investigated whether murine TFF3 directly regulated DC function. mTFF3-treated DC and non-treated DC did not differ phenotypically or functionally. Both populations expressed, both before and after LPS-stimulation, similar levels of co-stimulatory molecules and cytokines, and were both efficient stimulators of T-cells. Our results suggest that mTFF3 does not govern immune responses on the level of DC function.

Background & Aims: Obtaining antigen-specific immune suppression is an important goal in developing treatments of autoimmune, inflammatory, and allergic gastrointestinal diseases. Oral tolerance is a powerful means for inducing tolerance to a particular antigen, but implementing this strategy in humans has been difficult. Active delivery of recombinant autoantigens or allergens at the intestinal mucosa by genetically modified Lactococcus lactis (L lactis) provides a novel therapeutic approach for inducing tolerance. Methods: We engineered the food grade bacterium L lactis to secrete ovalbumin (OVA) and evaluated its ability to induce OVA-specific tolerance in OVA T-cell receptor (TCR) transgenic mice (DO11.10). Tolerance induction was assessed by analysis of delayed-type hypersensitivity responses, measurement of cytokines and OVA-specific proliferation, phenotypic analysis, and adoptive transfer experiments. Results: Intragastric administration of OVA-secreting L lactis led to active delivery of OVA at the mucosa and suppression of local and systemic OVA-specific T-cell responses in DO11.10 mice. This suppression was mediated by induction of CD4(+)CD25(-) regulatory T cells that function through a transforming growth factor beta-dependent mechanism. Restimulation of splenocytes and gut-associated lymph node tissue from these mice resulted in a significant OVA-specific decrease in interferon gamma and a significant increase in interleukin-10 production. Furthermore, Foxp3 and CTLA-4 were significantly up-regulated in the CD4(+)CD25(-) population. Conclusions: Mucosal antigen delivery by oral administration of genetically engineered L lactis leads to antigen-specific tolerance. This approach can be used to develop effective therapeutics for systemic and intestinal immune-mediated inflammatory diseases

Signaling via TLRs results in dendritic cell (DC) activation/maturation and plays a critical role in the outcome of primary immune responses. So far, no data exist concerning TLR expression by liver DC, generally regarded as less immunostimulatory than secondary lymphoid tissue DC. Because the liver lies directly downstream from the gut, it is constantly exposed to bacterial LPS, a TLR4 ligand. We examined TLR4 expression by freshly isolated, flow-sorted C57BL/10 mouse liver DC compared with spleen DC. Real-time PCR revealed that liver CD11c+CD8α− (myeloid) and CD11c+CD8α+ (“lymphoid-related”) DC expressed lower TLR4 mRNA compared with their splenic counterparts. Lower TLR4 expression correlated with reduced capacity of LPS (10 ng/ml) but not anti-CD40-stimulated liver DC to induce naive allogeneic (C3H/HeJ) T cell proliferation. By contrast to LPS-stimulated splenic DC, these LPS-activated hepatic DC induced alloantigen-specific T cell hyporesponsiveness in vitro, correlated with deficient Th1 (IFN-γ) and Th2 (IL-4) responses. When higher LPS concentrations (≥100 ng/ml) were tested, the capacity of liver DC to induce proliferation of T cells and Th1-type responses was enhanced, but remained inferior to that of splenic DC. Hepatic DC activated by LPS in vivo were inferior allogeneic T cell stimulators compared with splenic DC, whereas adoptive transfer of LPS-stimulated (10 ng/ml) liver DC induced skewing toward Th2 responses. These data suggest that comparatively low expression of TLR4 by liver DC may limit their response to specific ligands, resulting in reduced or altered activation of hepatic adaptive immune responses.

Using a new antibody, we found previously that contrary to adult natural killer (NK) cells, fetal NK cells have a unique phenotype, as they exclusively express Ly49E. This can be explained by an intrinsic different NK differentiation potential of fetal versus adult lymphoid progenitors, by immaturity of fetal NK cells or by instability of Ly49E expression. Here, we show that adult progenitor cells were still capable of differentiating into Ly49E-expressing NK cells but at a much lower frequency. Surprisingly, Ly49E expression in vitro did not require stromal cells. Kinetic analysis in vivo showed that Ly49E was expressed early, together with CD94/NKG2 and Ly49G2, followed by Ly49C, and finally Ly49D. Transfer of sorted Ly49E-positive fetal NK cells showed stable Ly49E expression, and later, part of these cells up-regulated other Ly49 members. These data indicate that although there are intrinsic differences, there is no strict fetal and adult wave of NK cell differentiation.