Development of a New Antileishmanial Aziridine-2,3-Dicarboxylate-Based Inhibitor with High Selectivity for Parasite Cysteine Proteases.

Leishmaniasis is one of the major neglected tropical diseases of the world. Druggable targets are the parasite cysteine proteases (CPs) of clan CA, family C1 (CAC1). In previous studies, we identified two peptidomimetic compounds, the aziridine-2,3-dicarboxylate compounds 13b and 13e, in a series of inhibitors of the cathepsin L (CL) subfamily of the papain clan CAC1. Both displayed antileishmanial activity in vitro while not showing cytotoxicity against host cells. In further investigations, the mode of action was characterized in Leishmania major. It was demonstrated that aziridines 13b and 13e mainly inhibited the parasitic cathepsin B (CB)-like CPC enzyme and, additionally, mammalian CL. Although these compounds induced cell death of Leishmania promastigotes and amastigotes in vitro, the induction of a proleishmanial T helper type 2 (Th2) response caused by host CL inhibition was observed in vivo. Therefore, we describe here the synthesis of a new library of more selective peptidomimetic aziridine-2,3-dicarboxylates discriminating between host and parasite CPs. The new compounds are based on 13b and 13e as lead structures. One of the most promising compounds of this series is compound s9, showing selective inhibition of the parasite CPs LmaCatB (a CB-like enzyme of L. major; also named L. major CPC) and LmCPB2.8 (a CL-like enzyme of Leishmania mexicana) while not affecting mammalian CL and CB. It displayed excellent leishmanicidal activities against L. major promastigotes (50% inhibitory concentration [IC50] = 37.4 µM) and amastigotes (IC50 = 2.3 µM). In summary, we demonstrate a new selective aziridine-2,3-dicarboxylate, compound s9, which might be a good candidate for future in vivo studies.

Inhibitory effect of phenothiazine- and phenoxazine-derived chloroacetamides on Leishmania major growth and Trypanosoma brucei trypanothione reductase.

A number of phenothiazine-, phenoxazine- and related tricyclics-derived chloroacetamides were synthesized and evaluated in vitro for antiprotozoal activities against Leishmania major (L. major) promastigotes. Several analogs were remarkably potent inhibitors, with antileishmanial activities being comparable or superior to those of the reference antiprotozoal drugs. Furthermore, we explored the structure-activity relationships of N-10 haloacetamides that influence the potency of such analogs toward inhibition of L. major promastigote growth in vitro. With respect to the mechanism of action, selected compounds were evaluated for time-dependent inactivation of Trypanosoma brucei trypanothione reductase. Our results are indicative of a covalent interaction which could account for potent antiprotozoal activities.

Pathogen- and Host-Directed Antileishmanial Effects Mediated by Polyhexanide (PHMB).

BACKGROUND: Cutaneous leishmaniasis (CL) is a neglected tropical disease caused by protozoan parasites of the genus Leishmania. CL causes enormous suffering in many countries worldwide. There is no licensed vaccine against CL, and the chemotherapy options show limited efficacy and high toxicity. Localization of the parasites inside host cells is a barrier to most standard chemo- and immune-based interventions. Hence, novel drugs, which are safe, effective and readily accessible to third-world countries and/or drug delivery technologies for effective CL treatments are desperately needed. METHODOLOGY/PRINCIPAL FINDINGS: Here we evaluated the antileishmanial properties and delivery potential of polyhexamethylene biguanide (PHMB; polyhexanide), a widely used antimicrobial and wound antiseptic, in the Leishmania model. PHMB showed an inherent antileishmanial activity at submicromolar concentrations. Our data revealed that PHMB kills Leishmania major (L. major) via a dual mechanism involving disruption of membrane integrity and selective chromosome condensation and damage. PHMB's DNA binding and host cell entry properties were further exploited to improve the delivery and immunomodulatory activities of unmethylated cytosine-phosphate-guanine oligodeoxynucleotides (CpG ODN). PHMB spontaneously bound CpG ODN, forming stable nanopolyplexes that enhanced uptake of CpG ODN, potentiated antimicrobial killing and reduced host cell toxicity of PHMB. CONCLUSIONS: Given its low cost and long history of safe topical use, PHMB holds promise as a drug for CL therapy and delivery vehicle for nucleic acid immunomodulators.

Antileishmanial and cytotoxic compounds from Valeriana wallichii and identification of a novel nepetolactone derivative.

The chloroform extract of Valeriana wallichii (V. wallichii) rhizomes was investigated to elucidate the structures responsible for reported antileishmanial activity. Besides bornyl caffeate (1, already been reported by us previously), bioassay-guided fractionation resulted in two additional cinnamic acid derivatives 2-3 with moderate leishmanicidal activity. The structure of a novel nepetolactone derivative 4 having a cinnamic acid moiety was elucidated by means of spectral analysis. To the best of our knowledge villoside aglycone (5) was isolated from this plant for the first time. The bioassay-guided fractionation yielded two new (compounds 6-7) and two known valtrates (compounds 8-9) with leishmanicidal potential against Leishmania major (L. major) promastigotes. In addition, ß-bisabolol (10), α-kessyl alcohol (11), valeranone (12), bornyl isovalerate (13) and linarin-2-O-methylbutyrate (14) were identified. This is the first report on the isolation of 4'-demethylpodophyllotoxin (15), podophyllotoxin (16) and pinoresinol (17) in V. wallichii. In total thirteen known and four new compounds were identified from the extract and their cytotoxic and antileishmanial properties were evaluated.

Cathepsin B in antigen-presenting cells controls mediators of the Th1 immune response during Leishmania major infection.

Resistance and susceptibility to Leishmania major infection in the murine model is determined by the capacity of the host to mount either a protective Th1 response or a Th2 response associated with disease progression. Previous reports involving the use of cysteine cathepsin inhibitors indicated that cathepsins B (Ctsb) and L (Ctsl) play important roles in Th1/Th2 polarization during L. major infection in both susceptible and resistant mouse strains. Although it was hypothesized that these effects are a consequence of differential patterns of antigen processing, the mechanisms underlying these differences were not further investigated. Given the pivotal roles that dendritic cells and macrophages play during Leishmania infection, we generated bone-marrow derived dendritic cells (BMDC) and macrophages (BMM) from Ctsb-/- and Ctsl-/- mice, and studied the effects of Ctsb and Ctsl deficiency on the survival of L. major in infected cells. Furthermore, the signals used by dendritic cells to instruct Th cell polarization were addressed: the expression of MHC class II and co-stimulatory molecules, and cytokine production. We found that Ctsb-/- BMDC express higher levels of MHC class II molecules than wild-type (WT) and Ctsl-/- BMDC, while there were no significant differences in the expression of co-stimulatory molecules between cathepsin-deficient and WT cells. Moreover, both BMDC and BMM from Ctsb-/- mice significantly up-regulated the levels of interleukin 12 (IL-12) expression, a key Th1-inducing cytokine. These findings indicate that Ctsb-/- BMDC display more pro-Th1 properties than their WT and Ctsl-/- counterparts, and therefore suggest that Ctsb down-regulates the Th1 response to L. major. Moreover, they propose a novel role for Ctsb as a regulator of cytokine expression.

Identification of multiple cellular uptake pathways of polystyrene nanoparticles and factors affecting the uptake: relevance for drug delivery systems.

Nanoparticles may address challenges by human diseases through improving diagnosis, vaccination and treatment. The uptake mechanism regulates the type of threat a particle poses on the host cells and how a cell responds to it. Hence, understanding the uptake mechanisms and cellular interactions of nanoparticles at the cellular and subcellular level is a prerequisite for their effective biomedical applications. The present study shows the uptake mechanisms of polystyrene nanoparticles and factors affecting their uptake in bone marrow-derived macrophages, 293T kidney epithelial cells and L929 fibroblasts. Labeling with the endocytic marker FM4-64 and transmission electron microscopy studies show that the nanoparticles were internalized rapidly via endocytosis and accumulated in intracellular vesicles. Soon after their internalizations, nanoparticles trafficked to organelles with acidic pH. Analysis of the ultrastructural morphology of the plasma membrane invaginations or extravasations provides clear evidence for the involvement of several uptake routes in parallel to internalize a given type of nanoparticles by mammalian cells, highlighting the complexity of the nanoparticle-cell interactions. Blocking the specific endocytic pathways by different pharmacological inhibitors shows similar outcomes. The potential to take up nanoparticles varies highly among different cell types in a particle sizes-, time- and energy-dependent manner. Furthermore, infection and the activation status of bone marrow-derived macrophages significantly affect the uptake potential of the cells, indicating the need to understand the diseases' pathogenesis to establish effective and rational drug-delivery systems. This study enhances our understanding of the application of nanotechnology in biomedical sciences.

Antileishmanial lead structures from nature: analysis of structure-activity relationships of a compound library derived from caffeic Acid bornyl ester.

Bioassay-guided fractionation of a chloroform extract of Valeriana wallichii (V. wallichii) rhizomes lead to the isolation and identification of caffeic acid bornyl ester (1) as the active component against Leishmania major (L. major) promastigotes (IC50 = 48.8 µM). To investigate the structure-activity relationship (SAR), a library of compounds based on 1 was synthesized and tested in vitro against L. major and L. donovani promastigotes, and L. major amastigotes. Cytotoxicity was determined using a murine J774.1 cell line and bone marrow derived macrophages (BMDM). Some compounds showed antileishmanial activity in the concentration range of pentamidine and miltefosine which are the standard drugs in use. In the L. major amastigote assay compounds 15, 19 and 20 showed good activity with relatively low cytotoxicity against BMDM, resulting in acceptable selectivity indices. Molecules with adjacent phenolic hydroxyl groups exhibited elevated cytotoxicity against murine cell lines J774.1 and BMDM. The Michael system seems not to be essential for antileishmanial activity. Based on the results compound 27 can be regarded as new lead structure for further structure optimization.

T cell-derived IL-10 determines leishmaniasis disease outcome and is suppressed by a dendritic cell based vaccine.

In the murine model of Leishmania major infection, resistance or susceptibility to the parasite has been associated with the development of a Th1 or Th2 type of immune response. Recently, however, the immunosuppressive effects of IL-10 have been ascribed a crucial role in the development of the different clinical correlates of Leishmania infection in humans. Since T cells and professional APC are important cellular sources of IL-10, we compared leishmaniasis disease progression in T cell-specific, macrophage/neutrophil-specific and complete IL-10-deficient C57BL/6 as well as T cell-specific and complete IL-10-deficient BALB/c mice. As early as two weeks after infection of these mice with L. major, T cell-specific and complete IL-10-deficient animals showed significantly increased lesion development accompanied by a markedly elevated secretion of IFN-γ or IFN-γ and IL-4 in the lymph nodes draining the lesions of the C57BL/6 or BALB/c mutants, respectively. In contrast, macrophage/neutrophil-specific IL-10-deficient C57BL/6 mice did not show any altered phenotype. During the further course of disease, the T cell-specific as well as the complete IL-10-deficient BALB/c mice were able to control the infection. Furthermore, a dendritic cell-based vaccination against leishmaniasis efficiently suppresses the early secretion of IL-10, thus contributing to the control of parasite spread. Taken together, IL-10 secretion by T cells has an influence on immune activation early after infection and is sufficient to render BALB/c mice susceptible to an uncontrolled Leishmania major infection.

A novel Leishmania major amastigote assay in 96-well format for rapid drug screening and its use for discovery and evaluation of a new class of leishmanicidal quinolinium salts.

In most laboratories, the screening for leishmanicidal compounds is carried out with Leishmania promastigotes or axenic amastigotes. However, the best approach to identify leishmanicidal compounds is the use of amastigotes residing in macrophages. Reporter gene-based assays are relatively new tools in the search for drugs against eucaryotic protozoa, permitting the development of faster, more automated assays. In this paper, we report on the establishment of a rapid screening assay in a 96-well format. A luciferase-transgenic (Luc-tg) Leishmania major strain was generated and used to infect bone marrow-derived macrophages (BMDM). Amastigote-infected BMDM were treated with different compound concentrations. Cells were lysed with a luciferin-containing buffer, and the resulting luminescence was measured to determine the half-maximal inhibitory concentration (IC50). To validate this new amastigote screening assay, a library of a new class of quinolinium salts was synthesized and tested for leishmanicidal activity. Some of the quinolinium salts showed very promising activities, with IC50s against intracellular amastigotes (IC50 < 1 µg/ml) and selectivity indices (SI > 20) that match the criteria of World Health Organization (WHO) for hits. Compound 21c (IC50 = 0.03 µg/ml; SI = 358) could become a new lead structure for the development of improved chemotherapeutic drugs against L. major. In summary, we describe the establishment of a new 96-well format assay with Luc-transgenic L. major for the rapid screening of compounds for leishmanicidal activity against intracellular amastigotes and its application to the identification of a new class of quinolinium salts with most promising leishmanicidal activity.

Dendritic cell-mediated vaccination relies on interleukin-4 receptor signaling to avoid tissue damage after Leishmania major infection of BALB/c mice.

Prevention of tissue damages at the site of Leishmania major inoculation can be achieved if the BALB/c mice are systemically given L. major antigen (LmAg)-loaded bone marrow-derived dendritic cells (DC) that had been exposed to CpG-containing oligodeoxynucleotides (CpG ODN). As previous studies allowed establishing that interleukin-4 (IL-4) is involved in the redirection of the immune response towards a type 1 profile, we were interested in further exploring the role of IL-4. Thus, wild-type (wt) BALB/c mice or DC-specific IL-4 receptor alpha (IL-4Rα)-deficient (CD11c(cre)IL-4Rα(-/lox)) BALB/c mice were given either wt or IL-4Rα-deficient LmAg-loaded bone marrow-derived DC exposed or not to CpG ODN prior to inoculation of 2×105 stationary-phase L. major promastigotes into the BALB/c footpad. The results provide evidence that IL4/IL-4Rα-mediated signaling in the vaccinating DC is required to prevent tissue damage at the site of L. major inoculation, as properly conditioned wt DC but not IL-4Rα-deficient DC were able to confer resistance. Furthermore, uncontrolled L. major population size expansion was observed in the footpad and the footpad draining lymph nodes of CD11c(cre)IL-4Rα(-/lox) mice immunized with CpG ODN-exposed LmAg-loaded IL-4Rα-deficient DC, indicating the influence of IL-4Rα-mediated signaling in host DC to control parasite replication. In addition, no footpad damage occurred in BALB/c mice that were systemically immunized with LmAg-loaded wt DC doubly exposed to CpG ODN and recombinant IL-4. We discuss these findings and suggest that the IL4/IL4Rα signaling pathway could be a key pathway to trigger when designing vaccines aimed to prevent damaging processes in tissues hosting intracellular microorganisms.

Valeriana wallichii root extracts and fractions with activity against Leishmania spp.

Leishmanial diseases, posing a public health problem worldwide, are caused by Leishmania parasites with a dimorphic life cycle alternating between the promastigote and amastigote forms. Promastigotes transmitted by the vector are transformed into amastigotes residing in the host tissue macrophages. Presently, new antiparasitic agents are needed against Leishmania donovani and Leishmania major, the respective organisms causing visceral and cutaneous leishmaniasis, since the available treatments are unsatisfactory due to toxicity, high cost, and emerging drug resistance. Over the years, traditional medicinal flora throughout the world enriched the modern pharmacopeia. Hence, roots of 'Indian Valerian' (Valeriana wallichii DC) were studied for its antileishmanial activity for the first time. The methanol and chloroform extracts showed activity against L. donovani promastigotes and both promastigotes and amastigotes of L. major. The most active fraction, F3, obtained from the chloroform extract, showed IC(50) at ∼ 3-7 µg/ml against both the promastigotes and 0.3 µg/ml against L. major amastigotes. On investigation of the mechanism of cytotoxicity in L. donovani promastigotes, the 'hall-mark' events of morphological degeneration, DNA fragmentation, externalization of phosphatidyl serine, and mitochondrial membrane depolarization indicated that F3 could induce apoptotic death in leishmanial cells. Therefore, the present study revealed a novel and unconventional property of V. wallichii root as a prospective source of effective antileishmanial agents.

Phenolic analogs of the N,C-coupled naphthylisoquinoline alkaloid ancistrocladinium A, from Ancistrocladus cochinchinensis (Ancistrocladaceae), with improved antiprotozoal activities.

The first N,8'-coupled naphthylisoquinoline alkaloids with free phenolic OH groups, 4'-O-demethylancistrocladinium A and 6,4'-O-didemethylancistrocladinium A, have been isolated from the leaves and bark of the Vietnamese liana Ancistrocladus cochinchinensis, along with its known, non-phenolic parent compound, ancistrocladinium A, and four C,C-coupled representatives. The structure elucidation was achieved by chemical, spectroscopic, and chiroptical methods. The mono-phenolic alkaloid showed excellent activities in particular against the pathogen causing Chagas' disease, Trypanosoma cruzi.

QSAR guided synthesis of simplified antiplasmodial analogs of naphthylisoquinoline alkaloids.

Naphthylisoquinoline alkaloids have attracted considerable interest because of their intriguing structure, their unique biosynthetic origin, and their biological activities against several pathogens causing tropical diseases. Their promising pharmacologic properties make them suitable lead structures for new agents, in particular against malaria. Since these natural products are not easy to isolate in sufficient quantities or to synthesize stereoselectively, quantitative structure-activity relationship studies were accomplished to find new antiplasmodial analogs that are structurally related to the naturally occurring naphthylisoquinoline alkaloids, but more easily accessible, more active against Plasmodium falciparum, and, last but not least, less toxic. We report on the synthesis of several simplified compounds by a Suzuki coupling between the naphthalene and the isoquinoline moieties and on their activities against different pathogens causing infectious diseases. Some structures were found to exhibit excellent--and selective--activities against P. falciparum in vitro.

Aziridine-2,3-dicarboxylate-based cysteine cathepsin inhibitors induce cell death in Leishmania major associated with accumulation of debris in autophagy-related lysosome-like vacuoles.

The papain-like cysteine cathepsins expressed by Leishmania play a key role in the life cycle of these parasites, turning them into attractive targets for the development of new drugs. We previously demonstrated that two compounds of a series of peptidomimetic aziridine-2,3-dicarboxylate [Azi(OBn)(2)]-based inhibitors, Boc-(S)-Leu-(R)-Pro-(S,S)-Azi(OBn)(2) (compound 13b) and Boc-(R)-Leu-(S)-Pro-(S,S)-Azi(OBn)(2) (compound 13e), reduced the growth and viability of Leishmania major and the infection rate of macrophages while not showing cytotoxicity against host cells. In the present study, we characterized the mode of action of inhibitors 13b and 13e in L. major. Both compounds targeted leishmanial cathepsin B-like cysteine cathepsin cysteine proteinase C, as shown by fluorescence proteinase activity assays and active-site labeling with biotin-tagged inhibitors. Furthermore, compounds 13b and 13e were potent inducers of cell death in promastigotes, characterized by cell shrinkage, reduction of mitochondrial transmembrane potential, and increased DNA fragmentation. Transmission electron microscopic studies revealed the enrichment of undigested debris in lysosome-like organelles participating in micro- and macroautophagy-like processes. The release of digestive enzymes into the cytoplasm after rupture of membranes of lysosome-like vacuoles resulted in the significant digestion of intracellular compartments. However, the plasma membrane integrity of compound-treated promastigotes was maintained for several hours. Taken together, our results suggest that the induction of cell death in Leishmania by cysteine cathepsin inhibitors 13b and 13e is different from mammalian apoptosis and is caused by incomplete digestion in autophagy-related lysosome-like vacuoles.

Natural killer cells support the induction of protective immunity during dendritic cell-mediated vaccination against Leishmania major.

Dendritic cell (DC)-mediated vaccination against Leishmania major induces a parasite-specific T helper 1 (Th1) response and long-lasting protective immunity in susceptible mice. As the cytokine interleukin-12 required for induction of this Th1 response is not derived from the transferred DC, but has to be produced by the vaccinated host, we examined cross-presentation of transferred DC via resident DC of the host and cross-activation with natural killer (NK) cells as mechanisms supporting the induction of protective immunity after DC-mediated vaccination. Co-culture with DC that had been conditioned ex vivo by loading with L. major lysate and stimulation with CpG-containing oligodeoxynucleotides did not result in the activation of naive DC in vitro. Furthermore, L. major antigen from conditioned DC was not cross-presented to a significant extent in vivo. In contrast, co-culture of DC with NK cells led to cross-activation of both cell populations with induction of interferon-γ, which was dependent on the activation status of the conditioned DC. Transient depletion of NK cells during vaccination of L. major-susceptible mice with conditioned DC resulted in reduced protection. Our findings indicate that cross-presentation of conditioned DC after DC-based vaccination against L. major plays a minor role in the induction of protective immunity. However, we demonstrated for the first time that the capacity of DC to mediate protection against L. major is supported by cross-activation with NK cells of the host and NK-cell-derived interferon-γ.

Fragments of antigen-loaded dendritic cells (DC) and DC-derived exosomes induce protective immunity against Leishmania major.

Upon loading with parasite antigen and adoptive transfer, dendritic cells (DC) are able to confer protection against the protozoan parasite Leishmania major. In the present study, we investigated whether viable DC are required for inducing protection. We provide evidence that L. major antigen-loaded DC that had been fixed with paraformaldehyde or exposed to UV irradiation, and even disrupted cells, are able to serve as an effective vaccine. Furthermore, we demonstrate the potential of DC-derived exosomes to mediate protective immunity against cutaneous leishmaniasis. The route of antigen presentation to recipient T cells involves uptake of intravenously injected DC fragments into late endosomal compartments of splenic DC in the recipient. In vitro studies showed that DC fragments induce T-cell proliferation and interleukin 12 secretion by splenocytes. Together, these findings suggest that the development of a cell-free vaccine for immunoprophylaxis against leishmaniasis and other infectious diseases is feasible.

Anti-parasitic compounds from Streptomyces sp. strains isolated from Mediterranean sponges.

Actinomycetes are prolific producers of pharmacologically important compounds accounting for about 70% of the naturally derived antibiotics that are currently in clinical use. In this study, we report on the isolation of Streptomyces sp. strains from Mediterranean sponges, on their secondary metabolite production and on their screening for anti-infective activities. Bioassay-guided isolation and purification yielded three previously known compounds namely, cyclic depsipeptide valinomycin, indolocarbazole alkaloid staurosporine and butenolide. This is the first report of the isolation of valinomycin from a marine source. These compounds exhibited novel anti-parasitic activities specifically against Leishmania major (valinomycin IC(50) < 0.11 microM; staurosporine IC(50) 5.30 microM) and Trypanosoma brucei brucei (valinomycin IC(50) 0.0032 microM; staurosporine IC(50) 0.022 microM; butenolide IC(50) 31.77 microM). These results underscore the potential of marine actinomycetes to produce bioactive compounds as well as the re-evaluation of previously known compounds for novel anti-infective activities.

Alterations to the structure of Leishmania major induced by N-arylisoquinolines correlate with compound accumulation and disposition.

Naphthylisoquinoline alkaloids equipped with a N,C-hetero-'biaryl' axis, and, in particular, simplified synthetic analogues thereof, kill intracellular Leishmania major at concentrations in the low submicromolar range, while being significantly less toxic to their major host cell, the macrophage, at the same concentrations. To further investigate their mechanism of action we evaluated the morphological and ultrastructural changes induced by specific N-arylisoquinolines in L. major, and the correlation of these changes with compound accumulation and disposition by the parasite. After 24 h of treatment with the synthetic arylisoquinolinium salts 3 or 4, dramatic structural changes and cell death were observed. Furthermore, the auto-fluorescent derivative salt 3 accumulates continually in intracellular compartments. Our results thus suggest that the leishmanicidal effect of arylisoquinolinium salts may involve their ability to accumulate and precipitate in intracellular organelles, form a huge vacuole and eventually promote cell lysis.

Split immune response after oral vaccination of mice with recombinant Escherichia coli Nissle 1917 expressing fimbrial adhesin K88.

Enterotoxigenic Escherichia coli (ETEC) are a leading cause of diarrhoea in piglets and newborn calves. Massive efforts have therefore been made to develop a vaccine for the induction of protective mucosal immunity against ETEC. Since it has been shown that the probiotic strain E. coli Nissle 1917 (EcN) can serve as a safe carrier for targeted delivery of recombinant molecules to the intestinal mucosa, we constructed the recombinant strain EcN pMut2-kanK88 (EcN-K88) stably expressing the determinant for the K88 fimbrial adhesin of ETEC on the bacterial surface. After oral application of EcN-K88 to mice for one week, EcN-K88 as well as wild-type EcN and EcN mock-transformed with the plasmid vector only could be detected in faecal samples for a minimum of 7 days after the last feeding, indicating that EcN can transiently colonise the murine intestine. Oral application of EcN-K88 resulted in significant IgG serum titres against K88 as early as 7 days after the initial feeding with EcN-K88, but no significant IgA titres. In contrast, we failed to detect any specific T cell responses towards the K88 antigen both in spleen and mesenteric lymph nodes. Although dendritic cells readily upregulated maturation and activation markers in response to K88 stimulation, accompanied by secretion of interleukin (IL)-12, IL-6, IL-10, and tumour necrosis factor, restimulation of T cells from mice having received EcN-K88 with K88-loaded dendritic cells did not result in detectable T cell proliferation and IL-2 secretion, but rather induced an IL-10 bias. While the serum antibody responses clearly demonstrate that K88 is recognized by the humoral immune system, our findings indicate that oral application of probiotic EcN expressing the K88 fimbrial adhesin does not induce a selective T cell response towards the antigen.