Search

Colonization factor antigen I (CFA/I) fimbria, an adhesin from enterotoxigenic Escherichia coli, confers protection in murine autoimmune models for type 1 diabetes (T1D), multiple sclerosis, and rheumatoid arthritis. Although CFA/I fimbriae's initial mode of action is in a bystander or in an antigen (Ag)-independent fashion, protection is ultimately dependent upon the induction and/or activation of auto-Ag-specific regulatory T cells (Tregs). However, little is known about how protection transitions from bystander suppression to Ag-specific Tregs. Since dendritic cells (DCs) play an integral role in fate decisions for T cells becoming inflammatory or tolerogenic, the described study tests the hypothesis that Lactococcus lactis expressing CFA/I (LL-CFA/I) stimulates DCs to establish a regulatory microenvironment. To this end, bone marrow-derived dendritic cells (BMDCs) were infected in vitro with LL-CFA/I. Results revealed increased production of IL-10, TGF-β, and indoleamine 2,3-deoxygenase (IDO). Although co-culture of LL-CFA/I infected BMDCs with naive T cells did not promote Foxp3 expression, TNF-α and IFN-γ production was suppressed. NOD mice orally dosed with LL-CFA/I showed an increase in regulatory plasmacytoid DCs (pDCs) expressing IDO and TGF-β in pancreatic lymph nodes (PaLNs) and spleen three days post-treatment. However, Tregs did not appear in the mucosal inductive sites until much later. These findings show that LL-CFA/I influences specific DC populations to establish tolerance.

Calcium is an essential element for human health, with key roles in the prevention and therapy of multifactorial conditions. Calcium dietary intake is often insufficient in the general population. The aim of this study was to perform a clinical audit for general practitioners (GPs) to understand the efficacy of training intervention on doctors' awareness about dietary calcium and supplements.General practice outpatients were enrolled (Before Clinical Audit, BCA) from the same sanitary district, and calcium dietary intake was evaluated with a validated questionnaire, also collecting information about the consumption of calcium and vitamin D supplements. Then, a training intervention with a frontal lesson and discussion with GPs involved was performed. After one month of this intervention, a second outpatient enrolment was performed (Post Clinical Audit, PCA) in the same general practices to evaluate differences in nutritional suggestions and supplement prescription by GPs. In BCA, the calcium dietary intake was low, with nobody reaching 1000 mg as suggested by the guidelines. Only 6.6% and 24.5% took calcium and vitamin D supplements, respectively; in the PCA, these percentages increased to 28% and 78% for calcium and vitamin D supplements, respectively (p 0.01 PCA vs BCA). There were no differences in calcium dietary intake between BCA and PCA.Training intervention on GPs was successful to sensitize them regarding calcium intake problems; GPs tended to increase the prescription of supplements but not to suggest changes in dietary habits.

Antigen (Ag)-specific tolerization prevents type 1 diabetes (T1D) in non-obese diabetic (NOD) mice but proved less effective in humans. Several auto-Ags are fundamental to disease development, suggesting T1D etiology is heterogeneous and may limit the effectiveness of Ag-specific therapies to distinct disease endotypes. Colonization factor antigen I (CFA/I) fimbriae from Escherichia coli can inhibit autoimmune diseases in murine models by inducing bystander tolerance. To test if Ag-independent stimulation of regulatory T cells (Tregs) can prevent T1D onset, groups of NOD mice were orally treated with Lactococcus lactis (LL) expressing CFA/I. LL-CFA/I treatment beginning at 6 weeks of age reduced disease incidence by 50% (p p +CD8+ T cells 6-fold at 11 weeks (p +CD4+ T cells were suppressed 10-fold (p +CD4+ T cells were suppressed (p

Background Sjögren’s syndrome (SjS), one of the most common autoimmune diseases, impacts millions of people annually. SjS results from autoimmune attack on exocrine (salivary and lacrimal) glands, and women are nine times more likely to be affected than men. To date, no vaccine or therapeutic exists to treat SjS, and patients must rely on lifelong therapies to alleviate symptoms. Methods Oral treatment with the adhesin from enterotoxigenic Escherichia coli colonization factor antigen I (CFA/I) fimbriae protects against several autoimmune diseases in an antigen (Ag)-independent manner. Lactococcus lactis, which was recently adapted to express CFA/I fimbriae (LL-CFA/I), effectively suppresses inflammation by the induction of infectious tolerance via Ag-specific regulatory T cells (Tregs), that produce IL-10 and TGF-β. To test the hypothesis that CFA/I fimbriae can offset the development of inflammatory T cells via Treg induction, oral treatments with LL-CFA/I were performed on the spontaneous, genetically defined model for SjS, C57BL/6.NOD-Aec1Aec2 mice to maintain salivary flow. Results Six-week (wk)-old C57BL/6.NOD-Aec1Aec2 mice were orally dosed with LL-CFA/I and treated every 3 wks; control groups were given L. lactis vector or PBS. LL-CFA/I-treated mice retained salivary flow up to 28 wks of age and showed significantly reduced incidence of inflammatory infiltration into the submandibular and lacrimal glands relative to PBS-treated mice. A significant increase in Foxp3+ and IL-10- and TGF-β-producing Tregs was observed. Moreover, LL-CFA/I significantly reduced the expression of proinflammatory cytokines, IL-6, IL-17, GM-CSF, and IFN-γ. Adoptive transfer of CD4+ T cells from LL-CFA/I-treated, not LL vector-treated mice, restored salivary flow in diseased SjS mice. Conclusion These data demonstrate that oral LL-CFA/I reduce or halts SjS progression, and these studies will provide the basis for future testing in SjS patients.

The benefits of mucosal vaccines over injected vaccines are difficult to ascertain since mucosally administered vaccines often induce serum antibody responses of lower magnitude than those induced by injected vaccines. This study aimed to determine if mucosal vaccination using a modified vaccinia Ankara expressing HIV-1 gp120 (MVA-g120) prime and HIV-1 gp120 protein boost could be optimized to induce serum antibody responses similar to those induced by an intramuscularly (IM) administered MVA prime/gp120 boost to allow comparison of an IM immunization regimen to a mucosal vaccination regimen for their ability to protect against a low dose rectal SHIV challenge while inducing similar serum anti-HIV-1 antibody responses. A 3-fold higher antigen dose was required for intranasal (IN) immunization with gp120 to induce serum anti-gp120 IgG responses not significantly different than those induced by IM immunization. Gp120 fused to the Adenovirus type 2 fiber binding domain (gp120-Ad2F), a mucosal targeting ligand, exhibited enhanced IN immunogenicity when compared to gp120 alone. MVA-gp120 was more immunogenic after IN delivery than gastric or rectal delivery, although serum antibodies induced by IN immunization were lower than those induced by intramuscular immunization. Using these optimized vaccines, an IN MVA-gp120 prime, combined IM (gp120) and IN (gp120-Ad2F) boost regimen (IN/IM+IN) induced serum anti-gp120 antibody titers similar to those induced by the intramuscular prime/boost regimen (IM/IM) in rabbits and non-human primates. Despite the induction of similar systemic anti-HIV-1 antibody responses, neither the IM/IM nor the IN/IM+IN regimen induced elevated anti-HIV-1 mucosal IgA responses nor protected against a repeated low-dose rectal SHIV challenge. These results demonstrate that immunization regimens utilizing the IN route are able to induce serum antigen-specific antibody responses similar to those induced by systemic immunizationIMPORTANCEMucosal vaccination is proposed as a method of immunization able to induce protection against mucosal pathogens that is superior to protection provided by parenteral immunization. However, mucosal vaccination often induces serum antigen-specific immune responses of lower magnitude than those induced by parenteral immunization, making the comparison of mucosal and parenteral immunization difficult. We identified vaccine parameters that allowed an immunization regimen consisting of an IN prime followed with boosters administered by both IN and IM routes to induce serum antibody responses similar to those induced by IM prime/boost vaccination. Additional studies are needed to determine the potential benefit of mucosal immunization for HIV-1 and other mucosally-transmitted pathogens.

Regulatory T cells (Tregs) induced during autoimmunity often become quiescent and unable to resolve disease, suggesting inadequate activation. Resolution of established experimental autoimmune encephalomyelitis (EAE) can be achieved with myelin oligodendrocyte glycoprotein (MOG) fused to reovirus protein σ1 (MOG-pσ1), which activates Tregs, restoring protection, but requiring other regulatory cells to revitalize them. B cells have a dichotomous role in both the pathogenesis and recovery from EAE. Although inflammatory B cells contribute to EAE’s pathogenesis, treatment of EAE mice with MOG-pσ1, but not OVA-pσ1, resulted in an influx of IL-10–producing B220+CD5+ B regulatory cells (Bregs) enabling Tregs to recover their inhibitory activity, and in turn, leading to the rapid amelioration of EAE. These findings implicate direct interactions between Bregs and Tregs to facilitate this recovery. Adoptive transfer of B220+CD5− B cells from MOG-pσ1–treated EAE or Bregs from PBS-treated EAE mice did not resolve disease, whereas the adoptive transfer of MOG-pσ1–induced B220+CD5+ Bregs greatly ameliorated EAE. MOG-pσ1–, but not OVA-pσ1–induced IL-10–producing Bregs, expressed elevated levels of B and T lymphocyte attenuator (BTLA) relative to CD5− B cells, as opposed to Tregs or effector T (Teff) cells, whose BTLA expression was not affected. These induced Bregs restored EAE Treg function in a BTLA-dependent manner. BTLA−/− mice showed more pronounced EAE with fewer Tregs, but upon adoptive transfer of MOG-pσ1–induced BTLA+ Bregs, BTLA−/− mice were protected against EAE. Hence, this evidence shows the importance of BTLA in activating Tregs to facilitate recovery from EAE.

IL-35, a relatively newly discovered cytokine belonging to the larger IL-12 family, shows unique anti-inflammatory properties, believed to be associated with dedicated receptors and signaling pathways. IL-35 plays a pivotal role in the development and the function of both regulatory B (Bregs) and T cells (Tregs). In order to further its therapeutic potential, a dairy Lactococcus lactis strain was engineered to express murine IL-35 (LL-IL35), and this recombinant strain was applied to suppress collagen-induced arthritis (CIA). Oral administration of LL-IL35 effectively reduced the incidence and disease severity of CIA. When administered therapeutically, LL-IL35 abruptly halted CIA progression with no increase in disease severity by reducing neutrophil influx into the joints. LL-IL35 treatment reduced IFN-γ and IL-17 3.7- and 8.5-fold, respectively, and increased IL-10 production compared to diseased mice. Foxp3+ and Foxp3− CD39+ CD4+ T cells were previously shown to be the Tregs responsible for conferring protection against CIA. Inquiry into their induction revealed that both CCR6+ and CCR6− Foxp3+or− CD39+ CD4+ T cells act as the source of the IL-10 induced by LL-IL35. Thus, this study demonstrates the feasibility and benefits of engineered probiotics for treating autoimmune diseases.

Recently we reported that IL-35 is an inflammation-induced ant-inflammatory cytokine; thus, function as a HAMP (homeostasis associated molecular patterns). However, whether IL-35 affects endothelial cell activation and subsequent development of atherosclerosis is not known. Hence, we studied the expression of IL-35 during early atherosclerosis; and its roles and mechanisms in suppressing EC activation. Initially, we analyzed whether endogenous IL-35 levels and its receptor expression was increased in early atherosclerosis. Our data revealed that IL-35 level was significantly increased in the plasma of hyperlipidemic patients and apolipoprotein E (ApoE-/-) deficient mice. Further, the expression of IL-35 receptor components were increased in ApoE-/- mouse aortas. IL-35 inhibited monocyte recruitment on to human aortic endothelial cells (EC) activated by pro-atherogenic lysophosphatidylcholine (LPC). Furthermore, our RNA-Seq analysis showed that IL-35 selectively inhibited ICAM-1 expression that mediates EC activation. Further analysis by flowcytometry and electron spin resonance analysis revealed that IL-35 blocked LPC-mediated mitochondrial-reactive oxygen species (mtROS) production. The subsequent mass spectrometry, ChIP-Seq analysis and EMSA showed that LPC-mediated mtROS promotes acetylation of histone 3 lysine 14 (H3K14). Acetylation of histones facilitate the transcription of EC activation-related genes. We found that acetylated H3K14 increased the binding of pro-inflammatory transcription factor Ap-1 on to ICAM-1 promoter region and induction of ICAM-1 gene transcription. IL-35 mediated Inhibition of mtROS attenuated H3K14 acetylation, inhibited AP-1 binding to ICAM-1 promoter and suppressed ICAM-1 transcription. Finally, IL-35 cytokine treatment inhibited the progression and development of atherosclerosis in ApoE-/- mice. Our study concluded that IL-35 is induced during atherosclerosis and plays an anti-atherogenic role by suppressing EC activation through mtROS and H3K14 acetylation dependent mechanism.

Vascular response is an essential pathological mechanism underlying various inflammatory diseases. This study determines whether IL-35, a novel responsive anti-inflammatory cytokine, inhibits vascular response in acute inflammation. Using a mouse model of LPS-induced acute inflammation and plasma samples from sepsis patients, we found that IL-35 was induced in the plasma of mice after LPS injection as well as in the plasma of sepsis patients. In addition, IL-35 decreased LPS-induced proinflammatory cytokines and chemokines in the plasma of mice. Furthermore, IL-35 inhibited leukocyte adhesion to the endothelium in the vessels of lung and cremaster muscle and decreased the numbers of inflammatory cells in bronchoalveolar lavage fluid. Mechanistically, IL-35 inhibited the LPS-induced up-regulation of endothelial cell (EC) adhesion molecule VCAM-1 through IL-35 receptors gp130 and IL-12Rβ2 via inhibition of the MAPK-activator protein-1 (AP-1) signaling pathway. We also found that IL-27, which shares the EBI3 subunit with IL-35, promoted LPS-induced VCAM-1 in human aortic ECs and that EBI3-deficient mice had similar vascular response to LPS when compared with that of WT mice. These results demonstrated for the first time that inflammation-induced IL-35 inhibits LPS-induced EC activation by suppressing MAPK-AP1-mediated VCAM-1 expression and attenuates LPS-induced secretion of proinflammatory cytokines/chemokines. Our results provide insight into the control of vascular inflammation by IL-35 and suggest that IL-35 is an attractive novel therapeutic reagent for sepsis and cardiovascular diseases.

Objective— IL-35 (interleukin-35) is an anti-inflammatory cytokine, which inhibits immune responses by inducing regulatory T cells and regulatory B cells and suppressing effector T cells and macrophages. It remains unknown whether atherogenic stimuli induce IL-35 and whether IL-35 inhibits atherogenic lipid-induced endothelial cell (EC) activation and atherosclerosis. EC activation induced by hyperlipidemia stimuli, including lysophosphatidylcholine is considered as an initiation step for monocyte recruitment and atherosclerosis. In this study, we examined the expression of IL-35 during early atherosclerosis and the roles and mechanisms of IL-35 in suppressing lysophosphatidylcholine-induced EC activation. Approach and Results— Using microarray and ELISA, we found that IL-35 and its receptor are significantly induced during early atherosclerosis in the aortas and plasma of ApoE (apolipoprotein E) knockout mice—an atherosclerotic mouse model—and in the plasma of hypercholesterolemic patients. In addition, we found that IL-35 suppresses lysophosphatidylcholine-induced monocyte adhesion to human aortic ECs. Furthermore, our RNA-sequencing analysis shows that IL-35 selectively inhibits lysophosphatidylcholine-induced EC activation-related genes, such as ICAM-1 (intercellular adhesion molecule-1). Mechanistically, using flow cytometry, mass spectrometry, electron spin resonance analyses, and chromatin immunoprecipitation-sequencing analyses, we found that IL-35 blocks lysophosphatidylcholine-induced mitochondrial reactive oxygen species, which are required for the induction of site-specific H3K14 (histone 3 lysine 14) acetylation, increased binding of proinflammatory transcription factor AP-1 in the promoter of ICAM-1, and induction of ICAM-1 transcription in human aortic EC. Finally, IL-35 cytokine therapy suppresses atherosclerotic lesion development in ApoE knockout mice. Conclusions— IL-35 is induced during atherosclerosis development and inhibits mitochondrial reactive oxygen species-H3K14 acetylation-AP-1–mediated EC activation.

Sjögren’s syndrome (SjS) is one of the most common autoimmune diseases impacting several million people every year in the US. SjS results from autoimmune attack of exocrine (salivary and lacrimal) glands, and women are nine times more likely to be affected than men. To date, no vaccine or therapeutic exists to cure SjS, and patients must rely on lifelong therapies to treat symptoms. Oral treatment with the adhesin from enterotoxigenic E. coli CFA/I fimbriae has been shown to protect against several autoimmune diseases in an antigen (Ag)-independent manner. Lactococcus lactis was recently adapted to express CFA/I (LL-CFA/I), and found effective in suppressing inflammation by the induction of infectious tolerance via Ag-specific regulatory T cells (Tregs) producing IL-10 and TGF-β. We hypothesized that in the spontaneous, genetically defined model for SjS in C57BL/6.NOD-Aec1Aec2 mice, oral treatments with LL-CFA/I would offset the development of inflammatory T cells via Treg induction. To test this, 6 wk-old C57BL/6.NOD-Aec1Aec2 mice were orally dosed with LL-CFA/I and treated every 3 wks; control groups were given L. lactis vector or PBS. We found a significant recovery in salivary flow following treatment with LL-CFA/I by 28 wks of age. Treatment also significantly reduced the incidence of inflammatory infiltrates into the submandibular and lacrimal glands compared to PBS-treated mice. Treg analysis revealed a significant 4-fold increase in Foxp3+ Tregs expressing CD25, not CD39, and produced a 3-fold increase in IL-10. These data show that orally dosing with LL-CFA/I weakens or halts SjS progression via activation of Tregs. These studies will provide the basis for future testing in SjS patients. Work supported by NIH DE026450.

Sublingual (s.l.) vaccination is an efficient way to induce elevated levels of systemic and mucosal immune responses. To mediate mucosal uptake, ovalbumin (OVA) was genetically fused to adenovirus 2 fiber protein (OVA-Ad2F) to assess whether s.l. immunization was as effective as an alternative route of vaccination. Ad2F-delivered vaccines were efficiently taken up by dendritic cells and migrated mostly to submaxillary gland lymph nodes, which could readily stimulate OVA-specific CD4(+) T cells. OVA-Ad2F + cholera toxin (CT)-immunized mice elicited significantly higher OVA-specific serum IgG, IgA and mucosal IgA antibodies among the tested immunization groups. These were supported by elevated OVA-specific IgG and IgA antibody-forming cells. A mixed T(h)-cell response was induced as evident by the enhanced IL-4, IL-10, IFN-γ and TNF-α-specific cytokine-forming cells. To assess whether this approach can stimulate neutralizing antibodies, immunizations were performed with the protein encumbering the β-trefoil domain of C-terminus heavy chain (Hcβtre) from botulinum neurotoxin A (BoNT/A) as well as when fused to Ad2F. Hcβtre-Ad2F + CT-dosed mice showed the greatest serum IgG, IgA and mucosal IgA titers among the immunization groups. Hcβtre-Ad2F alone also induced elevated antibody production in contrast to Hcβtre alone. Plasma from Hcβtre + CT- and Hcβtre-Ad2F + CT-immunized groups neutralized BoNT/A and protected mice from BoNT/A intoxication. Most importantly, Hcβtre-Ad2F + CT-immunized mice were protected from BoNT/A intoxication relative to Hcβtre + CT-immunized mice, which only showed ∼60% protection. This study shows that s.l. immunization with Ad2F-based vaccines is effective in conferring protective immunity.

The presence of illicit drugs and their metabolites in surface waters has to be considered a new type of hazard, still unknown, for the aquatic ecosystem, due to the potent pharmacological activities of all the illicit drugs. Our research was therefore aimed at evaluating the impact of illicit drugs on the aquatic fauna, till now still undervalued. To this aim, we verified the ability of the European eel (Anguilla anguilla), a well-known biomonitor of environmental contamination, to bioaccumulate cocaine, one of the most abundant illicit drugs found in surface waters. Silver eels were exposed to a nominal cocaine concentration of 20 ng/l for one month; at the same time, control, carrier and post-exposure recovery groups were made. Brains, gills, liver, kidney, muscle, gonads, spleen, digestive tract and sections of dorsal skin were assayed by high-pressure liquid chromatography (HPLC). Cocaine was found in the tissues of the treated eels, and, at low concentrations, in almost all tissues of post-exposure recovery eels. These results indicate that cocaine is able to accumulate into the eel tissues; its presence suggests potential risks for eels, since cocaine could affect their physiology and contribute to their decline, and for humans, consuming contaminated fish

Stimulation of Ag-specific inducible Treg can enhance resolution of autoimmune disease. Conventional methods to induce Treg often require induction of autoimmune disease or subjection to infection. Reovirus adhesin, protein σ1 (pσ1), can successfully facilitate tolerance when fused to a tolerogen. We tested whether myelin oligodendrocyte glycoprotein (MOG) fused to pσ1 (MOG-pσ1) can stimulate Ag-specific Treg. We show that C57BL/6 mice treated nasally with MOG-pσ1 fail to induce MOG-specific Abs and delayed-type hypersensitivity (DTH) responses and resist EAE. Such resistance was attributed to stimulation of Foxp3(+) Treg, as well as Th2 cells. MOG-pσ1's protective capacity was abrogated in IL-10(-/-) mice, but restored when adoptively transferred with MOG-pσ1-induced Treg. As a therapeutic, MOG-pσ1 diminished EAE within 24 h of nasal application, unlike recombinant MOG (rMOG), pσ1, or pσ1+rMOG, implicating the importance of Ag specificity by pσ1-based therapeutics. MOG-pσ1-treated mice showed elevated IL-4, IL-10, and IL-28 production by CD4(+) T cells, unlike rMOG treated or control mice that produced elevated IFN-γ or IL-17, respectively. These data show the feasibility of using pσ1 as a tolerogen platform for Ag-specific tolerance induction and highlight its potential use as an immunotherapeutic for autoimmunity.

Type 1 diabetes (T1D) is an autoimmune disease in which insulin-producing pancreatic β cells are destroyed by auto-aggressive T cells. Tregs in T1D have been shown to lose their suppressive function and/or become inflammatory, complicating Treg immunotherapy. Oral treatment with the adhesin from enterotoxigenic E. coli, CFA/I fimbriae, protects against autoimmunity. Although CFA/I fimbriae act in a bystander or in an antigen (Ag)-independent fashion, protection is ultimately dependent upon the induction and/or activation of auto-Ag-specific Tregs. Our initial findings showed that oral dosing with a Lactococcus lactis vector expressing CFA/I fimbriae (LL-CFA/I) reduces incidence of T1D in NOD mice by 45%. We hypothesized that dosing NOD mice more frequently would induce Foxp3+ Tregs in the pancreas and PaLNs to provide more robust protection. To test this, 4 wk-old NOD mice were orally dosed with LL-CFA/I varying the dosage and frequency of treatment. When dosed every 2 wks, the frequency of insulitis was reduced by more than half at 11 wks of age, and tetramer staining revealed a 2-fold reduction of insulin-specific CD4+ and CD8+ T cells within the PaLNs. Additional groups were followed until 16 wks of age, and examined for Treg and inflammatory T cell phenotypes. Treatment with LL-CFA/I reduced 2-fold IFN-γ-producing CD8+ T cells in the spleen and PaLNs. PaLN Foxp3+ T cells from the PBS and vector control groups, but not the LL-CFA/I-treated mice, co-expressed Tbet and IFN-γ, suggesting that Tregs from LL-CFA/I treated mice maintained their regulatory function at 16 wks. These data show that orally dosing with LL-CFA/I diminishes or halts T1D progression via activation of stably functional Foxp3+ Tregs. Work supported by AI121745.

The chemokine, lymphotactin (LTN), was tested as a molecular adjuvant using bicistronic DNA vaccines encoding the protective Yersinia capsular (F1) antigen and virulence antigen (V-Ag) as a F1-V fusion protein. The LTN-encoding F1-V or V-Ag vaccines were given by the intranasal (i.n.) or intramuscular (i.m.) routes, and although serum IgG and mucosal IgA antibodies (Abs) were induced, F1-Ag boosts were required for robust anti-F1-Ag Abs. Optimal efficacy against pneumonic plague was obtained in mice i.m.-, not i.n.-immunized with these DNA vaccines. These vaccines stimulated elevated Ag-specific Ab-forming cells and mixed Th cell responses, with Th17 cells markedly enhanced by i.m. immunization. These results show that LTN can be used as a molecular adjuvant to enhance protective immunity against plague., Vaccine, 28(18), pp.3219-3230; 2010

Previous studies have shown that mucosal application of interleukin-12 (IL-12) can stimulate elevated secretory immunoglobulin A (IgA) responses. Since possible exposure to plague is viaYersinia pestis-laden aerosols that results in pneumonic plague, arming both the mucosal and systemic immune systems may offer an added benefit for protective immunity. Two bicistronic plasmids were constructed that encoded the protective plague epitopes, capsular antigen (F1-Ag) and virulence antigen (V-Ag) as a F1-V fusion protein but differed in the amounts of IL-12 produced. When applied nasally, serum IgG and mucosal IgA anti-F1-Ag and anti-V-Ag titers were detectable beginning at week 6 after three weekly doses, and recombinant F1-Ag boosts were required to elevate the F1-Ag-specific antibody (Ab) titers. Following pneumonic challenge, the best efficacy was obtained in mice primed with IL-12(Low)/F1-V vaccine with 80% survival compared to mice immunized with IL-12(Low)/F1, IL-12(Low)/V, or IL-12(Low) vector DNA vaccines. Improved expression of IL-12 resulted in lost efficacy when using the IL-12(High)/F1-V DNA vaccine. Despite differences in the amount of IL-12 produced by the two F1-V DNA vaccines, Ab responses and Th cell responses to F1- and V-Ags were similar. These results show that IL-12 can be used as a molecular adjuvant to enhance protective immunity against pneumonic plague, but in a dose-dependent fashion.

Background & Aims: The follicle-associated epithelium (FAE) plays key roles in antigen uptake and subsequent induction of mucosal immunity. In this study, we examined whether M-cell targeting using a protein antigen (Ag) delivery system would induce oral tolerance instead of enhancement of Ag-specific mucosal antibody (Ab) responses. Methods: Mice were fed different doses of a recombinant protein sigma 1 of reovirus genetically conjugated to ovalbumin (OVA-pσ1), pσ1 only, or phosphate-buffered saline (PBS) before oral challenge with OVA plus cholera toxin as mucosal adjuvant. OVA-specific Ab and CD4-positive (CD4+) T-cell responses were determined. Results: A low dose of OVA-pσ1 reduced anti-OVA Ab and CD4+ T-cell responses in both mucosal and systemic lymphoid tissues. OVA/MHC I-Ad tetramer staining showed that the numbers of OVA-specific CD4+ T cells were significantly reduced in lamina propria of mice fed OVA-pσ1 than those fed pσ1 only or PBS only. In fact, Foxp3 expressing CD25+ CD4+ T cells were markedly increased in this tissue. Nonetheless, CD25+ CD4+ T cells from the spleen, mesenteric lymph nodes, and Peyer's patches of orally tolerized mice showed increased transforming growth factor β1 (TGF-β1) and interleukin-10 (IL-10) production compared with nontolerized mice. Conclusions: These results show that an FAE M-cell targeting protein Ag delivery system facilitates oral tolerance induction because of a reduction in Ag-specific CD4+ T cells and increased levels of TGF-β1 and IL-10 producing, CD25+ CD4+ regulatory T cells in both systemic and mucosal lymphoid tissues.

Mucosal tolerance induction generally requires multiple or large Ag doses. Because microfold (M) cells have been implicated as being important for mucosal tolerance induction and because reovirus attachment protein σ1 (pσ1) is capable of binding M cells, we postulated that targeting a model Ag to M cells via pσ1 could induce a state of unresponsiveness. Accordingly, a genetic fusion between OVA and the M cell ligand, reovirus pσ1, termed OVA-pσ1, was developed to enhance tolerogen uptake. When applied nasally, not parenterally, as little as a single dose of OVA-pσ1 failed to induce OVA-specific Abs even in the presence of adjuvant. Moreover, the mice remained unresponsive to peripheral OVA challenge, unlike mice given multiple nasal OVA doses that rendered them responsive to OVA. The observed unresponsiveness to OVA-pσ1 could be adoptively transferred using cervical lymph node CD4+ T cells, which failed to undergo proliferative or delayed-type hypersensitivity responses in recipients. To discern the cytokines responsible as a mechanism for this unresponsiveness, restimulation assays revealed increased production of regulatory cytokines, IL-4, IL-10, and TGF-β1, with greatly reduced IL-17 and IFN-γ. The induced IL-10 was derived predominantly from FoxP3+CD25+CD4+ T cells. No FoxP3+CD25+CD4+ T cells were induced in OVA-pσ1-dosed IL-10-deficient (IL-10−/−) mice, and despite showing increased TGF-β1 synthesis, these mice were responsive to OVA. These data demonstrate the feasibility of using pσ1 as a mucosal delivery platform specifically for low-dose tolerance induction.

Bacteria associated with varroa mites were cultivated and genotyped by 16S RNA. Under our experimental conditions, the cultivable bacteria were few in number, and most of them proved to be fastidious to grow. Cultivation with seven different media under O2/CO2 conditions and selection for colony morphology yielded a panel of species belonging to 13 different genera grouped in two different phyla, proteobacteria and actinobacteria. This study identified one species of actinobacteria that is a known commensal of the honey bee. Some isolates are oxalotrophic, a finding that may carry ramifications into the use of oxalic acid to control the number of phoretic mites in the managed colonies of honey bees. Significance and Impact of the Study Oxalic acid, legally or brevi manu, is widely used to control phoretic Varroa destructor mites, a major drive of current honey bees' colony losses. Unsubstantiated by sanctioned research are rumours that in certain instances oxalic acid is losing efficacy, forcing beekeepers to increase the frequency of treatments. This investigation fathoms the hypothesis that V. destructor associates with bacteria capable of degrading oxalic acid. The data show that indeed oxalotrophy, a rare trait among bacteria, is common in bacteria that we isolated from V. destructor mites. This finding may have ramifications in the use of oxalic acid as a control agent.

A/B toxins, produced by bacteria and plants, are among the deadliest molecules known. The B chain binds the cell, whereas the A chain exerts the toxic effect. Both anti-A chain and anti-B chain Abs can neutralize toxins in vivo and in vitro. B chain Abs block binding of the toxin to the cell. It is not known how anti-A chain Abs function. Working with ricin toxin, we demonstrate that immunization with A chain induces greater protection than immunization with B chain. A panel of mAbs, binding to A chain, B chain, or both chains, has been produced and characterized. Immunologic characteristics evaluated include isotype, relative avidity, and epitope specificity. The ability to inhibit ricin enzymatic or cell binding activity was studied, as was the ability to block ricin-mediated cellular cytotoxicity on human and murine cell lines. Finally, the in vivo protective efficacy of the Abs in mice was studied. The Ab providing the greatest in vivo protective efficacy was directed against the A chain. It had the greatest relative avidity and the greatest ability to block enzymatic function and neutralize cytotoxicity. Interestingly, we also obtained an anti-A chain Ab that bound with high avidity, blocked enzymatic activity, did not neutralize cytotoxicity, and actually enhanced the in vivo toxicity of ricin. Anti-A chain Abs with moderate avidity had no in vivo effect, nor did any anti-B chain Abs.

Oral treatment with the adhesin from enterotoxigenic E. coli, CFA/I fimbriae protect in murine models of multiple sclerosis and rheumatoid arthritis. Although CFA/I fimbriae initial mode of action is in a bystander or in an antigen (Ag)-independent fashion, protection was found to be ultimately dependent upon the induction and/or activation of auto-Ag-specific regulatory T cells (Tregs). Our recent findings show that oral dosing with a L. lactis vector expressing CFA/I fimbriae (LL-CFA/I) reduces incidence of T1D in NOD mice by 45% with a concomitant 8-fold increase in their splenic Foxp3+CD25+ Tregs. However, little is known about how protection transitions from bystander suppression to Ag-specific Tregs. We hypothesized that LL-CFA/I stimulates dendritic cells (DCs) to establish a regulatory microenvironment since DCs play an integral role in fate decisions for T cells becoming inflammatory or tolerogenic. In this study, 4 wk-old NOD mice were orally dosed with LL-CFA/I and treated every 2 wks; control groups were given L. lactis vector or PBS. At 11 wks of age, frequency of insulitis was reduced by more than half, and insulin-specific T cells were reduced in the pancreatic lymph nodes (PaLNs; P < 0.05). To discern how this was mediated, groups of mice were examined at 3, 7, 14, and 21 days post-treatment for changes in DC and T cell phenotypes. As early as 3 days post-treatment, DCs exhibited significantly less expression of costimulatory molecules, CD40 and CD86, and stably maintained this phenotype for at least 7 wks. Splenic DCs showed reduced IL-6 production. These data show that orally dosing with LL-CFA/I ameliorates T1D in NOD mice by establishing a regulatory microenvironment via DCs. Work is supported by AI121745.

The presence of illicit drugs and their metabolites in surface waters has to be considered a new type of hazard, still unknown, for both the human health and the aquatic ecosystem, due to the potent pharmacological activities of all the illicit drugs. Our research was aimed at evaluating the presence of illicit drugs in the Sarno River (Campania region, Italy), crossing a densely populated area, the basin of the Sarno River, one of the largest and most important economic areas in Campania region, famous for the presence of zones with high landscape-environmental value. The drugs selected for this study were the most used in Campania region. The presence of illicit drugs in surface water was analyzed by a selective multi-residue assay based on liquid chromatographytandem mass spectrometry. The analysis showed the presence of all the illicit drugs investigated: cocaine and its main metabolites (benzoylecgonine, nor-benzoylecgonine), morphine, THC-COOH and codeine; cocaine was the most abundant illicit drug. The presence of illicit drugs and their metabolites in the Sarno River suggests new potential risk for the basin’s inhabitants, using water in the food chain, via field irrigation and animal feed, and for the health of the aquatic fauna.

The maize b-32 protein is a functional ribosome-inactivating protein (RIP), inhibiting in vitro translation in the cell-free reticulocyte-derived system and having specific N-glycosidase activity on 28S rRNA. Previous results indicated that opaque-2 (o2) mutant kernels, lacking b-32, show an increased susceptibility to fungal attack and insect feeding and that ectopic expression in plants of a barley and a pokeweed RIP leads to increased tolerance to fungal and viral infection. This prompted us to test whether b-32 might functi on as a protectant against pathogens. The b32.66 cDNA clone under the control of the potato wun1 gene promoter was introduced into tobacco by Agrobacterium tumefaciens-mediated transformation. Out of 23 kanamycin resistant regenerated shoots, 16 contained a PCR fragment of the corrrect size spanning the boundary between the promoter used and the coding region of the b-32 gene. Eight independently transformed tobacco lines were randomly chosen for protein analysis: all of them expressed b-32 protein. The data presented indicate that transgenic tobacco plants expressing b-32 show an increased tolerance against infection by the soil-borne fungal pathogen Rhizoctonia solani Kuhn

A Salmonella therapeutic expressing enterotoxigenic Escherichia coli colonization factor Ag I (CFA/I) fimbriae protects against collagen-induced arthritis (CIA) by eliciting two regulatory T cell (Treg) subsets: TGF-β–producing Foxp3−CD39+CD4+ T cells and IL-10–producing Foxp3+CD39+CD4+ T cells. However, it is unclear whether CFA/I fimbriae alone are protective and whether other regulatory cytokines are involved, especially in the context for the EBI3-sharing cytokines, Treg-derived IL-35 and APC-derived IL-27, both capable of suppressing Th17 cells and regulating autoimmune diseases. Subsequent evaluation revealed that a single oral dose of purified, soluble CFA/I fimbriae protected against CIA as effectively as did Salmonella-CFA/I and found that Foxp3+CD39+CD4+ T cells were the source of secreted IL-35, whereas IL-27 production by CD11c+ cells was inhibited. Inquiring into their relevance, CFA/I fimbriae–treated IL-27R–deficient (WSX-1−/−) mice were equally protected against CIA as were wild-type mice, suggesting a limited role for IL-27. In contrast, CFA/I fimbriae–mediated protection was abated in EBI3−/− mice, accompanied by the loss of TGF-β– and IL-10–producing Tregs. Adoptive transfer of C57BL/6 CD39+CD4+ T cells to EBI3−/− mice with concurrent CFA/I plus IL-35 treatment effectively stimulated Tregs suppressing proinflammatory collagen II–specific Th cells. In contrast, recipients cotransferred with C57BL/6 and EBI3−/− CD39+CD4+ T cells and treated with CFA/I plus IL-35 were not protected, implicating the importance of endogenous IL-35 for conferring CFA/I-mediated protection. Thus, CFA/I fimbriae stimulate IL-35 required for the coinduction of TGF-β and IL-10.

The aim of the present study was to verify if cocaine, at environmental concentrations, influences the endocrine system of the European eel. Silver eels (a stage of the eel life cycle preparing the fish for the oceanic reproductive migration) were exposed to a nominal cocaine concentration of 20 ng/l during 30 days; at the same time, control, carrier, and postexposure recovery groups were made. The effects of cocaine were observed in (1) brain dopamine content, (2) plasma catecholamine levels (dopamine, norepinephrine, and epinephrine), (3) pituitary–adrenal axis activity [plasma adrenocorticotropic hormone (ACTH), corticosterone, cortisol, and aldosterone levels], and (4) pituitary–thyroid axis activity [plasma thyroid-stimulating hormone (TSH), triiodothyronine, and thyroxine levels]. In the treated group, brain dopamine, plasma catecholamines, cortisol, and TSH levels were higher, whereas ACTH, corticosterone, and triiodothyronine levels were lower than controls. In the postexposure recovery group, brain dopamine, plasma dopamine and epinephrine, and thyroxine levels further increased, whereas plasma norepinephrine, cortisol, and corticosterone levels were similar to treated values. Finally, ACTH and TSH were similar, whereas triiodothyronine levels were lower than controls. Aldosterone levels were unaffected by cocaine exposure. The results of the present study show that cocaine, at environmental concentrations, behaves like an endocrine disruptor changing brain dopamine and plasma catecholamine levels and the activity of pituitary–adrenal/thyroid axes. Since the endocrine system plays a key role in the metabolic and reproductive processes of the eel, our results suggest that environmental cocaine could be considered another cause for the decline in the European eel.

The maize Opaque-2 (O2) protein is a transcription factor of the basic/leucine-zipper class, involved in the regulation of endosperm proteins including the 22kDa alpha-zein storage proteins and b32 protein. In this study we have focussed our attention on the relationship between O2 and the cyPPDK1 gene, which encodes a cytoplasmic pyruvate orthophosphate dikinase (PPDK) isoform. The results of this study showed that PPDK activity is detectable in wild-type maize endosperms, while in o2 mutant endosperms, the levels of PPDK protein, mRNA and enzymatic activity are reduced, indicating that O2 is involved in the regulation of cyPPDK1 in this tissue. By employing transient expression experiments in tobacco mesophyll protoplasts, we have demonstrated that the O2 protein can activate expression of a chloramphenicol acetyl transferase reporter gene placed under the control of the cyPPDK1 promoter. An in vitro binding assay and DNaseI footprint analysis demonstrated that a specific sequence in the cyPPDK1 promoter can be recognized and protected by maize O2 protein. The regulation by the O2 locus of cyPPDK1 reported here, and control of alpha-zein synthesis by O2 suggest that the O2 protein may play a more general role in maize endosperm development than previously thought.

The Glossy2 (Gl2) locus of maize is required for the formation of the epicuticular wax layer of young plants. gl2 mutant seedlings can be visually identified because of their glossy leaf surface which is different from the dull surface of wild-type seedlings. The Gl2 locus was isolated by transposon tagging. Seven unstable mutations, gl2-m2 to gl2-m8, were induced in a parental strain carrying an active transposable Activator (Ac) element in the unstable wx-m7 allele. Genetic tests on the gl2-m2 allele indicated that it was not caused by the Ac element but by the insertion of the transposable element Enhancer/Suppressor-Mutator (En/Spm). A Sa/l restriction fragment segregating with the mutant phenotype was identified, by Southern analysis, using sequences from the En/Spm element as a probe. Part of the fragment was cloned and was shown to carry part of the unstable gl2-m2 allele. These gl2 sequences were used to identify a genomic fragment carrying the wild-type allele and to isolate its corresponding cDNA sequence. The predicted Glossy2 protein consists of 426 amino acids. No similar amino acid sequence was found in protein data banks and the biochemical function of the Gl2 gene product is still unknown. The wild-type Gl2 transcript is found predominantly in juvenile leaves. The transcript level in the leaves of seedlings homozygous for a stable recessive gl2-ref allele is hardly detectable.

Summary The major seed storage proteins of cereals are deposited in the maturation phase of endosperm development, starting at 12 days after pollination (dap) in maize and continuing up to around 30dap. Cell types-pecific expression in the endosperm is conferred by a multicomponent promoter motif, the endosperm box sequence. Proteins interacting at this motif include the gene product of the regulatory gene Opaque-2 (O2). O2 encodes a b2IP transcription factor, which is capable of activating its target promoters, 22kD α-zein and b-32, in a number of experimental systems. Storage protein accumulation, and storage protein promoter activity, are modulated according to available levels of nitrogen (N). In barley, nitrogen regulation is mediated by the endosperm box via interaction of the endosperm and GCN 4 motifs. The nitrogen- responsive components of the 22kd zein promoter have been shown to include the O2-binding sites Z1–Z3 but to display N-responsiveness even in the absence of O2 protein.

The aim of our study was to verify whether environmental concentrations of nonylphenol influenced the adrenal gland of Triturus carnifex. Newts were exposed to 19 µg/l nominal concentration of nonylphenol throughout the periods of December-January and March-April, corresponding to different stages of the chromaffin cell functional cycle. The morphological features of the steroidogenic and chromaffin tissues, and the serum levels of ACTH, aldosterone, corticosterone, norepinephrine and epinephrine were evaluated. Nonylphenol did not influence ACTH serum levels. During the two periods examined, the steroidogenic tissue had the same reaction: the quantity of cytoplasmic lipids, and the corticosteroid serum levels, decreased, suggesting the inhibition of synthesis and release of corticosteroids. During the two periods examined, the chromaffin tissue reacted differently to nonylphenol. During December-January, the numeric ratio of norepinephrine granules to epinephrine granules, and the epinephrine serum levels, increased, suggesting the stimulation of epinephrine release. During March-April, the numeric ratio of norepinephrine granules to epinephrine granules did not change, and the norepinephrine serum levels decreased, suggesting the inhibition of norepinephrine release. Our results show that nonylphenol influences the activity of the newt adrenal gland; considering the physiological role of this gland, our results suggest that nonylphenol may contribute to amphibian decline.

Natural killer (NK) cells and dendritic cells (DCs) have been shown to link the innate and adaptive immune systems. Likewise, a new innate cell subset, interferon-producing killer DCs (IKDCs), shares phenotypic and functional characteristics with both DCs and NK cells. Here, we show IKDCs play an essential role in the resolution of experimental autoimmune encephalomyelitis (EAE) upon treatment with the tolerizing agent, myelin oligodendrocyte glycoprotein (MOG), genetically fused to reovirus protein σ1 (termed MOG-pσ1). Activated IKDCs were recruited subsequent MOG-pσ1 treatment of EAE, and disease resolution was abated upon NK1.1 cell depletion. These IKDCs were able to kill activated CD4(+) T cells and mature dendritic DCs, thus, contributing to EAE remission. In addition, IKDCs were responsible for MOG-pσ1-mediated MOG-specific regulatory T cell recruitment to the CNS. The IKDCs induced by MOG-pσ1 expressed elevated levels of HVEM for interactions with cognate ligand-positive cells: LIGHT(+) NK and T(eff) cells and BTLA(+) B cells. Further characterization revealed these activated IKDCs being MHC class II(high), and upon their adoptive transfer (CD11c(+)NK1.1(+)MHC class II(high)), IKDCs, but not CD11c(+)NK1.1(+)MHC class II(intermediate/low) (unactivated) cells, conferred protection against EAE. These activated IKDCs showed enhanced CD107a, PD-L1, and granzyme B expression and could present OVA, unlike unactivated IKDCs. Thus, these results demonstrate the interventional potency induced HVEM(+) IKDCs to resolve autoimmune disease.

Background We previously reported that the immunogenicity of Hcβtre, a botulinum neurotoxin A (BoNT/A) immunogen, was enhanced by fusion to an epithelial cell binding domain, Ad2F, when nasally delivered to mice with cholera toxin (CT). This study was performed to determine if Ad2F would enhance the nasal immunogenicity of Hcβtre in rabbits, an animal model with a nasal cavity anatomy similar to humans. Since CT is not safe for human use, we also tested the adjuvant activity of compound 48/80 (C48/80), a mast cell activating compound previously determined to safely exhibit nasal adjuvant activity in mice. Methods New Zealand White or Dutch Belted rabbits were nasally immunized with Hcβtre or Hcβtre-Ad2F alone or combined with CT or C48/80, and serum samples were tested for the presence of Hcβtre-specific binding (ELISA) or BoNT/A neutralizing antibodies. Results Hcβtre-Ad2F nasally administered with CT induced serum anti-Hcβtre IgG ELISA and BoNT/A neutralizing antibody titers greater than those induced by Hcβtre + CT. C48/80 provided significant nasal adjuvant activity and induced BoNT/A-neutralizing antibodies similar to those induced by CT. Conclusions Ad2F enhanced the nasal immunogenicity of Hcβtre, and the mast cell activator C48/80 was an effective adjuvant for nasal immunization in rabbits, an animal model with a nasal cavity anatomy similar to that in humans.

The protein encoded by the Opaque-2 (O2) gene is a transcription factor, translated from an mRNA that possesses an unusually long 5' leader sequence containing three upstream open reading frames (uORFs). The efficiency of translation of O2 mRNA has been tested in vivo by a transient assay in which the level of activation of the b32 promoter, a natural target of O2 protein, is measured. We show that uORF-less O2 alleles possess a higher transactivation value than the wild-type allele and that the reduction in transactivation due to the uORFs is a cis-dominant effect. The data presented indicate that both uORF1 and uORF2 are involved in the reducing effect and suggest that both are likely to be translated.

Background Immunization with recombinant carboxyl-terminal domain of the heavy chain (Hc domain) of botulinum neurotoxin (BoNT) stimulates protective immunity against native BoNT challenge. Most studies developing a botulism vaccine have focused on the whole Hc; however, since the principal protective epitopes are located within β-trefoil domain (Hcβtre), we hypothesize that immunization with the Hcβtre domain is sufficient to confer protective immunity. In addition, enhancing its uptake subsequent to nasal delivery prompted development of an alternative vaccine strategy, and we hypothesize that the addition of targeting moiety adenovirus 2 fiber protein (Ad2F) may enhance such uptake during vaccination. Results The Hcβtre serotype B immunogen was genetically fused to Ad2F (Hcβtre/B-Ad2F), and its immunogenicity was tested in mice. In combination with the mucosal adjuvant, cholera toxin (CT), enhanced mucosal IgA and serum IgG Ab titers were induced by nasal Hcβtre-Ad2F relative to Hcβtre alone; however, similar Ab titers were obtained upon intramuscular immunization. These BoNT/B-specific Abs induced by nasal immunization were generally supported in large part by Th2 cells, as opposed to Hcβtre-immunized mice that showed more mixed Th1 and Th2 cells. Using a mouse neutralization assay, sera from animals immunized with Hcβtre and Hcβtre-Ad2F protected mice against 2.0 LD50. Conclusion These results demonstrate that Hcβtre-based immunogens are highly immunogenic, especially when genetically fused to Ad2F, and Ad2F can be exploited as a vaccine delivery platform to the mucosa.

Pneumonic plague remains problematic in endemic areas, and because it can be readily transmitted and has high mortality, the development of efficacious vaccines is warranted. To test whether stimulation of cell-mediated immunity with IL-12 will improve protective immunity against plague, we constructed two IL-12 DNA vaccines using a bicistronic plasmid encoding the protective plague epitopes, capsular (F1) antigen and virulence antigen (V-Ag) as F1-V fusion protein and V-Ag only, respectively. When applied intramuscularly, antibody responses to F1- and V-Ag were detectable beginning at week 6 after three weekly doses, and F1-Ag protein boosts were required to induce elevated Ab responses. These Ab responses were supported by mixed Th cell responses, and the IL-12/V-Ag DNA vaccine showed greater cell-mediated immune bias than IL-12/F1-V DNA vaccine. Following pneumonic challenge, both IL-12 DNA vaccines showed similar efficacy despite differences in Th cells simulated. These results show that IL-12 can be used as a molecular adjuvant to enhance protective immunity against pneumonic plague.

We previously reported that the inclusion of the trimerizing and knob domains from adenovirus type 2 fiber (Ad2F) in protein immunogens enhanced their mucosal binding and immunogenicity when delivered nasally. This study was performed to determine if Ad2F would enhance the nasal immunogenicity of HIV-1 gp120 from the Clade C Env C.1086. A fusion protein with Ad2F at the C-terminus of HIV-1 gp120 was used as gp120-Ad2F. NZW rabbits were nasally immunized with 100, 200 or 300 µg of gp120 or equimolar doses of gp120-Ad2F fusion proteins adjuvanted with the cationic mast cell activating peptide mastoparan 7 on days 0 and boosted on days 28 - 34. Intramuscular immunization with 100 µg gp120 adjuvanted with an MF59-like adjuvant served as the positive control. Serum anti-HIV-1 gp120 IgG titers were measured 2 weeks after the final boost. While only the 200 and 300 µg doses of gp120 delivered intranasally induced serum anti-gp120 IgG similar to those induced by IM immunization, all doses of gp120-Ad2F fusion proteins induced serum anti-gp120 IgG titers similar to those induced by IM immunization. While the 100 µg dose of gp120 delivered nasally induced serum anti-gp120 IgG titers significantly lower than that induced by 300 µg of gp120 delivered nasally, all doses of gp120-Ad2F induced similar anti-gp120 IgG titers. Our results demonstrate that the addition of Ad2F to HIV-1 gp120 enhances its nasal immunogenicity, lessening the amount of gp120 required for vaccination.

Autoimmune diseases arise from the loss of tolerance to self, and because the etiologies of such diseases are largely unknown, symptomatic treatments rely on anti-inflammatory and analgesic agents. Tolerogenic treatments that can reverse disease are preferred, but again, often thwarted by not knowing the responsible auto-antigens (auto-Ags). Hence, a viable alternative to stimulating regulatory T cells (Tregs) is to induce bystander tolerance. Colonization factor antigen I (CFA/I) has been shown to evoke bystander immunity and to hasten Ag-specific Treg development independent of auto-Ag. To translate in treating human autoimmune diseases, the food-based Lactococcus was engineered to express CFA/I fimbriae, and Lactococcus-CFA/I fermented milk fed to arthritic mice proved highly efficacious. Protection occurred via CD39+ Tregs producing TGF-β and IL-10 to potently suppress TNF-α production and neutrophil influx into the joints. Thus, these data demonstrate the feasibility of oral nutraceuticals for treating arthritis, and potency of protection against arthritis was improved relative to that obtained with Salmonella-CFA/I.

Absence of suitable mucosal adjuvants for humans prompted us to consider alternative vaccine designs for mucosal immunization. Because adenovirus is adept in binding to the respiratory epithelium, we tested the adenovirus 2 fiber protein (Ad2F) as a potential vaccine-targeting molecule to mediate vaccine uptake. The vaccine component (the host cell-binding domain to botulinum toxin (BoNT) serotype A) was genetically fused to Ad2F to enable epithelial binding. The binding domain for BoNT was selected because it lies within the immunodominant H chain as a β-trefoil (Hcβtre) structure; we hypothesize that induced neutralizing Abs should be protective. Mice were nasally immunized with the Hcβtre or Hcβtre-Ad2F, with or without cholera toxin (CT). Without CT, mice immunized with Hcβtre produced weak secretory IgA (sIgA) and plasma IgG Ab response. Hcβtre-Ad2F-immunized mice produced a sIgA response equivalent to mice coimmunized with CT. With CT, Hcβtre-Ad2F-immunized mice showed a more rapid onset of sIgA and plasma IgG Ab responses that were supported by a mixed Th1/Th2 cells, as opposed to mostly Th2 cells by Hcβtre-dosed mice. Mice immunized with adjuvanted Hcβtre-Ad2F or Hcβtre were protected against lethal BoNT serotype A challenge. Using a mouse neutralization assay, fecal Abs from Hcβtre-Ad2F or Hcβtre plus CT-dosed mice could confer protection. Parenteral immunization showed that the inclusion of Ad2F enhances anti-Hcβtre Ab titers even in the absence of adjuvant. This study shows that the Hcβtre structure can confer protective immunity and that use of Hcβtre-Ad2F gives more rapid and sustained mucosal and plasma Ab responses.

In an effort to develop a non-lethal model of ricin toxicosis, we studied the biochemical effects of the administration of ricin in mice.Mice received an intraperitoneal injection of ricin toxin, after which a panel of 21 biochemical parameters was determined. Effect of ricin administration on blood glucose concentration was studied in greater detail. To determine whether results of biochemical analyses correlated with therapeutic manipulations known to protect against ricin toxicosis, the effect of immunization on blood glucose values after ricin administration was studied.Of the biochemical parameters studied, only blood glucose and amylase values were affected by ricin administration. Because blood glucose concentration can be easily and instantaneously measured on 25 to 50 microl of blood, using handheld instruments, this parameter was further evaluated. A dose- and time-related decrease in blood glucose concentration was documented. Mice immunized with ricin had smaller changes in blood glucose concentration than did non-immunized animals after ricin administration.Blood glucose concentration may be used as a surrogate for lethal challenge as a measure of ricin toxicosis after its systemic administration.

To date, efforts to treat autoimmune diseases have primarily focused on the disease symptoms rather than on the cause of the disease. In large part, this is attributed to not knowing the responsible auto-antigens (auto-Ags) for driving the self-reactivity coupled with the poor success of treating autoimmune diseases using oral tolerance methods. Nonetheless, if tolerogenic approaches or methods that stimulate regulatory T (Treg) cells can be devised, these could subdue autoimmune diseases. To forward such efforts, our approach with colonization factor antigen I (CFA/I) fimbriae is to establish bystander immunity to ultimately drive the development of auto-Ag-specific Treg cells. Using an attenuated Salmonella vaccine expressing CFA/I fimbriae, fimbriae-specific Treg cells were induced without compromising the vaccine's capacity to protect against travelers' diarrhea or salmonellosis. By adapting the vaccine's anti-inflammatory properties, it was found that it could also dampen experimental inflammatory diseases resembling multiple sclerosis (MS) and rheumatoid arthritis. Because of this bystander effect, disease-specific Treg cells are eventually induced to resolve disease. Interestingly, this same vaccine could elicit the required Treg cell subset for each disease. For MS-like disease, conventional CD25(+) Treg cells are stimulated, but for arthritis CD39(+) Treg cells are induced instead. This review article will examine the potential of treating autoimmune diseases without having previous knowledge of the auto-Ag using an innocuous antigen to stimulate Treg cells via the production of transforming growth factor-β and interleukin-10.

The maize b-32 protein is a functional ribosome-inactivating protein (RIP), inhibiting in vitro translation in the cell-free reticulocyte-derived system and having specific N-glycosidase activity on 28S rRNA. previous results indicated that opaque-2 (o2) mutant kernels, lacking b-32, show an increased susceptibility to fungal attack and insect feeding and that ectopic expression in plants of a barley and a pokeweed RIP leads to increased tolerance to fungal and viral infection. This prompted us to test whether b-32 might function as a protectant against pathogens. The b32.66 cDNA clone under the control of the potato wunl gene promoter was introduced into tobacco by Agrobacterium tumefaciens-mediated transformation. Out of 23 kanamycin resistant regenerated shoots, 16 contained a PCR fragment of the corrrect size spanning the boundary between the promoter used and the coding region of the b-32 gene. Eight independently transformed tobacco lines were randomly chosen for protein analysis: all of them expressed b-32 protein. The data presented indicate that transgenic tobacco plants expressing b-32 show an increased tolerance against infection by the soil-borne fungal pathogen Rhizoctonia solani Kuhn. The exploitation of b-32 protein in immunoconjugates directed against HIV-infected cells is also discussed.

Orally delivered colonization factor antigen I (CFA/I) fimbriae stimulate IL-35 producing Foxp3+CD39+CD4+ regulatory T cells (Tregs) essential against CIA in C57BL/6 mice. To begin to understand how CFA/I-induced IL-35 mediates protection, recent studies have begun to examine immunosuppressive mediators by dendritic cells (DCs). It was found that IDO was induced in splenic, mesenteric lymph nodes (MLNs), and inguinal, axillary, and iliac lymph nodes (LNs) DCs in protected mice. We hypothesize that CFA/I-induced IL-35 could directly regulate development of IDO+ DCs to sustain anti-inflammatory responses. By flow cytometry, recombinant IL-35 was found capable of binding DCs in vitro suggesting direct action of IL-35 upon DCs. Mice deficient in IL-35 (EBI3-/-) were unresponsive to CFA/I fimbriae and unable to induce IDO. Similar results were observed in CD39-/- mice correlating with the lack of clinical interventions. To inhibit IL-35's impact, CFA/I- or PBS-dosed C57BL/6 mice were treated in vivo with rabbit IgG anti-mouse IL-35 during arthritis manifestation resulting in 50% suppression of intracellular IDO expression along with significantly enhanced CIA severity when compared to nonimmune IgG-treated mice. Adoptive transfer of CFA/I-induced CD39+CD4+ Tregs to mice induced with CIA elevated IDO expression by DCs. Thus, our findings demonstrate the effect of induced IL-35 by Tregs upon IDO+ DCs in conferring protection against CIA. Supported by NIH P01 AT-04986.