Oral administration of modified live canine parvovirus type 2b induces systemic immune response.

Different strategies have been proposed to overcome maternally derived antibody (MDA) interference with canine parvovirus type 2 (CPV-2) immunisation, including intranasal vaccination, which presents some practical limitations. In the present study, the results of the oral administration of a commercial CPV-2b modified live virus (MLV) vaccine in pups with MDA are reported. The CPV-2b vaccine was orally administered to 14 6-week-old pups with a bait. Blood samples and rectal swabs were collected at different days post-vaccination (dpv) to determine CPV-2 antibody titres and DNA loads. Thirteen pups were positive to serological and virological tests after the first vaccination and one pup became positive after the second vaccine administration. The findings of this study suggest that systemic immunity against CPV-2 may be achieved by the use of an MLV CPV-2b vaccine administered orally even in the presence of MDA titres that usually interfere with vaccination.

In vitro virucidal activity of sodium hypochlorite against canine parvovirus type 2.

Canine parvovirosis is a very contagious, severe and often lethal infectious disease of dogs caused by canine parvovirus type 2 (CPV-2). Parvoviruses are very resistant to several disinfectants while are sensitive to halogens such as sodium hypochlorite which is often used for decontamination of veterinary clinics and animal housing facilities due to its broad spectrum of activity. If compliance with vaccination programmes and with proper disinfection plans is ensured, there should be no continuous, nor frequent, CPV-2 outbreaks in kennels and veterinary clinics. However, a continuous spread of CPV-2 infections is observed, even in kennels where an appropriate vaccination programme is applied, and this imposes a re-evaluation of disinfection protocols using sodium hypochlorite. The aim of the present study was to determine the effect of concentration, contact time and presence of organic matter on the virucidal activity of sodium hypochlorite against several CPV-2 strains. A sensitive in vitro assay capable of measuring the infectivity of CPV-2 was employed to determine the efficacy of three different concentrations of sodium hypochlorite. The data indicate that using a 0.75% sodium hypochlorite solution for a short contact time (1 min) can reduce significantly the CPV-2 titres and that even lower concentrations, i.e. 0.37%, can efficiently inactivate the viruses provided that the contact time is extended to 15 min. Results also confirm the importance of cleaning before disinfection since the presence of organic matter totally abrogated the virucidal activity of sodium hypochlorite solutions against the three CPV-2 strains.

Importance of Reaction Kinetics and Oxygen Crossover in aprotic Li-O2 Batteries Based on a Dimethyl Sulfoxide Electrolyte.

Although still in their embryonic state, aprotic rechargeable Li-O2 batteries have, theoretically, the capabilities of reaching higher specific energy densities than Li-ion batteries. There are, however, significant drawbacks that must be addressed to allow stable electrochemical performance; these will ultimately be solved by a deeper understanding of the chemical and electrochemical processes occurring during battery operations. We report a study on the electrochemical and chemical stability of Li-O2 batteries comprising Au-coated carbon cathodes, a dimethyl sulfoxide (DMSO)-based electrolyte and Li metal negative electrodes. The use of the aforementioned Au-coated cathodes in combination with a 1 M lithium bis(trifluoromethane)sulfonimide (LiTFSI)-DMSO electrolyte guarantees very good cycling stability (>300 cycles) by minimizing eventual side reactions. The main drawbacks arise from the high reactivity of the Li metal electrode when in contact with the O2 -saturated DMSO-based electrolyte.

Serum transforming growth factor ß1 during diabetes development in non-obese diabetic mice and humans.

Recent data show that regulatory cells with transforming growth factor (TGF)-ß1-dependent activity are able to restore self-tolerance in overtly diabetic non-obese diabetic (NOD) mice. Thus, TGF-ß1 seems to have a relevant role in protection from autoimmune diabetes. Our aim was to investigate the possible significance of serum TGF-ß1 measurement in the natural history of diabetes in NOD mice, as well as in children positive for at least one islet-related antibody. Serum TGF-ß1 (both total and active) was measured by enzyme-linked immunosorbent assay at monthly intervals in 26 NOD mice during the spontaneous development of diabetes and, on a yearly basis, in nine siblings of patients with type 1 diabetes (T1D) with a follow-up of 4 years. Diabetes appeared between the 12th week of age and the end of the study period (36 weeks) in 17 mice. TGF-ß1 serum level variations occurred in the prediabetic period in both NOD mice and humans and diabetes diagnosis followed a continuing reduction of active TGF-ß1 (aTGF-ß1) serum levels. In mice, aTGF-ß1 serum levels measured at 4 weeks of age correlated positively with severity of insulitis, and negatively with percentage of insulin-positive cells. Our findings suggest that in NOD mice serum TGF-ß1 levels during the natural history of the diabetes reflect the course of islet inflammation. The measurement of aTGF-ß1 in islet-related antibody-positive subjects may provide insights into the natural history of prediabetic phase of T1D.

Methicillin-resistant staphylococci carriage in the oral cavity: a study conducted in Bari (Italy).

OBJECTIVES: The oral cavity may represent a site of colonization by antibiotic-resistant bacteria, such as methicillin-resistant staphylococci (MRS). To define the prevalence of staphylococci and MRS in the oral cavity, an observational study was carried out in the city of Bari (Italy). METHODS: Sixty subjects were asked to provide oral samples and a questionnaire about risk factors of colonization by MRS. An enrichment medium specific for staphylococci was used for the isolation. RESULTS: Swabs and corresponding questionnaires were available from 36 out of 60 patients. Staphylococci were isolated from seven out of 36 samples (prevalence 19.4%). Among the seven staphylococcal isolates, three were Staphylococcus aureus, and one strain, belonging to S. epidermidis species, was found to be MR (1.7%). No methicillin-resistant S. aureus were isolated. Five out of seven staphylococcal isolates exhibited resistance to more than two classes of non-beta-lactams antimicrobials. None of the risk factors analysed correlated with the status of MRS carriers, except the presence of oral disease. CONCLUSIONS: The results underline the potential role of the oral cavity as a reservoir of staphylococci.

Intestinal permeability in patients with adverse reactions to food.

BACKGROUND: An abnormal intestinal permeability could contribute to establish an altered sensitivity to food-allergen. AIM: To evaluate the intestinal permeability in subjects with adverse reactions to food on allergen-free diet. SUBJECTS: Twenty-one patients with food allergy and 20 with food hypersensitivity on allergen-free diet were enrolled and divided in four groups according to the seriousness of their referred clinical symptoms when they were on a free diet. METHODS: Intestinal permeability was evaluated by Lactulose/Mannitol ratio urinary detection determined by anion-exchange chromatography. RESULTS: Statistically significant different Lactulose/Mannitol ratio was evidenced in subjects with food allergy (p=0.003) or hypersensitivity (p=0.0008) compared to control patients. The correlation between Lactulose/Mannitol ratio and the seriousness of clinical symptoms, by using Spearman test, was statistically significant for food allergy (p=0.0195) and hypersensitivity (p=0.005) patients. CONCLUSIONS: The present data demonstrate that impaired intestinal permeability, measured in our conditions, is present in all subjects with adverse reactions to food. In addition, for the first time, we report a statistically significant association between the severity of referred clinical symptoms and the increasing of Intestinal Permeability Index. These data reveal that intestinal permeability is not strictly dependent on IgE-mediated processes but could better be related to other mechanisms involved in early food sensitisation, as breast-feeding, or microbial environment that influence the development of oral tolerance in early infancy.

Oral probiotic administration induces interleukin-10 production and prevents spontaneous autoimmune diabetes in the non-obese diabetic mouse.

AIMS/HYPOTHESIS: Recent observations suggest the involvement of the gastrointestinal tract in the pathogenesis of islet autoimmunity. Thus, the modulation of gut-associated lymphoid tissue may represent a means to affect the natural history of the disease. Oral administration of probiotic bacteria can modulate local and systemic immune responses; consequently, we investigated the effects of oral administration of the probiotic compound VSL#3 on the occurrence of diabetes in non-obese diabetic (NOD) mice. METHODS: VSL#3 was administered to female NOD mice three times a week starting from 4 weeks of age. A control group received PBS. Whole blood glucose was measured twice a week. IFN-gamma and IL-10 production/expression was evaluated by ELISA in culture supernatants of mononuclear cells isolated from Peyer's patches and the spleen, and by real-time PCR in the pancreas. Insulitis was characterised by immunohistochemistry and histomorphometric studies. RESULTS: Early oral administration of VSL#3 prevented diabetes development in NOD mice. Protected mice showed reduced insulitis and a decreased rate of beta cell destruction. Prevention was associated with an increased production of IL-10 from Peyer's patches and the spleen and with increased IL-10 expression in the pancreas, where IL-10-positive islet-infiltrating mononuclear cells were detected. The protective effect of VSL#3 was transferable to irradiated mice receiving diabetogenic cells and splenocytes from VSL#3-treated mice. CONCLUSIONS/INTERPRETATION: Orally administered VSL#3 prevents autoimmune diabetes and induces immunomodulation by a reduction in insulitis severity. Our results provide a sound rationale for future clinical trials of the primary prevention of type 1 diabetes by oral VSL#3 administration.

The recombinant major allergen of Parietaria judaica and its hypoallergenic variant: in vivo evaluation in a murine model of allergic sensitization.

BACKGROUND: Par j 1 represents the major allergenic component of Parietaria judaica pollen. Its three-dimensional structure is stabilized by four disulphide bridges. A family of three-dimensional mutants of the recombinant Par j 1 (rPar j 1) allergen, showing reduced allergenicity and retained T cell recognition has been recently developed by site-directed mutagenesis. OBJECTIVE: To develop and characterize a murine model of IgE sensitization to rPar j 1. To evaluate similarities between the murine model and the human IgE response. To investigate in this model the recognition of a hypoallergenic mutant of Par j 1, and to study the immune responses elicited in mice by the mutant itself. METHODS: BALB/c mice were sensitized by two intraperitoneal immunizations with rPar j 1 in alum on days 0 and 21. Allergen-specific serum IgE and IgG responses were studied by direct ELISA and immunoblotting, ELISA inhibition and competitive ELISA. Cell proliferation was evaluated in splenocyte cultures. RESULTS: Sensitization with rPar j 1 induced high levels of IgE and IgG1 vs. low levels of IgG2a. Mouse antibodies specific to rPar j 1 were able to compete with human IgE for recognition of rPar j 1. IgE from mice immunized with rPar j 1 showed a significantly reduced binding activity towards the hypoallergenic variant rPjC, which lacks three disulphide bridges. On the contrary, rPjC was recognized by IgG1 and IgG2a antibodies as well as rPar j 1. The proliferative response to rPjC by splenocytes from mice immunized with rPar j 1 was comparable to that stimulated by rPar j 1. Immunization with rPjC induced low levels of IgE antibodies to the rPjC itself, while IgG and proliferative responses were similar to those induced by rPar j 1. CONCLUSION: Conformational variants of allergens, displaying reduced allergenicity accompanied by retained IgG and T cell recognition, offer a safe, specific and flexible approach to immunotherapy of type I allergy. Our mouse model of IgE sensitization to a recombinant allergen, mimicking the human response to its native counterpart, could provide valuable information for pre-clinical testing of such hypoallergenic molecules.

Immunological aspects in migraine: increase of IL-10 plasma levels during attack.

In the present study, 23 patients with migraine without aura were monitored during a migraine attack. Plasma levels of interleukin (IL)-4, IL-5, IL-10, and interferon-gamma were measured by enzyme-linked immunosorbent assay techniques. Interestingly, we observed low to undetectable IL-5 and IL-4 levels, whereas high IL-10 levels were seen in 52.2% of the patients. Interferon-gamma plasma levels were undetectable in all patients. After treatment with sumatriptan, 10 patients showed a subsequent decrease in IL-10 and an increase in both IL-4 and IL-5 plasma levels. Although these findings are derived from a limited number of patients, the apparent return to the IL-4 and IL-5 cytokine profile observed during the interictal period leads us to speculate that a preferential enhancement of TH2-type cytokine production may contribute to the pathogenesis of migraine.

Immunoglobulin A (IgA) responses and IgE-associated inflammation along the respiratory tract after mucosal but not systemic immunization.

The purpose of the present study was to determine the extent of immunologic responses, particularly immunopathologic responses, within the upper and lower respiratory tracts after intranasal immunization using the mucosal adjuvant cholera toxin (CT). BALB/c mice were nasally immunized with influenza virus vaccine combined with CT. The inclusion of the mucosal adjuvant CT clearly enhanced generation of antibody responses in both the nasal passages and lungs. After nasal immunization, antigen-specific immunoglobulin A (IgA) antibody-forming cells dominated antibody responses throughout the respiratory tract. However, IgG responses were significant in lungs but not in nasal passages. Furthermore, parenteral immunization did not enhance humoral immunity in the upper respiratory tract even after a nasal challenge, whereas extrapulmonary lymphoid responses enhanced responses in the lung. After nasal immunization, inflammatory reactions, characterized by mononuclear cell infiltration, developed within the lungs of mice but not in nasal passages. Lowering dosages of CT reduced, but did not eliminate, these adverse reactions without compromising adjuvancy. Serum IgE responses were also enhanced in a dose-dependent manner by inclusion of CT. In summary, there are differences in the generation of humoral immunity between the upper respiratory tract and the lung. As the upper respiratory tract is in a separate compartment of the immune system from that stimulated by parenteral immunization, nasal immunization is an optimal approach to generate immunity throughout the respiratory tract. Despite the promise of nasal immunization, there is also the potential to develop adverse immunopathologic reactions characterized by pulmonary airway inflammation and IgE production.

Oral QS-21 requires early IL-4 help for induction of mucosal and systemic immunity.

The highly purified saponin derivative, QS-21, from the Quillaja saponaria Molina tree has been proved to be safe for parenteral administration and represents a potential alternative to bacterial enterotoxin derivatives as a mucosal adjuvant. Here we report that p.o. administration of QS-21 with the vaccine protein tetanus toxoid elicited strong serum IgM and IgG Ab responses, which were only slightly enhanced by further oral immunization. The IgG Ab subclass responses were predominantly IgG1 followed by IgG2b for the 50-microg p.o. dose of QS-21, whereas the 250-microg p.o. dose also induced IgG2a and IgG3 Abs. Low oral QS-21 doses induced transient IgE Ab responses 7 days after the primary immunization, whereas no IgE Ab responses were seen in mice given the higher QS-21 dose. Further, low but not high p.o. QS-21 doses triggered Ag-specific secretory IgA (S-IgA) Ab responses. Th cell responses showed higher IFN-gamma (Th1-type) and lower IL-5, IL-6, and IL-10 (Th2-type) secretion after the high QS-21 p.o. dose than after low doses. Interestingly, the mucosal adjuvant activity of low oral QS-21 doses was diminished in IL-4(-/-) mice, suggesting a role for this cytokine in the initiation of mucosal immunity by oral QS-21. In summary, our results show that oral QS-21 enhances immunity to coadministered Ag and that different doses of QS-21 lead to distinct patterns of cytokine and serum Ab responses. We also show that an early IL-4 response is required for the induction of mucosal immunity by oral QS-21 as adjuvant.

Human nasopharyngeal-associated lymphoreticular tissues. Functional analysis of subepithelial and intraepithelial B and T cells from adenoids and tonsils.

Subepithelial and intraepithelial lymphocytes of human adenoids and tonsils were characterized and directly compared to determine the potential contribution of these tissues to mucosal and systemic immune responses. The distribution of T and B cell subsets, cytokine patterns, and antibody (Ab) isotype profiles were similar for adenoids and tonsils. Both tissues contained predominantly B cells ( approximately 65%), approximately 5% macrophages, and 30% CD3(+) T cells. The T cells were primarily of the CD4(+) subset ( approximately 80%). Tonsillar intraepithelial lymphocytes were also enriched in B cells. The analysis of dispersed cells revealed a higher frequency of cells secreting IgG than IgA and the predominant Ig subclass profiles were IgG1 > IgG3 and IgA1 > IgA2, respectively. In situ analysis also revealed higher numbers of IgG- than IgA-positive cells. These IgG-positive cells were present in the epithelium and in the subepithelial zones of both tonsils and adenoids. Mitogen-triggered T cells from tonsils and adenoids produced both Th1- and Th2-type cytokines, clearly exhibiting their pluripotentiality for support of cell-mediated and Ab responses. Interestingly, antigen-specific T cells produced interferon-gamma and lower levels of interleukin-5. These results suggest that adenoids and tonsils of the nasopharyngeal-associated lymphoreticular tissues represent a distinct component of the mucosal-associated lymphoreticular tissues with features of both systemic and mucosal compartments.

Approximation of continuous and discontinuous mappings by a growing neural RBF-based algorithm.

In this paper a neural network for approximating continuous and discontinuous mappings is described. The activation functions of the hidden nodes are the Radial Basis Functions (RBF) whose variances are learnt by means of an evolutionary optimization strategy. A new incremental learning strategy is used in order to improve the net performances. The learning strategy is able to save computational time because of the selective growing of the net structure and the capability of the learning algorithm to keep the effects of the activation functions local. Further, it does not require high order derivatives. An analysis of the learning capabilities and a comparison of the net performances with other approaches reported in literature have been performed. It is shown that the resulting network improves the approximation results reported for continuous mappings and for those exhibiting a finite number of discontinuities.

Cholera toxin induces maturation of human dendritic cells and licences them for Th2 priming.

Cholera toxin (CT) is a potent mucosal adjuvant that amplifies B and T cell responses to mucosally co-administered antigens, stimulating predominant Th2-type responses. However, little is known about the mechanism of adjuvanticity of CT and on the influence this toxin may have on Th2 cell development during the priming of an immune response. We analyzed the effect of CT on dendritic cells (DC), which are responsible for the priming of immune responses at the systemic as well as at the mucosal level. We found that CT induces phenotypic and functional maturation of blood monocyte-derived DC. Indeed, CT-treated DC up-regulate expression of HLA-DR molecules, B7. 1 and B7.2 co-stimulatory molecules, and are able to prime naive CD4(+)CD45RA(+) T cells in vitro, driving their polarization towards the Th2 phenotype. Furthermore, CT-matured DC express functional chemokine receptors CCR7 and CXCR4 which may render them responsive to migratory stimuli towards secondary lymphoid organs. Interestingly, the maturation program induced by CT is unique since CT does not induce but rather inhibits cytokine (IL-12p70 and TNF-alpha) and chemokine (RANTES, MIP-1alpha and MIP-1beta) secretion by lipopolysaccharide- or CD40 ligand-activated DC. Our results help to elucidate the mechanism of action of CT as an adjuvant and highlight a new stimulus of bacterial origin that promotes maturation of DC.

IL-4 promotes the migration of circulating B cells to the spleen and increases splenic B cell survival.

We report that IL-4 causes a redistribution of B cells and modestly increases B cell life span. Intravenous injection of a long-acting formulation of IL-4 induces increases in both spleen cell number and the percentage of splenic B cells. These effects are observed within 1 day of IL-4 administration and plateau after approximately 3 days if IL-4 treatment is continued. The increase in splenic B cell number is IL-4 dose dependent, CD4+ T cell independent, FcgammaRII/FcgammaRIII independent, and Stat6 independent. Decreases in the number of B cells in the blood and the percentage of mature B cells in the bone marrow, concomitant with the increase in splenic B cell number, suggest that redistribution of circulating B cells to the spleen is partially responsible for IL-4 induction of splenic B cell hyperplasia. Considerable reduction in the effect of 5 days of IL-4 treatment on splenic B cell number when B lymphopoiesis is blocked with anti-IL-7 mAb suggests that generation of new B cells is also involved in IL-4-induced splenic B cell hyperplasia. 5-Bromo-2'-deoxyuridine labeling experiments demonstrate that IL-4 modestly prolongs the life span of newly generated splenic B cells, and experiments that measure B cell HSA (CD24) expression as an indicator of B cell age suggest that IL-4 may also prolong the life span of mature splenic B cells. Thus, IL-4 increases splenic B cell number through two Stat6-independent effects: increased net migration of circulating B cells to the spleen and increased B cell life span. Both effects may promote Ab responses to a systemic Ag challenge.

On-line learning in RBF neural networks: a stochastic approach.

The on-line learning of Radial Basis Function neural networks (RBFNs) is analyzed. Our approach makes use of a master equation that describes the dynamics of the weight space probability density. An approximate solution of the master equation is obtained in the limit of a small learning rate. In this limit, the on line learning dynamics is analyzed and it is shown that, since fluctuations are small, dynamics can be well described in terms of evolution of the mean. This allows us to analyze the learning process of RBFNs in which the number of hidden nodes K is larger than the typically small number of input nodes N. The work represents a complementary analysis of on-line RBFNs, with respect to the previous works (Phys. Rev. E 56 (1997a) 907; Neur. Comput. 9 (1997) 1601), in which RBFNs with N >> K have been analyzed. The generalization error equation and the equations of motion of the weights are derived for generic RBF architectures, and numerically integrated in specific cases. Analytical results are then confirmed by numerical simulations. Unlike the case of large N > K we find that the dynamics in the case N < K is not affected by the problems of symmetric phases and subsequent symmetry breaking.

A double-blind, placebo-controlled study of Mycobacterium-specific human immune responses induced by intradermal bacille Calmette-Guérin vaccination.

Recent studies have indicated that type 1 T cell responses (potent interferon-gamma and cytolytic responses, with absence of interleukin-4 production) are important for protective immunity against mycobacteria. These observations suggest that assays of type 1 T cell responses may be useful as surrogate markers of protective immunity in the evaluation of new tuberculosis vaccines. To be useful as surrogate markers, immunologic assays must distinguish between vaccine recipients and control subjects in clinical trials. Previous studies have shown that bacille Calmette-Guérin (BCG) vaccination can induce human type 1 T cell responses, but randomized trials have not been done to determine whether measurement of these responses can distinguish between BCG recipients and control subjects. We have conducted a double-blind, placebo-controlled trial of intradermal vaccination with two different BCG strains. We compared the mean lymphoproliferative, cytotoxic, Th1 and Th2 cytokine, and antibody responses detected in BCG and placebo recipients. These studies demonstrated that significant increases in Mycobacterium-specific T cell proliferative responses and type 1 cytokine responses were induced by BCG when compared with results with a placebo. In addition, BCG induced significant increases in Mycobacterium-specific antibody responses with an isotype profile characteristic of a type 1 cytokine bias. T cell and antibody assays involving the use of mycobacterial whole cell lysates or live BCG were able to discriminate between BCG and placebo recipients better than were assays using mycobacterial culture filtrates. These studies provide important information for the development of immunologic assays that might be useful as surrogate markers of protective immunity in future trials of new tuberculosis vaccines.

Strategies for mucosal vaccine development.

Vaccines able to induce both secretory IgA for protection of mucosal surfaces and systemic immunity to pathogens invading the host are of great interest in the war against infectious diseases. Mucosal vaccines trigger immune cells in mucosal inductive sites and thus can induce immunity in both the mucosal and systemic compartments. This review presents a critical survey of adjuvants and delivery systems currently being tested for mucosal immunization. A better understanding of cellular and molecular factors involved in the regulation of mucosal immunity will help in the design of safer mucosal vaccines to elicit the appropriate protective immune response to a given pathogen.

Zonula occludens toxin is a powerful mucosal adjuvant for intranasally delivered antigens.

Zonula occludens toxin (Zot) is produced by toxigenic strains of Vibrio cholerae and has the ability to reversibly alter intestinal epithelial tight junctions, allowing the passage of macromolecules through the mucosal barrier. In the present study, we investigated whether Zot could be exploited to deliver soluble antigens through the nasal mucosa for the induction of antigen-specific systemic and mucosal immune responses. Intranasal immunization of mice with ovalbumin (Ova) and recombinant Zot, either fused to the maltose-binding protein (MBP-Zot) or with a hexahistidine tag (His-Zot), induced anti-Ova serum immunoglobulin G (IgG) titers that were approximately 40-fold higher than those induced by immunization with antigen alone. Interestingly, Zot also stimulated high anti-Ova IgA titers in serum, as well as in vaginal and intestinal secretions. A comparison with Escherichia coli heat-labile enterotoxin (LT) revealed that the adjuvant activity of Zot was only sevenfold lower than that of LT. Moreover, Zot and LT induced similar patterns of Ova-specific IgG subclasses. The subtypes IgG1, IgG2a, and IgG2b were all stimulated, with a predominance of IgG1 and IgG2b. In conclusion, our results highlight Zot as a novel potent mucosal adjuvant of microbial origin.

Use of intranasal IL-12 to target predominantly Th1 responses to nasal and Th2 responses to oral vaccines given with cholera toxin.

We have investigated the effects of IL-12 and cholera toxin (CT) on the immune response to tetanus toxoid (TT) given by intranasal or oral routes. CT inhibited IL-12-induced IFN-gamma secretion both in vivo and in vitro. Intranasal administration of IL-12 to mice nasally immunized with the combined vaccine of TT and CT resulted in increased TT-specific IgG2a and IgG3 Abs, while IgG1 and IgE Ab responses were markedly reduced. This shift of the CT-induced immune response toward Th1 type was associated with TT-specific CD4+ T cells secreting IFN-gamma and reduced levels of Th2-type cytokines (i.e., IL-4, IL-5, IL-6, and IL-10). In contrast, intranasal IL-12 enhanced the CT-induced serum IgG1 and IgE Ab responses in mice given the combined vaccine orally. IFN-gamma secretion by TT-specific CD4+ T cells was also enhanced; however, Th2-type cytokine responses were predominant. Mucosal secretory IgA responses to oral or nasal vaccines were not affected by intranasal IL-12. Thus, intranasal IL-12 delivery influences Th cell subset development in mucosal inductive sites that are dependent on the route of vaccine delivery.