Peptidoglycan-treated tumor antigen-pulsed dendritic cells impart complete resistance against tumor rechallenge.

Solid tumors elicit suppressive T cell responses which impair antigen-presenting cell (APC) functions. Such immune suppression results in uncontrolled tumor growth and mortality. Addressing APC dysfunction, dendritic cell (DC)-mediated anti-tumor vaccination was extensively investigated in both mice and humans. These studies never achieved full resistance to tumor relapse. Herein, we describe a repetitive RM-1 murine tumor rechallenge model for recurrence in humans. Using this newly developed model, we show that priming with tumor antigen-pulsed, Toll-like receptor (TLR)2 ligand-activated DCs elicits a host-protective anti-tumor immune response in C57BL/6 mice. Upon stimulation with the TLR2 ligand peptidoglycan (PGN), the tumor antigen-pulsed DCs induce complete resistance to repetitive tumor challenges. Intra-tumoral injection of PGN reduces tumor growth. The tumor resistance is accompanied by increased expression of interleukin (IL)-27, T-box transcription factor TBX21 (T-bet), IL-12, tumor necrosis factor (TNF)-α and interferon (IFN)-γ, along with heightened cytotoxic T lymphocyte (CTL) functions. Mice primed four times with PGN-stimulated tumor antigen-pulsed DCs remain entirely resistant to repeat challenges with RM-1 tumor cells, suggesting complete prevention of relapse and recurrence of tumor. Adoptive transfer of T cells from these mice, which were fully protected from RM-1 rechallenge, confers anti-tumor immunity to syngeneic naive recipient mice upon RM-1 challenge. These observations indicate that PGN-activated DCs induce robust host-protective anti-tumor T cells that completely resist tumor growth and recurrence.

Superior immunogenicity of HCV envelope glycoproteins when adjuvanted with cyclic-di-AMP, a STING activator or archaeosomes.

Three decades after the discovery, hepatitis C virus (HCV) is still the leading cause of liver transplantation and poses a major threat to global health. In spite of recent advances in the development of direct acting antivirals, there is still a need for a prophylactic vaccine to limit the virus spread and protect at-risk populations, especially in developing countries, where the cost of the new treatments may severely limit access. The use of recombinant HCV glycoproteins E1E2 (rE1E2) in combination with the MF59, an oil-in-water emulsion-based adjuvant, has previously been shown to reduce the rate of chronicity in chimpanzees and to induce production of cross-neutralizing antibodies and cellular immune responses in human volunteers. To further improve neutralizing antibody responses in recipients along with robust T cell responses, we have explored the immunogenicity of different adjuvants when formulated with the HCV rE1E2 vaccine in mice. Our data show that cyclic di-adenosine monophosphate (c-di-AMP) and archaeosomes elicit strong neutralizing antibodies similar to those elicited using aluminum hydroxide/monophosphoryl lipid A (Alum/monophos. /MPLA) and MF59. However, both c-di-AMP and archaeosomes induced a more robust cellular immune response, which was confirmed by the detection of vaccine-specific poly-functional CD4+ T cells. We conclude that these adjuvants may substantially boost the immunogenicity of our E1E2 vaccine. In addition, our data also indicates that use of a partial or exclusive intranasal immunization regimen may also be feasible using c-di-AMP as adjuvant.

A rapid method for breath analysis in cystic fibrosis patients.

For easy handling and speed of lung diseases diagnostics, approaches based on volatile organic compounds (VOCs), including those emitted by pathogenic microorganisms, are considered but currently require considerable sampling efforts. We tested whether easy-to-handle and fast detection of lung infections is possible using solid-phase microextraction (SPME) of 100 ml of exhaled breath. An analytical procedure for the detection of VOCs from the headspace of epithelial lung cells infected with four human pathogens was developed. The feasibility of this method was tested in a cystic fibrosis (CF) outpatient clinic in vivo. Exhaled breath was extracted by SPME and analyzed by gas chromatography-mass spectrometry (GC-MS). The compositions of VOCs released in the infection model were characteristic for all individual pathogens tested. Exhaled breath of CF patients allowed clear distinction of CF patients and controls by their VOC compositions using multivariate analyses. Interestingly, the major specific VOCs detected in the exhaled breath of infected CF patients in vivo differed from those monitored during bacterial in vitro growth. SPME extraction of VOCs from 100 ml of human breath allowed the distinction between CF patients and healthy probands. Our results highlight the importance of assessing the entire pattern of VOCs instead of selected biomarkers for diagnostic purposes, as well as the need to use clinical samples to identify reliable biomarkers. This study provides the proof-of-concept for the approach using the composition of exhaled VOCs in human breath for the rapid identification of infectious agents in patients with lower respiratory tract infections.

Local treatment with BPPcysMPEG reduces allergic airway inflammation in sensitized mice.

According to the hygiene hypothesis, triggering the immune system with microbial components during childhood balances the inherent Th2 bias. In contrast, specific immunotherapy involves exposure of the patient to the allergen in order to achieve desensitization to subsequent contact. In a human in vitro allergy model the potential of the TLR2/6 agonist BPPcysMPEG to modulate antigen presenting cells and allergen-specific immune responses was evaluated. Specific immunomodulation via co-administration of the allergen and BPPcysMPEG enhanced expression of co-stimulatory molecules on DC and increased secretion of the proinflammatory cytokine TNF-α. Acting as an adjuvant, BPPcysMPEG elevated allergen-specific immune responses in co-culture with autologous lymphocytes. Although administration of BPPcysMPEG alone enhanced expression of co-stimulatory molecules on DC, proliferation of autologous lymphocytes was not induced. Based on this finding, the potential of BPPcysMPEG to reduce allergic airway inflammation by preventive modulation of the innate immune system via TLR2/6 agonization was investigated in mice. Local administration of BPPcysMPEG altered cellular influx and cell composition in BAL fluid. Furthermore, the Th2-associated cytokines IL-4 and IL-5 were diminished. Allergen-specific restimulation of cells from mediastinal lymph nodes and splenocytes suggested an alteration of immune responses. The treatment with BPPcysMPEG induced a Th1-dominated cytokine milieu in mediastinal lymph nodes, while allergen-specific immune responses in splenocytes were diminished. The co-administration of allergen and BPPcysMPEG reduced cytokine secretion upon restimulation in mediastinal lymph nodes and splenocytes. From these data we conclude that BPPcysMPEG was able to influence the immune system with regard to subsequent allergen contact by TLR2/6 agonization.

Natural innate cytokine response to immunomodulators and adjuvants in human precision-cut lung slices.

Prediction of lung innate immune responses is critical for developing new drugs. Well-established immune modulators like lipopolysaccharides (LPS) can elicit a wide range of immunological effects. They are involved in acute lung diseases such as infections or chronic airway diseases such as COPD. LPS has a strong adjuvant activity, but its pyrogenicity has precluded therapeutic use. The bacterial lipopeptide MALP-2 and its synthetic derivative BPPcysMPEG are better tolerated. We have compared the effects of LPS and BPPcysMPEG on the innate immune response in human precision-cut lung slices. Cytokine responses were quantified by ELISA, Luminex, and Meso Scale Discovery technology. The initial response to LPS and BPPcysMPEG was marked by coordinated and significant release of the mediators IL-1ß, MIP-1ß, and IL-10 in viable PCLS. Stimulation of lung tissue with BPPcysMPEG, however, induced a differential response. While LPS upregulated IFN-γ, BPPcysMPEG did not. This traces back to their signaling pathways via TLR4 and TLR2/6. The calculated exposure doses selected for LPS covered ranges occurring in clinical studies with human beings. Correlation of obtained data with data from human BAL fluid after segmental provocation with endotoxin showed highly comparable effects, resulting in a coefficient of correlation >0.9. Furthermore, we were interested in modulating the response to LPS. Using dexamethasone as an immunosuppressive drug for anti-inflammatory therapy, we found a significant reduction of GM-CSF, IL-1ß, and IFN-γ. The PCLS-model offers the unique opportunity to test the efficacy and toxicity of biological agents intended for use by inhalation in a complex setting in humans.

Immune responses of a liposome/ISCOM vaccine adjuvant against streptococcal fibronectin binding protein 1 (Sfb1) in mice.

BACKGROUND & OBJECTIVES: The fibronectin binding protein Sfb1 of Streptococcus pyogenes is a well characterised antigen which induces protection against lethal challenge with group A streptococcus (GAS) when adjuvanted with cholera toxin B-subunit (CTB). As an alternative to CTB adjuvanted intranasal immunisations we investigated the immune responses generated in mice using Sfb1 incorporated in to the skin and mucosal adjuvant SAMA4. METHODS: Mice (BALB/c) were vaccinated intradermally with 100 microl of either SAMA4 (adjuvant only group) or SAMA4/Sfb1 and were boosted 7 days later. Mice vaccinated with CTB based vaccines were immunised by intranasal inoculation with a mixture containing 30 microg Sfb1 and 10 microg CTB on days 1, 3, 5 and 15. At 14 days after the last booster immunisation the immune response was characterised and mice were challenged with 10(8) CFU of S. pyogenes. RESULTS: Mice vaccinated with SAMA4/Sfb1 elicited a Sfb1-specific IgG response in the sera that was significantly higher than that seen in control mice and mice immunised with the adjuvant only (P<0.05). No significant differences were seen for specific IgA antibodies in the sera in all groups examined. Compared with non-immunised and adjuvant only immunised controls, mice immunised with the Sfb1/SAMA4 vaccine exhibited a significant increase (P<0.05) in the number of Sfb1 reactive spleen cells in lymphoproliferation assays which were three fold higher than those seen for mice vaccinated with the Sfb1/CTB vaccine. Mice vaccinated with CTB/Sfb1 had the highest level of protection (80%) as where mice vaccinated with SAMA4 and SAMA4/Sfb1 displayed no protection (20% and 40%). INTERPRETATION & CONCLUSION: These data suggest that the SAMA4 adjuvant used in this study fails to elicit protective immunity in BALB/c mice when used to adjuvant the known protective antigen Sfb1.

Role played by the response regulator Ris in Bordetella bronchiseptica resistance to macrophage killing.

Previous studies suggested that the persistence in eukaryotic cells of a Bordetella bronchiseptica mutant carrying an insertion in the locus encoding the response regulator RisAS is impaired. This suggested that ris-dependent products are required for the intracellular survival of bacteria. In this study we demonstrate that ris-regulated products play a role in B. bronchiseptica resistance against both phagosomal acidification and reactive oxygen intermediates.

Elicitation of predictable immune responses by using live bacterial vectors.

There is an increasing need for novel vaccines able to stimulate efficient and long-lasting responses, which have also low production costs. To confer protective immunity following vaccination, the adequate type of response should be elicited. Vaccines based on attenuated bacterial carriers have contained production and delivery costs, and are able to stimulate more potent immune responses than non-replicating formulations. The improved knowledge on carrier physiology and host response, the availability of different mutants and highly sophisticated expression tools, and the possibility of co-administering modulators enable to trigger predictable responses according to the specific needs. Recent studies support the use of attenuated bacteria not only as conventional carriers, but also as a delivery system for DNA vaccines against infectious agents and tumors. In this review we discuss the most widely used bacterial carrier systems for either antigens or nucleic acid vaccines, and the strategies which have been successfully exploited to modulate the immune responses elicited.

Characterization of the domain of fibronectin-binding protein I of Streptococcus pyogenes responsible for elicitation of a protective immune response.

Fibronectin-binding protein I (SfbI) represents a major adhesin of Streptococcus pyogenes. Mice were intranasally immunized with recombinant proteins spanning different portions of SfbI to identify the minimal fragment able to elicit a protective response against a lethal challenge with S. pyogenes. The strongest cellular responses and the highest levels of antigen-specific secretory immunoglobulin A (IgA) were detected in mice immunized with the fibronectin-binding region of SfbI. In contrast, animals vaccinated with a polypeptide spanning the aromatic and proline-rich regions showed the highest titers and fastest IgG response in serum. Vaccination with either SfbI without a membrane anchor and signal peptide or a polypeptide encompassing its fibronectin-binding regions resulted in efficient protection against heterologous challenge (60% and 80%, respectively), whereas the use of a polypeptide lacking this region conferred marginal protection (10%) with respect to the control group (0%). These results demonstrate that the fibronectin-binding region of SfbI is a promising candidate antigen for developing anti-S. pyogenes vaccines.

In vivo correction of genetic defects of monocyte/macrophages using attenuated Salmonella as oral vectors for targeted gene delivery.

Macrophages are normal targets for Salmonella during natural infections, and it has been demonstrated that attenuated bacteria can deliver nucleic acid vaccine constructs. Therefore, we assessed if attenuated Salmonella can be used for the in vivo delivery of transgenes to their natural cellular target, in an attempt to correct genetic defects associated with monocytes/macrophages. This system would offer the distinct advantage of achieving a specific targeting of defective cells in a non-invasive form. Using a reporter gene, we demonstrated that attenuated Salmonella could be used as an effective in vitro delivery system to transfer genetic material into nondividing cells like murine macrophages. In vivo, the oral administration of attenuated Salmonella allows targeted delivery of transgenes to macrophages and subsequently expression of transgenes at a systemic level. IFNgamma-deficient mice (GKO) were thus selected as a model for the in vivo validation of the Salmonella-based delivery approach. Attenuated Salmonella, used as the carrier for a eukaryotic expression vector encoding the murine IFNgamma gene, was able to restore the production of this cytokine in GKO macrophages. Their oral administration to IFNgamma-deficient mice also re-established, in these immunocompromised animals, the natural resistance to bacterial infections. These results demonstrate, for the first time, that attenuated Salmonella can be successfully used in vivo as a DNA delivery system for the correction of a genetic defect associated with monocyte/macrophages.

Characterization of SepL of enterohemorrhagic Escherichia coli.

The sepL gene is expressed in the locus of enterocyte effacement and therefore is most likely implicated in the attaching and effacing process, as are the products encoded by open reading frames located up- and downstream of this gene. In this study, the sepL gene of the enterohemorrhagic Escherichia coli (EHEC) strain EDL933 was analyzed and the corresponding polypeptide was characterized. We found that sepL is transcribed monocistronically and independently from the esp operon located downstream, which codes for the secreted proteins EspA, -D, and -B. Primer extension analysis allowed us to identify a single start of transcription 83 bp upstream of the sepL start codon. The analysis of the upstream regions led to the identification of canonical promoter sequences between positions -5 and -36. Translational fusions using lacZ as a reporter gene demonstrated that sepL is activated in the exponential growth phase by stimuli that are characteristic for the intestinal niche, e.g., a temperature of 37 degrees C, a nutrient-rich environment, high osmolarity, and the presence of Mn(2+). Protein localization studies showed that SepL was present in the cytoplasm and associated with the bacterial membrane fraction. To analyze the functional role of the SepL protein during infection of eukaryotic cells, an in-frame deletion mutant was generated. This sepL mutant was strongly impaired in its ability to attach to HeLa cells and induce a local accumulation of actin. These defects were partially restored by providing the sepL gene in trans. The EDL933DeltasepL mutant also exhibited an impaired secretion but not biosynthesis of Esp proteins, which was fully complemented by providing sepL in trans. These results demonstrate the crucial role played by SepL in the biological cycle of EHEC.

Wild-type HFE protein normalizes transferrin iron accumulation in macrophages from subjects with hereditary hemochromatosis.

Hereditary hemochromatosis (HC) is one of the most common single-gene hereditary diseases. A phenotypic hallmark of HC is low iron in reticuloendothelial cells in spite of body iron overload. Most patients with HC have the same mutation, a change of cysteine at position 282 to tyrosine (C282Y) in the HFE protein. The role of HFE in iron metabolism and the basis for the phenotypic abnormalities of HC are not understood. To clarify the role of HFE in the phenotypic expression of HC, we studied monocytes-macrophages from subjects carrying the C282Y mutation in the HFE protein and clinically expressing HC and transfected them with wild-type HFE by using an attenuated Salmonella typhimurium strain as a gene carrier. The Salmonella system allowed us to deliver genes of interest specifically to monocytes-macrophages with high transduction efficiency. The accumulation of (55)Fe delivered by (55)Fe-Tf was significantly lower in macrophages from patients with HC than from controls expressing wild-type HFE. Transfection of HC macrophages with the HFE gene resulted in a high level of expression of HFE protein at the cell surface. The accumulation of (55)Fe delivered by (55)Fe-Tf was raised by 40% to 60%, and this was reflected by an increase in the (55)Fe-ferritin pool within the HFE-transfected cells. These results suggest that the iron-deficient phenotype of HC macrophages is a direct effect of the HFE mutation, and they demonstrate a role for HFE in the accumulation of iron in these cells.

Role of phosphoglucomutase of Bordetella bronchiseptica in lipopolysaccharide biosynthesis and virulence.

The phosphoglucomutase (PGM)-encoding gene of Bordetella bronchiseptica is required for lipopolysaccharide (LPS) biosynthesis. An insertion mutant of the wild-type B. bronchiseptica strain BB7865 which disrupted LPS biosynthesis was created and characterized (BB7865pgm). Genetic analysis of the mutated gene showed it shares high identity with PGM genes of various bacterial species and forms part of an operon which also encompasses the gene encoding phosphoglucose isomerase. Functional assays for PGM revealed that enzyme activity is expressed in both bvg-positive and bvg-negative strains of B. bronchiseptica and is substantially reduced in BB7865pgm. Complementation of the mutated PGM gene with that from BB7865 restored the wild-type condition for all phenotypes tested. The ability of the mutant BB7865pgm to survive within J774. A1 cells was significantly reduced at 2 h (40% reduction) and 24 h (56% reduction) postinfection. BB7865pgm was also significantly attenuated in its ability to survive in vivo following intranasal infection of mice, being effectively cleared from the lungs within 4 days, whereas the wild-type strain persisted at least 35 days. The activities of superoxide dismutase, urease, and acid phosphatase were unaffected in the PGM-deficient strain. In contrast, the inability to produce wild-type LPS resulted in a reduced bacterial resistance to oxidative stress and a higher susceptibility to the antimicrobial peptide cecropin P.

Modulation of host immune responses stimulated by Salmonella vaccine carrier strains by using different promoters to drive the expression of the recombinant antigen.

We evaluated whether immune responses stimulated by Salmonella vaccine carriers can be modulated by using different promoters to drive antigen expression. Mice were orally immunized with strains transfected with plasmids carrying beta-galactosidase (beta-gal) under the control of either a constitutive or an in vivo-activated promoter. While alpha-gal-reactive IgG1, IgG2a, IgG2b and IgG3 were detected in sera of mice immunized with Salmonella expressing constitutively beta-gal, higher titers dominated by IgG2a and IgG2b were detected in sera when the in vivo-activated promoter was used. beta-gal-specific proliferative responses of spleen-derived CD4+ T lymphocytes were similar in both groups. However, CD4+ T lymphocytes from mice immunized with the constitutive promoter secreted IL-4, IL-5, IL-6, IL-10 and IFN-gamma (Th1/Th2 pattern), whereas CD4+ cells mainly secreted IFN-gamma (Th1 pattern) when the second construct was used. The spleens of all immunized mice contained beta-gal-reactive CD8+ CTL precursors. The vaccine prototypes were tested for their capacity to control seeding and/or development within the lung of an intravenously delivered aggressive fibrosarcoma transfected with beta-gal. Reduced metastasis and significantly increased mean survival times were observed in all vaccinated mice. However, protection was improved when the carrier expressed beta-gal upon infection (80 % versus 50% survival, p < 0.05).

Two distinct pathways for the invasion of Streptococcus pyogenes in non-phagocytic cells.

Adherence to and invasion of epithelial cells represent important pathogenic mechanisms of Streptococcus pyogenes. A fibronectin-binding surface protein of S. pyogenes, Sfbl protein, has been implicated in both adherence and invasion processes. Invasion of Sfbl-containing strains has been suspected to be responsible for the failure of antibiotics treatment to eradicate S. pyogenes. In this study, we tested the adherence and invasion properties of two well-characterized clinical isolates: A40, which expresses Sfbl; and A8, which is Sfbl negative and is unable to bind fibronectin. In strain A40, Sfbl was the main factor required for attachment and invasion by using fibronectin as a bridging molecule and the alpha5beta1 integrin as cellular receptor. The uptake process was characterized by the generation of large membrane invaginations at the bacteria-cell interface without evidence of actin recruitment or cellular injury. A40 cells were located in phagosomes and, only 24 h after infection, a consistent part of the bacterial population reached the cytoplasm. In contrast, uptake of strain A8 required major rearrangements of cytoskeletal proteins underneath attached bacteria. In A8, a proteinaceous moiety was involved, which does not interact with alpha5beta1 or need any known bridging molecule. Bacterial attachment stimulated elongation and massive recruitment of neighbouring microvilli, which fused to surround streptococcal chains. They led to the generation of large pseudopod-like structures, which engulfed bacteria that were rapidly released and replicated in the cytoplasm. The identification of two completely different uptake pathways reported here provided further evidence regarding the diversity of S. pyogenes isolates and might contribute towards understanding the pathogenesis and persistence of S. pyogenes.

Co-operative binding of human fibronectin to Sfbl protein triggers streptococcal invasion into respiratory epithelial cells.

Streptococcal fibronectin binding protein I (SfbI) mediates adherence to and invasion of Streptococcus pyogenes into human epithelial cells. In this study, we analysed the binding activity of distinct domains of SfbI protein towards its ligand, the extracellular matrix component fibronectin, as well as the biological implication of the binding events during the infection process. By using purified recombinant SfbI derivatives as well as in vivo expressed SfbI domains on the surface of heterologous organism Streptococcus gordonii, we were able to dissociate the two major streptococcal target domains on the human fibronectin molecule. The SfbI repeat region exclusively bound to the 30 kDa N-terminal fragment of fibronectin, whereas the SfbI spacer region exclusively bound to the 45 kDa collagen-binding fragment of fibronectin. In the case of native surface-expressed SfbI protein, an induced fit mode of bacteria-fibronectin interaction was identified. We demonstrate that binding of the 30 kDa fibronectin fragment to the repeat region of SfbI protein co-operatively activates the adjacent SfbI spacer domain to bind the 45 kDa fibronectin fragment. The biological consequence arising from this novel mode of fibronectin targeting was analysed in eukaryotic cell invasion assays. The repeat region of SfbI protein is mediating adherence and constitutes a prerequisite for subsequent invasion, whereas the SfbI spacer domain efficiently triggers the invasion process of streptococci into the eukaryotic cell. Thus, we were able to dissect bacterial adhesion from invasion by manipulating one protein. SfbI protein therefore represents a highly evolved prokaryotic molecule that exploits the host factor fibronectin not only for extracellular targeting but also for its subsequent activation that leads to efficient cellular invasion.

Fc-mediated nonspecific binding between fibronectin-binding protein I of Streptococcus pyogenes and human immunoglobulins.

Fibronectin-binding protein I (SfbI) from Streptococcus pyogenes plays a key role in bacterial adhesion to, and invasion of, eukaryotic cells. In addition, SfbI exhibits a considerable potential as mucosal adjuvant and can trigger polyclonal activation of B cells. Here, we report that SfbI is also capable of binding human IgG in a nonimmune fashion. SfbI was reactive with IgG1, IgG2, IgG3, and IgG4 isotypes (type IIo IgG-binding profile). The affinity constant (Kd) of the SfbI-IgG interaction was in the range of 1-2 x 10(-5) M. Further studies demonstrated that the SfbI binding was mediated by the Fc component of the IgG molecule. Experiments performed using purified recombinant proteins spanning different domains of SfbI showed that the IgG-binding activity was restricted to the fibronectin-binding domains, and in particular to the fibronectin-binding repeats. Finally, the presence of recombinant SfbI resulted in an impairment of both phagocytosis of IgG-coated RBCs and Ab-dependent cell cytotoxicity by macrophages. These results demonstrated for the first time that, in addition to its major role during the colonization process, SfbI may also favor bacterial immune evasion after the onset of the infection by interfering with host clearance mechanisms.

Differential effects of dietary Oenothera, Zizyphus mistol, and corn oils, and essential fatty acid deficiency on the progression of a murine mammary gland adenocarcinoma.

The modulating effect of dietary enrichment in mistol seed oil (MO) containing 25% of alpha-linolenic acid (ALA), evening primrose oil (EPO) enriched in gamma-linolenic acid (GLA) and corn oil (CO) as sources of omega-6 and omega-9 fatty acids on the growth parameters of one transplantable mammary tumor were compared. Mice fed on different lipid formulae were inoculated with a mammary gland adenocarcinoma and different growth development tumor parameters were recorded. Results showed that corn oil feeding slowed down most of the tumor growth parameters, as did the EPO diet. MO also showed antitumor activity. Olein feeding, which induces an essential fatty acid deficiency (EFAD), increased the incidence and the multiplicity of metastases when compared with the controls. It may be concluded that a diet enriched in omega-6 fatty acids did not behave as a tumor promoter in this mammary gland tumor model. The antitumor activities of EPO and MO are corroborated in present experiments, suggesting that both oils may be of value in nutritional approaches of mammary gland tumor therapies. In addition, present data add further experimental proof about the proposed protumorigenic proneness induced by the EFAD state.

Salmonella vaccine carrier strains: effective delivery system to trigger anti-tumor immunity by oral route.

Recombinant Salmonella strains expressing heterologous antigens can be delivered by oral route triggering the elicitation of efficient antigen-specific humoral, T helper and cytotoxic responses. The potential of attenuated Salmonella spp. to trigger anti-tumor immunity was evaluated for the first time by using beta-galactosidase (beta-gal) as a model tumor-associated antigen (TAA). Beta-gal was expressed in a Salmonella typhimurium aroA vaccine carrier strain either constitutively or under the control of a promoter activated upon infection. Oral immunization with both vaccine prototypes resulted in the elicitation of beta-gal-specific humoral and cell-mediated immunity. Although both strains were able to trigger antigen-specific CTL, responses were more efficient when the expression was controlled by the promoter activated upon infection. The anti-tumor efficacy of the stimulated response was validated by challenging vaccinated animals with an aggressive fibrosarcoma transfected with beta-gal, which operationally acts as a TAA. Both groups of vaccinated mice exhibited a significant reduction in tumor take and growth with respect to animals vaccinated with plasmidless carrier (p < 0.05). However, the overall efficiency was better in the group in which beta-gal was controlled by the in vivo-activated promoter (85% versus 54%; p < 0.05).

Fibronectin-binding protein I of Streptococcus pyogenes promotes T cell-independent proliferation of murine B lymphocytes and enhances the expression of MHC class II molecules on antigen-presenting cells.

We have previously shown that fibronectin-binding protein I (SfbI) of Streptococcus pyogenes can act as an adjuvant for mucosal-delivered antigens (Medina, E., Talay, S. R., Chhatwal, G. S. and Guzmán, C. A. 1998. Eur. J. Immunol. 28:1069). To characterize the underlying mechanism of the adjuvancity, we investigated the in vitro stimulating activity of SfbI. The SfbI protein promoted a dose-dependent proliferation of mouse spleen cells. Studies performed using cellular subpopulations showed that proliferation involved B cells and was T cell- and macrophage-independent. SfbI also induced lg production by B cells in a T cell-independent manner. The kinetics of lg isotype accumulation in supernatant fluids and the analysis of Ig-secreting cells suggested that SfbI stimulates B cells expressing different Ig isotypes rather than promoting the isotype switching of single subpopulations. Experiments performed with recombinant proteins encompassing different functional domains of SfbI showed that the fibronectin-binding repeats were responsible for B cell activation. The sera from mice immunized by the intranasal route with SfbI did not react with either double-stranded DNA, cardiolipin or collagen. Interestingly, stimulation with Sfbl also resulted in the up-regulation of MHC class 11 molecules expression by B cells and macrophages. The elucidation of the underlying molecular events to the immunomodulatory effect exerted by SfbI will facilitate the exploitation of the potential of this molecule for the generation of mucosal vaccines.