Bifidobacterium Strains Present Distinct Effects on the Control of Alveolar Bone Loss in a Periodontitis Experimental Model.

Periodontitis is an inflammatory disease induced by a dysbiotic oral microbiome. Probiotics of the genus Bifidobacterium may restore the symbiotic microbiome and modulate the immune response, leading to periodontitis control. We evaluated the effect of two strains of Bifidobacterium able to inhibit Porphyromonas gingivalis interaction with host cells and biofilm formation, but with distinct immunomodulatory properties, in a mice periodontitis model. Experimental periodontitis (P+) was induced in C57Bl/6 mice by a microbial consortium of human oral organisms. B. bifidum 1622A [B+ (1622)] and B. breve 1101A [B+ (1101)] were orally inoculated for 45 days. Alveolar bone loss and inflammatory response in gingival tissues were determined. The microbial consortium induced alveolar bone loss in positive control (P + B-), as demonstrated by microtomography analysis, although P. gingivalis was undetected in oral biofilms at the end of the experimental period. TNF-α and IL-10 serum levels, and Treg and Th17 populations in gingiva of SHAM and P + B- groups did not differ. B. bifidum 1622A, but not B. breve 1101A, controlled bone destruction in P+ mice. B. breve 1101A upregulated transcription of Il-1ß, Tnf-α, Tlr2, Tlr4, and Nlrp3 in P-B+(1101), which was attenuated by the microbial consortium [P + B+(1101)]. All treatments downregulated transcription of Il-17, although treatment with B. breve 1101A did not yield such low levels of transcripts as seen for the other groups. B. breve 1101A increased Th17 population in gingival tissues [P-B+ (1101) and P + B+ (1101)] compared to SHAM and P + B-. Administration of both bifidobacteria resulted in serum IL-10 decreased levels. Our data indicated that the beneficial effect of Bifidobacterium is not a common trait of this genus, since B. breve 1101A induced an inflammatory profile in gingival tissues and did not prevent alveolar bone loss. However, the properties of B. bifidum 1622A suggest its potential to control periodontitis.

Neutrophil Cells Are Essential for The Efficacy of a Therapeutic Vaccine against Paracoccidioidomycosis.

Paracoccidioidomycosis (PCM), caused by the Paracoccidioides species, is a systemic disease endemic in several Latin American countries, mainly in Brazil, Colombia, Argentina, and Venezuela. Current treatment approaches are challenging as they require prolonged durations of antifungal drugs that have potential toxicities, and despite antifungals, relapses are common. Hence, new therapeutic approaches, such as vaccines, are being investigated. The therapeutic vaccine consisting of peptide P10 associated with lipid cationic DODAB (P10+DODAB) is effective in murine models of PCM. However, the specific immune mechanisms required for the protective response has not been fully elucidated. The present work aims at evaluating the participation of neutrophils in the immune response induced by P10+DODAB. We found that the vaccine reduced both the influx of pulmonary neutrophils and the fungal load in comparison to infected animals that did not receive this treatment. The parenchymal architecture of the lungs of P10+DODAB-treated animals was largely preserved with only a few granulomas present, and tissue cytokine analysis showed a Th1 cytokine profile with augmented levels of IL-12, IFN-γ and TNF-α, and low levels of IL-4. When neutrophils were depleted 24 h prior to each treatment, the effectiveness of the P10+DODAB vaccine was completely lost as the fungal burdens remained high and histological examination showed a marked inflammation and fungal dissemination with a dysregulated cytokine response. In conclusion, these findings indicate that neutrophils are vital to ensure the triggering of an effective immune response to P10+DODAB.

Identification of Potentially Therapeutic Immunogenic Peptides From Paracoccidioides lutzii Species.

Paracoccidioidomycosis (PCM) is an endemic mycosis in Latin America caused by the thermodimorphic fungi of the genus Paracoccidioides spp. Paracoccidioides lutzii (PL) is one of the 5 species that constitute the Paracoccidioides genus. PL expresses low amounts of glycoprotein (Gp) 43 (PLGp43) and PLGp43 displays few epitopes in common with the P. brasiliensis (PB) immunodominant antigen PBGp43, which is commonly used for serological diagnosis of PCM. This difference in structure between the glycoproteins markedly reduces the efficiency of serological diagnosis in patients infected with PL. We previously demonstrated that peptide 10 (P10) from the PBGp43 induces protective immune responses in in vitro and in vivo models of PB PCM. Since, P10 has proven to be a promising therapeutic to combat PB, we sought to identify peptides in PL that could similarly be applied for the treatment of PCM. PL yeast cell proteins were isolated from PL: dendritic cell co-cultures and subjected to immunoproteomics. This approach identified 18 PL peptides that demonstrated in silico predictions for immunogenicity. Eight of the most promising peptides were synthesized and applied to lymphocytes obtained from peptide-immunized or PL-infected mice as well as to in vitro cultures with peptides or dendritic cells pulsed the peptides. The peptides LBR5, LBR6 and LBR8 efficiently promoted CD4+ and CD8+ T cell proliferation and dendritic cells pulsed with LBR1, LBR3, LBR7 or LBR8 stimulated CD4+ T cell proliferation. We observed increases of IFN-γ in the supernatants from primed T cells for the conditions with peptides without or with dendritic cells, although IL-2 levels only increased in response to LBR8. These novel immunogenic peptides derived from PL will be employed to develop new peptide vaccine approaches and the proteins from which they are derived can be used to develop new diagnostic assays for PL and possibly other Paracoccidioides spp. These findings identify and characterize new peptides with a promising therapeutic profile for future against this important neglected systemic mycosis.

Experimental Therapy of Paracoccidioidomycosis Using P10-Primed Monocyte-Derived Dendritic Cells Isolated From Infected Mice.

Paracoccidioidomycosis (PCM) is an endemic mycosis in Latin American caused by the thermodimorphic fungi of the genus Paracoccidioides spp. Notably, a Th1 immune response is required to control PCM. In this context, dendritic cells (DCs) seem to be essential players in capture, processing and presentation of Paracoccidioides antigens to naïve T cells and their further activation. We have previously demonstrated that differentiated DCs from bone marrow cells, pulsed with the immunoprotective peptide 10 (P10), effectively control experimental PCM immunocompetent and immunosuppressed mice. However, this procedure may not be infeasible or it is limited for the therapy of human patients. Therefore, we have sought a less invasive but equally effective approach that would better mimics the autologous transplant of DC in a human patient. Here, we isolated and generated monocyte differentiated dendritic cells (MoDCs) from infected mice, pulsed them with P-10, and used them in the therapy of PCM in syngeneic mice. Similar to the results using BMDCs, the P10-pulsed MoDCs stimulated the proliferation of CD4+ T lymphocytes, induced a mixed production of Th1/Th2 cytokines and decreased the fungal burden in murine lungs in the setting of PCM. The process of differentiating MoDCs derived from an infected host, and subsequently used for therapy of PCM is much simpler than that for obtaining BMDCs, and represents a more reasonable approach to treat patients infected with Paracoccidioides. The results presented suggest that P10-primed MoDC may be a promising strategy to combat complicated PCM as well as to significantly shorten the lengthy requirements for treatment of patients with this fungal disease.

Dendritic Cells Primed with Paracoccidioides brasiliensis Peptide P10 Are Therapeutic in Immunosuppressed Mice with Paracoccidioidomycosis.

Paracoccidioidomycosis (PCM) is an endemic systemic mycosis in Latin America, with the highest prevalence in Brazil, Colombia, and Venezuela. Fungi of the Paracoccidioides genus are etiologic agents of the disease. The 15 amino acid peptide P10 is derived from gp43, the main diagnostic antigen of Paracoccidioides brasiliensis. We previously reported that P10-pulsed dendritic cells (DCs) induce a protective response against P. brasiliensis. Presently, dexamethasone-treated BALB/c mice were intratracheally infected with P. brasiliensis Pb18 to establish the therapeutic efficacy of P10-pulsed DCs. Immunosuppressed and infected animals that received DCs had a reduction in their fungal burden, and this result was most pronounced in mice receiving DCs primed with P10. The efficacy of therapeutic DCs was significantly augmented by concomitant treatment with trimethoprim-sulfamethoxazole. Additionally, primed-DCs with or without the antifungal drug induced a beneficial Th1-biased immune response and significantly reduced tissue damage. In conclusion, these studies with immunocompromised mice demonstrate that P10-pulsed DCs with or without concomitant antifungal drugs are potently effective in combating invasive PCM. These findings support further translational studies to validate the use of P10-primed DCs for PCM in immunocompetent and immunosuppressed hosts.