Reactivity of MEST-1 (antigalactofuranose) with Trypanosoma cruzi, glycosylinositol phosphorylceramides (GIPCs): Immunolocalization of GIPCs in acidic vesicles of epimastigotes

Using confocal microscopy, MEST-1-positive immunofluorescence was observed within various Trypanosoma cruzi forms, except in cell-derived trypomastigotes. Glycosylinositol phosphorylceramides were identified by thin-layer chromatography immunostaining as the antigens recognized by MEST-1 in these parasites. in epimastigotes, labeling of MEST-1 coincided with acidic vesicles, indicating an internal localization of these glycoconjugates.Universidade Federal de São Paulo, Escola Paulista Med, Dept Bioquim, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Bioquim, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, BR-04023900 São Paulo, BrazilWeb of Scienc

Effect of anti-glycosphingolipid monoclonal antibodies in pathogenic fungal growth and differentiation. Characterization of monoclonal antibody MEST-3 directed to Manpα1→3Manpα1→2IPC

<p>Abstract</p> <p>Background</p> <p>Studies carried out during the 1990's demonstrated the presence of fungal glycoinositol phosphorylceramides (GIPCs) with unique structures, some of them showed reactivity with sera of patients with histoplasmosis, paracoccidioidomycosis or aspergillosis. It was also observed that fungal GIPCs were able to inhibit T lymphocyte proliferation "in vitro", and studies regarding the importance of these molecules to fungal survival showed that many species of fungi are vulnerable to inhibitors of sphingolipid biosynthesis.</p> <p>Results</p> <p>In this paper, we describe a detailed characterization of an IgG2a monoclonal antibody (mAb), termed MEST-3, directed to the <it>Paracoccidioides brasiliensis </it>glycolipid antigen Pb-2 (Man<it>p</it>α1→3Man<it>p</it>α1→2IPC). mAb MEST-3 also recognizes GIPCs bearing the same structure in other fungi. Studies performed on fungal cultures clearly showed the strong inhibitory activity of MEST-3 on differentiation and colony formation of <it>Paracoccidioides brasiliensis</it>, <it>Histoplasma capsulatum </it>and <it>Sporothrix schenckii</it>. Similar inhibitory results were observed when these fungi where incubated with a different mAb, which recognizes GIPCs bearing terminal residues of β-D-galactofuranose linked to mannose (mAb MEST-1). On the other hand, mAb MEST-2 specifically directed to fungal glucosylceramide (GlcCer) was able to promote only a weak inhibition on fungal differentiation and colony formation.</p> <p>Conclusions</p> <p>These results strongly suggest that mAbs directed to specific glycosphingolipids are able to interfere on fungal growth and differentiation. Thus, studies on surface distribution of GIPCs in yeast and mycelium forms of fungi may yield valuable information regarding the relevance of glycosphingolipids in processes of fungal growth, morphological transition and infectivity.</p

Positron-emission tomography–based staging reduces the prognostic impact of early disease progression in patients with follicular lymphoma

Background: Previous studies reported that early progression of disease (POD) after initial therapy predicted poor overall survival (OS) in patients with follicular lymphoma (FL). Here, we investigated whether pre-treatment imaging modality had an impact on prognostic significance of POD. Methods: In this retrospective study, we identified 1088 patients with grade I–IIIA FL; of whom, 238 patients with stage II–IV disease were initially treated with rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP), and 346 patients were treated with rituximab-based chemotherapy. Patients (N = 484) from the FOLL05 study served as an independent validation cohort. We risk-stratified patients based on pre-treatment radiographic imaging (positron-emission tomography [PET] versus computed tomography [CT]) and early POD status using event-defining and landmark analyses. A competing risk analysis evaluated the association between early POD and histologic transformation. Results: In the discovery cohort, patients with POD within 24 months (PFS24) of initiating R-CHOP therapy had a 5-year OS of 57.6% for CT-staged patients compared with 70.6% for PET-staged patients. In the validation cohort, the 5-year OS for patients with early POD was 53.9% and 100% in CT- and PET-staged patients, respectively. The risk of histologic transformation in patients whose disease progressed within one year of initiating therapy was higher in CT-staged patients than in PET-staged patients (16.7% versus 6.3%, respectively), which was associated with a 9.7-fold higher risk of death. Conclusion: In FL, pre-treatment PET staging reduced the prognostic impact of early POD compared with CT staging. Patients with early POD and no histologic transformation have an extended OS with standard therapy

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Expression of Antibodies Directed to Paracoccidioides brasiliensis Glycosphingolipids during the Course of Paracoccidioidomycosis Treatment

Paracoccidioidomycosis (PCM) is a granulomatous disease caused by the dimorphic fungus Paracoccidioides brasiliensis. The immunoglobulin classes and isotypes of antibodies directed to acidic glycosphingolipids (GSLs) and glucosylceramide of P. brasiliensis were determined by enzyme-linked immunosorbent assay of sera from 31 PCM patients. The reactivities of 38 serum samples were analyzed by considering the stage of treatment: before antifungal treatment (n = 10), during 1 to 4 months of treatment (T1-4; n = 9), during 5 to 12 months of treatment (T5-12; n = 9), and posttreatment (PT; n = 10). Sera from healthy subjects (n = 12) were used as controls. Only the GSL Pb-1 antigen, which presents the carbohydrate structure Galfβ1-6(Manα1-3)Manβ1, was reactive with the PCM patient sera. The PCM patient sera did not react with Pb-2, which lacks the Galf residue and which is considered the biosynthetic precursor of Pb-1, indicating that the Galf residue is essential for antibody reactivity. The Pb-1 glycolipid from nontreated patients elicited a primary immune response with immunoglobulin M (IgM) production and subsequent switching to IgG1 production. The IgG1 titer increased after the start of antifungal treatment (T1-4 group), and general decreases in the anti-Pb-1 antibody titers were observed after 5 months of treatment (T5-12 and PT groups). The Pb-1 antigen, an acidic GSL with terminal Galf residue, has potential application as an elicitor of the host immune response in patients with PCM

Immunocytochemical localization of heparin in secretory granules of rat peritoneal mast cells using a monoclonal anti-heparin antibody (ST-1)

We performed immunogold labeling with an ST-1 monoclonal antibody (IgM), specific for intact heparin, to define the subcellular localization of heparin in mast cells. Rat peritoneal mast cells were fixed by a modified Karnovsky method and embedded in Araldite. Ultrathin sections were first treated with sodium periodate and then sequentially incubated with MAb ST-1, rabbit anti-mouse IgM, and protein A-gold. By transmission electron microscopy, gold particles were localized inside cytoplasmic granules of peritoneal mast cells. In contrast, with the same procedure, no labeling was observed in mast cells from rat intestinal mucosa. Control sections of rat peritoneal or intestinal mucosa mast Mast cells cells treated with an irrelevant MAb (IgM) did not show any labeling. Treatment with nitrous Heparin acid abolished the reactivity of MAb ST-1 with peritoneal mast cells. These results Granules show that different mast cells can be identified regarding their heparin content by immunochemical procedures using MAb ST-1.UNIV FED SAO PAULO,EPM,DEPT BIOCHEM,BR-04023900 SAO PAULO,BRAZILUNIV FED SAO PAULO,EPM,ELECTRON MICROSCOPY UNIT,BR-04023900 SAO PAULO,BRAZILUNESP,IBILCE,DEPT BIOL,S JOSE RIO PR,BRAZILUNIV FED SAO PAULO,EPM,DEPT BIOCHEM,BR-04023900 SAO PAULO,BRAZILUNIV FED SAO PAULO,EPM,ELECTRON MICROSCOPY UNIT,BR-04023900 SAO PAULO,BRAZILWeb of Scienc

Inhibition of Leishmania (Leishmania) amazonensis growth and infectivity by aureobasidin A

Objectives: To study the effect of aureobasidin A, an inhibitor of inositol phosphorylceramide (IPC) synthase, on Leishmania growth and infectivity.Methods: Effects of aureobasidin A were determined for: (i) promastigote growth in axenic culture; (ii) promastigote infectivity in macrophage monolayers; (iii) development of footpad lesions in BALB/c mice; (iv) differentiation of amastigotes into promastigotes.Results: Aureobasidin A (20 mu M) inhibited 90% of Leishmania (Leishmania) amazonensis promastigote growth in axenic culture, but the parasites remained viable, i.e. growth curves returned to normal after aureobasidin A was removed from culture medium. the aureobasidin A IC50 was determined by MTT assay as 4.1 mu M for L. (L.) amazonensis promastigotes, 12.6 mu M for Leishmania (Leishmania) major and 13.7 mu M for Leishmania (Viannia) braziliensis. There was a significant delay in infection when L. (L.) amazonensis promastigotes pre-treated with aureobasidin A were inoculated into BALB/c mouse footpads. When aureobasidin A was added to cultured macrophages infected with amastigotes, the number of infected macrophages was reduced by >90%.Conclusions: Aureobasidin A is an interesting pharmacological tool to investigate the effect of lipid metabolism inhibition in Leishmania spp.Universidade Federal de São Paulo, Dept Biochem, Escola Paulista Med, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biochem, Escola Paulista Med, BR-04023900 São Paulo, BrazilWeb of Scienc

Current relevance of fungal and trypanosomatid glycolipids and sphingolipids: studies defining structures conspicuously absent in mammals

Recently, glycosphingolipids have been attracting attention due to their role on biological systems as second messengers or modulators of signal transduction, affecting several events, which range from apoptosis to regulation of the cell cycle. In pathogenic fungi, glycolipids are expressed in two classes: neutral monohexosylceramides (glucosyl-or galactosylceramide) and acidic glycosylinositol phosphorylceramides (the latter class carries longer glycan chains). It is worth to mention that monohexosylceramides exhibit significant structural differences in their lipid moieties compared to their mammalian counterparts, whereas the glycosylinositol phosphorylceramides exhibit remarkable structural differences in their carbohydrate moieties in comparison to mammal glycosphingolipids counterpart. We observed that glycosylinositol phosphorylceramides are capable of promoting immune response in infected humans. In addition, inhibiting fungal glycosphingolipid biosynthetic pathways leads to an inhibition of colony formation, spore germination, cell cycle, dimorphism and hyphal growth. Other pathogens, such as trypanosomatids, also present unique glycolipids, which may have an important role for the parasite development and/or disease establishment. Regarding host-pathogen interaction, cell membrane rafts, which are enriched in sphingolipids and sterols, participate in parasite/fungal infection. In this review, it is discussed the different biological roles of (glyco) (sphingo)lipids of pathogenic/opportunistic fungi and trypanosomatids