Diagnostic application of sensitive and specific phage-exposed epitopes for visceral leishmaniasis and human immunodeficiency virus coinfection.

The diagnosis of visceral leishmaniasis (VL) has improved with the search of novel antigens; however, their performance is limited when samples from VL/human immunodeficiency virus (HIV)-coinfected patients are tested. In this context, studies conducted to identify more suitable antigens to detect both VL and VL/HIC coinfection cases should be performed. In the current study, phage display was performed using serum samples from healthy subjects and VL, HIV-infected and VL/HIV-coinfected patients; aiming to identify novel phage-exposed epitopes to be evaluated with this diagnostic purpose. Nine non-repetitive and valid sequences were identified, synthetized and tested as peptides in enzyme-linked immunosorbent assay experiments. Results showed that three (Pep2, Pep3 and Pep4) peptides showed excellent performance to diagnose VL and VL/HIV coinfection, with 100% sensitivity and specificity values. The other peptides showed sensitivity varying from 50.9 to 80.0%, as well as specificity ranging from 60.0 to 95.6%. Pep2, Pep3 and Pep4 also showed a potential prognostic effect, since specific serological reactivity was significantly decreased after patient treatment. Bioinformatics assays indicated that Leishmania trypanothione reductase protein was predicted to contain these three conformational epitopes. In conclusion, data suggest that Pep2, Pep3 and Pep4 could be tested for the diagnosis of VL and VL/HIV coinfection.

Leishmania infantum ß-Tubulin Identified by Reverse Engineering Technology through Phage Display Applied as Theranostic Marker for Human Visceral Leishmaniasis.

Two Leishmania infantum mimotopes (B10 and C01) identified by phage display showed to be antigenic and immunogenic for visceral (VL) and tegumentary (TL) leishmaniasis; however, their biological targets in the parasites have not been identified. The aim of the present study was to investigate the native antigens expressing both mimotopes, and to use them in distinct immunological assays. For this, a subtractive phage display technology was used, where a combinatorial library of single-chain variable fragments (scFv) was employed and the most reactive monoclonal antibodies for each target were captured, being the target antigens identified by mass spectrometry. Results in immunoblotting and immunoprecipitation assays showed that both monoclonal scFvs antibodies identified the ß-tubulin protein as the target antigen in L. infantum. To validate these findings, the recombinant protein was cloned, purified and tested for the serodiagnosis of human leishmaniasis, and its immunogenicity was evaluated in PBMC derived from healthy subjects and treated or untreated VL patients. Results showed high diagnostic efficacy, as well as the development of a specific Th1 immune response in the cell cultures, since higher IFN-γ and lower IL-10 production was found.

High-through identification of T cell-specific phage-exposed mimotopes using PBMCs from tegumentary leishmaniasis patients and their use as vaccine candidates against Leishmania amazonensis infection.

In the current study, phage-exposed mimotopes as targets against tegumentary leishmaniasis (TL) were selected by means of bio-panning cycles employing sera of TL patients and healthy subjects, besides the immune stimulation of peripheral blood mononuclear cells (PBMCs) collected from untreated and treated TL patients and healthy subjects. The clones were evaluated regarding their specific interferon-γ (IFN-γ) and interleukin-4 (IL-4) production in the in vitro cultures, and selectivity and specificity values were calculated, and those presenting the best results were selected for the in vivo experiments. Two clones, namely A4 and A8, were identified and used in immunization protocols from BALB/c mice to protect against Leishmania amazonensis infection. Results showed a polarized Th1 response generated after vaccination, being based on significantly higher levels of IFN-γ, IL-2, IL-12, tumour necrosis factor-α (TNF-α) and granulocyte-macrophage colony-stimulating factor (GM-CSF); which were associated with lower production of specific IL-4, IL-10 and immunoglobulin G1 (IgG1) antibodies. Vaccinated mice presented significant reductions in the parasite load in the infected tissue and distinct organs, when compared with controls. In conclusion, we presented a strategy to identify new mimotopes able to induce Th1 response in PBMCs from TL patients and healthy subjects, and that were successfully used to protect against L. amazonensis infection.

Extracellular vesicles as drivers of epithelial-mesenchymal transition and carcinogenic characteristics in normal prostate cells.

There is increasing evidence that cancer dissemination and metastasis establishment may not only be due to the movement of tumor cells. Content of extracellular vesicles (EVs) secreted by tumor cells may also reflect the origin of these cells. Some molecules that constitute these EVs have already been used as targets for detection of specific tumors. However, to the best of our knowledge, EVs from biopsies and plasma have not yet been compared nor thoroughly investigated as triggers of malignant transformation and metastatic niche formation. To evaluate the role of EVs in the cellular microenvironment, we have treated the normal epithelial prostate cell lines, RWPE-1 and PNT-2, with a pool of EVs from biopsies of prostate primary tumors (bEVs), biopsies of benign prostate hyperplasia (hEVs), plasma of prostate cancer (PCa) patients (pEVs) or plasma of healthy individuals (pnEVs). Each of the four pools consisted of isolated EVs from several subjects, of which PCa patients were in different stages of cancer. Migration and proliferation profiles, cytokine release, and a panel of PCa-associated genes' expression of epithelial-mesenchymal transition in the cell lines were evaluated after 24 h incubation with EVs. When compared to the control groups, cells treated with the pool of EVs isolated from tumor biopsies and plasma of PCa patients showed greater migration and proliferation, significant alterations in gene expression, and high levels of IL-8, factors that are associated with cancer development. Specifically, isolated bEVs and pEVs may induce malignant features in non-tumor cells by activating several cellular events associated with cancer progression, suggesting that future PCa therapy may target multiple elements found in tumor-derived EVs.

Altered Leukocyte Sphingolipid Pathway in Breast Cancer.

Sphingolipid metabolism pathway is essential in membrane homeostasis, and its dysfunction has been associated with favorable tumor microenvironment, disease progression, and chemotherapy resistance. Its major components have key functions on survival and proliferation, with opposing effects. We have profiled the components of the sphingolipid pathway on leukocytes of breast cancer (BC) patients undergoing chemotherapy treatment and without, including the five sphingosine 1-phosphate (S1P) receptors, the major functional genes, and cytokines, in order to better understand the S1P signaling in the immune cells of these patients. To the best of our knowledge, this is the first characterization of the sphingolipid pathway in whole blood of BC patients. Skewed gene profiles favoring high SPHK1 expression toward S1P production during BC development was observed, which was reversed by chemotherapy treatment, and reached similar levels to those found in healthy donors. Such levels were also correlated with high levels of TNF-α. Our data revealed an important role of the sphingolipid pathway in immune cells in BC with skewed signaling of S1P receptors, which favored cancer development even under chemotherapy, and may probably be a trigger of cancer resistance. Thus, these molecules must be considered as a target pathway for combined BC therapeutics.

Antitumoral effects of γCdcPLI, a PLA2 inhibitor from Crotalus durissus collilineatus via PI3K/Akt pathway on MDA-MB-231 breast cancer cell.

Phospholipases A2 (PLA2s) overexpression is closely associated with the malignant potential of breast cancers. Here, we showed for the first the antitumoral effects of γCdcPLI, a PLA2 inhibitor from Crotalus durissus collilineatus via PI3K/Akt pathway on MDA-MB-231 cell. Firstly, γCdcPLI was more cytotoxic to MDA-MB-231 breast cancer cells than other cell lines (MCF-7, HeLa, PC3 and A549) and did not affect the viability of non-tumorigenic breast cell (MCF 10A). In addition, γCdcPLI induced modulation of important mediators of apoptosis pathways such as p53, MAPK-ERK, BIRC5 and MDM2. γCdcPLI decreased MDA-MB-231 adhesion, migration and invasion. Interestingly, the γCdcPLI also inhibited the adhesion and migration of endothelial cells and blocked angiogenesis by inhibiting tube formation by HUVECs in vitro and sprouting elongation on aortic ring assay ex vivo. Furthermore, γCdcPLI reduced the production of vascular endothelial growth factor (VEGF). γCdcPLI was also able to decrease PGE2 levels in MDA-MB-231 and inhibited gene and protein expression of the PI3K/Akt pathway. In conclusion, γCdcPLI showed in vitro antitumoral, antimestatatic and anti-angiogenic potential effects and could be an attractive approach for futures studies in cancer therapy.

Leishmania infantum mimotopes and a phage-ELISA assay as tools for a sensitive and specific serodiagnosis of human visceral leishmaniasis.

Serological methods used to diagnose visceral leishmaniasis (VL) are considered minimally invasive, but they present problems related with their sensitivity and/or specificity. In this study, a subtractive selection using the phage display technology against antibodies from healthy subjects living in endemic and non-endemic areas of disease, as well as from Chagas disease patients and those developing active VL, was developed. The aim of this study was to select bacteriophage-fused epitopes to be used in the serodiagnosis of human VL. Eight phage clones were selected after the bio-panning rounds, and their reactivity was evaluated in a phage-ELISA assay against a human serological panel. A wild-type clone and the recombinant K39-based immunochromatographic test were used as controls. In the results, it was shown that all clones showed an excellent performance to serologically identify VL patients, demonstrating the feasibility of the isolated phages for developing a specific and sensitive serodiagnosis of human VL.

Antitumoral effects of gamma CdcPLI, a PLA(2) inhibitor from Crotalus durissus collilineatus via PI3K/Akt pathway on MDA-MB-231 breast cancer cell

Phospholipases A(2) (PLA(2)s) overexpression is closely associated with the malignant potential of breast cancers. Here, we showed for the first the antitumoral effects of gamma CdcPLI, a PLA2 inhibitor from Crotalus durissus collilineatus via PI3K/Akt pathway on MDA-MB-231 cell. Firstly, gamma CdcPLI was more cytotoxic to MDA-MB-231 breast cancer cells than other cell lines ( MCF-7, HeLa, PC3 and A549) and did not affect the viability of non-tumorigenic breast cell (MCF 10A). In addition, gamma CdcPLI induced modulation of important mediators of apoptosis pathways such as p53, MAPK-ERK, BIRC5 and MDM2.gamma CdcPLI decreased MDA-MB-231 adhesion, migration and invasion. Interestingly, the gamma CdcPLI also inhibited the adhesion and migration of endothelial cells and blocked angiogenesis by inhibiting tube formation by HUVECs in vitro and sprouting elongation on aortic ring assay ex vivo. Furthermore,gamma CdcPLI reduced the production of vascular endothelial growth factor (VEGF).gamma CdcPLI was also able to decrease PGE2 levels in MDA-MB-231 and inhibited gene and protein expression of the PI3K/Akt pathway. In conclusion,gamma CdcPLI showed in vitro antitumoral, antimestatatic and anti-angiogenic potential effects and could be an attractive approach for futures studies in cancer therapy.

New serological tools for improved diagnosis of human tegumentary leishmaniasis.

Human tegumentary leishmaniasis (HTL), characterized by skin ulcers that may spread and cause dreadful and massive tissue destruction of the nose and mouth, is considered a neglected tropical disease, and it is a serious threat to global health due to its continuous expansion, favored by the lifecycle of its causative organism that is maintained in domestic animal reservoirs and anthropophilic sand fly species. Serodiagnosis of HTL is a great challenge due to many biological factors, including hampered specificity and/or sensitivity. This investigation addresses the unmet need for new diagnostic markers of HTL, and describes a simple platform to improve the serodiagnosis. A constrained conformational phage display random peptide library combined with a magnetic microsphere-based subtraction strategy was used to identify ligands with potential diagnostic applications. Six clones were selected against IgG antibodies from HTL patients, characterized by sequencing and confirmed by a phage-ELISA using sera from patients developing visceral leishmaniasis (n=20), Chagas disease (n=10), mucosal (n=30) and cutaneous (n=20) leishmaniasis; as well as from healthy subjects living in endemic (n=20) and non-endemic (n=30) areas of leishmaniasis. A wild-type M13-phage clone and a soluble Leishmania antigenic extract were used as negative and positive controls, respectively. Three clones reached 100% sensitivity and specificity, without any cross-reactivity with sera from patients with leishmaniasis-related diseases. Briefly, we describe for the first time a set of serological markers based on three immunodominant mimotopes that showed 100% accuracy, and that could be used in a phage-ELISA assay for the HTL serodiagnosis.

3D Cell-SELEX: Development of RNA aptamers as molecular probes for PC-3 tumor cell line.

Human prostate cancer (PCa) is a highly heterogeneous and multifactorial disease. Current clinical biomarkers are not sufficiently accurate, thus being unable to predict the clinical outcome. Therefore, searching for new biomarkers aiming to improve diagnosis, prognosis and therapy is still required. In this study, we performed 3D Cell-SELEX against PC-3 prostate cancer cell line, a novel strategy to select specific nucleic acid ligands against spheroid cells in 3D cell culture. This original system combines Cell-SELEX, a process that exploits the cellular structure to generate specific ligands, and 3D cell culture, an approach that mimics the tissue microenvironment in vitro. In the first round of 3D Cell-SELEX, a negative selection against RWPE-1, non-tumor cell line, was performed to subtract non-tumor specific aptamers. The supernatant was used in eight additional rounds of selection, which were performed against PC-3 cell line. After nine selection cycles, eight PC-3 specific RNA aptamers were selected and sequenced. The aptamers presented sizes between 20 and 50 nucleotides-long, with low free energy (∆G<-13.6), which contributed for their spontaneous folding and high stability. Furthermore, our results showed the aptamer A4 as a specific ligand to prostate tumor cells, with dissociation constant in the nanomolar scale. Therefore, the novel 3D Cell-SELEX procedure improved the selection of PCa cell-surface ligands and the aptamer A4 has shown potential for the identification of prostate tumor cells, suggesting the application of this molecule in further screening assays for PCa.

Prostate-specific RNA aptamer: promising nucleic acid antibody-like cancer detection.

We described the selection of a novel nucleic acid antibody-like prostate cancer (PCa) that specifically binds to the single-stranded DNA molecule from a 277-nt fragment that may have been partially paired and bound to the PCA3 RNA conformational structure. PCA3-277 aptamer ligands were obtained, and the best binding molecule, named CG3, was synthesized for validation. Aiming to prove its diagnostic utility, we used an apta-qPCR assay with CG3-aptamer conjugated to magnetic beads to capture PCA3 transcripts, which were amplified 97-fold and 7-fold higher than conventional qPCR in blood and tissue, respectively. Histopathologic analysis of 161 prostate biopsies arranged in a TMA and marked with biotin-labeled CG3-aptamer showed moderate staining in both cytoplasm and nucleus of PCa samples; in contrast, benign prostatic hyperplasia (BPH) samples presented strong nuclear staining (78% of the cases). No staining was observed in stromal cells. In addition, using an apta-qPCR, we demonstrated that CG3-aptamer specifically recognizes the conformational PCA3-277 molecule and at least three other transcript variants, indicating that long non-coding RNA (lncRNA) is processed after transcription. We suggest that CG3-aptamer may be a useful PCa diagnostic tool. In addition, this molecule may be used in drug design and drug delivery for PCa therapy.

Dynamic dialog between cytokeratin 18 and annexin A1 in breast cancer: a transcriptional disequilibrium.

Cytokeratins (CKs) constitute the cytoskeletal network and are regulated by post-translational modifications, acting not only as a mechanical support, but also in cell signaling and regulatory processes. Signaling is mediated by CK-associated proteins, such as Annexin A1 (ANXA1), a ligand of the CK18/CK8 complex. ANXA1 has a pivotal role in cellular and immunological responses, and together with CK18 have been implicated in several processes related to malignant transformation in breast cancer (BC). Our aim was to demonstrate how their interaction might be linked to BC development. We investigated transcript levels, protein expression and distribution for both targets in breast tissues of 92 patients (42 BCs and 50 benign diseases) using qPCR and immunohistochemistry, respectively. ANXA1 and CK18 mRNAs were inversely correlated, and their ratio in each TNM stage significantly differentiated BC from benign diseases (OR=5.62). These differences did not mirror tissue protein levels, but a significant dichotomous protein distribution in tumor tissues was observed, differing from the expected co-localization observed during cell homeostasis. The disequilibrium of transcriptional levels between ANXA1/CK18 and alterations in their tissue distribution are present either in initial events or tumor progression, which suggest a critical event in BC. The broken dialog between ANXA1 and CK18 in normal breast tissues may play a critical role in BC development, and together may be used as combined targets for BC diagnostics.