Peptide Microarrays for Flavivirus Diagnosis.

Flavivirus are the most alarming prevalent viruses worldwide due to its vast impact on public health. Most early symptoms of diseases caused by Flavivirus are similar among each other and to other febrile illnesses making the clinical differential diagnosis challenging. In addition, due to cross-reactivity and a relatively limited persistence of viral RNA in infected individuals, the current available diagnosis strategies fail to efficiently provide a differential viral identification. In this context, virus-specific tests are essential to improve patient care, as well as to facilitate disease surveillance and the effective control of transmission. Here, we describe the use of protein microarrays as an effective tool for screening peptides differentially recognized by anti-Yellow Fever virus antibodies induced by vaccination or by natural viral infection.

The role of T-cells in head and neck squamous cell carcinoma: From immunity to immunotherapy.

Head and neck squamous cell carcinoma (HNSCC) encompass a group of complex entities of tumours affecting the aerodigestive upper tract. The main risk factors are strongly related to tobacco and alcohol consumption, but also HPV infection is often associated. Surgery, radiotherapy and/or chemotherapy are the standard treatments, though the 5-year overall survival is less than 50%. The advances in genomics, molecular medicine, immunology, and nanotechnology have shed a light on tumour biology which helps clinical researchers to obtain more efficacious and less toxic therapies. Head and neck tumours possess different immune escape mechanisms including diminishing the immune response through modulating immune checkpoints, in addition to the recruitment and differentiation of suppressive immune cells. The insights into the HNSCC biology and its strong interaction with the tumour microenvironment highlights the role of immunomodulating agents. Recently, the knowledge of the immunological features of these tumours has paved the way for the discovery of effective biomarkers that allow a better selection of patients with odds of improving overall survival through immunotherapy. Specially biomarkers regarding immune checkpoint inhibitors antibodies, such as anti-PD-1/PD-L1 and anti-CTLA-4 in combination with standard therapy or as monotherapy. New immunotherapies to treat head and neck cancer carcinomas, such as CAR T cells and nanoparticles have been the center of attention and in this review, we discuss the necessity of finding targets for the T cell in the cancer cells to generate CAR T cells, but also the relevance of evaluating specificity and safety of those therapies.

Mapping and Validation of Peptides Differentially Recognized by Antibodies from the Serum of Yellow Fever Virus-Infected or 17DD-Vaccinated Patients.

Yellow Fever disease is caused by the Yellow Fever virus (YFV), an arbovirus from the Flaviviridae family. The re-emergence of Yellow Fever (YF) was facilitated by the increasing urbanization of sylvatic areas, the wide distribution of the mosquito vector, and the low percentage of people immunized in the Americas, which caused severe outbreaks in recent years, with a high mortality rate. Therefore, serological approaches capable of discerning antibodies generated from the wild-type (YFV-WT) strain between the vaccinal strain (YFV-17DD) could facilitate vaccine coverage surveillance, enabling the development of strategies to avoid new outbreaks. In this study, peptides were designed and subjected to microarray procedures with sera collected from individuals infected by WT-YFV and 17DD-YFV of YFV during the Brazilian outbreak of YFV in 2017/2018. From 222 screened peptides, around ten could potentially integrate serological approaches aiming to differentiate vaccinated individuals from naturally infected individuals. Among those peptides, one was synthesized and validated through ELISA.

Immunotherapy for cancer: effects of iron oxide nanoparticles on polarization of tumor-associated macrophages.

Cancer immunotherapy is the most promising trend in oncology, focusing on helping or activating the patient's immune system to identify and fight against cancer. In the last decade, interest in metabolic reprogramming of tumor-associated macrophages from M2-like phenotype (promoting tumor progression) to M1-like phenotypes (suppressing tumor growth) as a therapeutic strategy against cancer has increased considerably. Iron metabolism has been standing out as a target for the reprogramming of tumor-associated macrophages to M1-like phenotype with therapeutic purposes against cancer. Due to the importance of the iron levels in macrophage polarization states, iron oxide nanoparticles can be used to change the activation state of tumor-associated macrophages for a tumor suppressor phenotype and as an anti-tumor strategy.

Molecular surveillance of dengue in Minas Gerais provides insights on dengue virus 1 and 4 circulation in Brazil.

Dengue, caused by any of the four types of Dengue virus (DENV) is the most important arbovirus in the world. In this study we performed a molecular surveillance of dengue during the greatest dengue outbreak that took place in Divinópolis, Minas Gerais state, Southeast Brazil, in 2013. Samples from 100 patients with clinical symptoms of dengue were studied and 26 were positive. The capsid/premembrane (CprM) and envelope gene sequences of some samples were amplified and sequenced. Molecular analyses demonstrated that two DENV-1 lineages, belonging to genotype V were introduced and co-circulated in Divinópolis. When compared to each other, those lineages presented high genetic diversity and showed unique amino acids substitutions in the envelope protein, including in domains I, II, and III. DENV-4 strains from Divinópolis clustered within genotype IIb and the most recent common ancestor was probably introduced into the city three years before the 2013 epidemic. Here we demonstrated for the first time the circulation of DENV-4 and the co-circulation of two DENV-1 lineages in Midwest region of Minas Gerais, Brazil. Moreover our analysis indicated the introduction of five DENV-1 lineages, genotype V into Brazil, in different times. J. Med. Virol. 89:966-973, 2017. © 2016 Wiley Periodicals, Inc.

Primary dengue haemorrhagic fever in patients from northeast of Brazil is associated with high levels of interferon-β during acute phase

Dengue is an acute febrile disease caused by the mosquito-borne dengue virus (DENV) that according to clinical manifestations can be classified as asymptomatic, mild or severe dengue. Severe dengue cases have been associated with an unbalanced immune response characterised by an over secretion of inflammatory cytokines. In the present study we measured type I interferon (IFN-I) transcript and circulating levels in primary and secondary DENV infected patients. We observed that dengue fever (DF) and dengue haemorrhagic fever (DHF) patients express IFN-I differently. While DF and DHF patients express interferon-α similarly (52,71 ± 7,40 and 49,05 ± 7,70, respectively), IFN- β were associated with primary DHF patients. On the other hand, secondary DHF patients were not able to secrete large amounts of IFN- β which in turn may have influenced the high-level of viraemia. Our results suggest that, in patients from our cohort, infection by DENV serotype 3 elicits an innate response characterised by higher levels of IFN- β in the DHF patients with primary infection, which could contribute to control infection evidenced by the low-level of viraemia in these patients. The present findings may contribute to shed light in the role of innate immune response in dengue pathogenesis.

RAP1 GTPase overexpression is associated with cervical intraepithelial neoplasia.

RAP1 (RAS proximate 1), a small GTP-binding protein of the RAS superfamily, is a putative oncogene that is highly expressed in several malignant cell lines and types of cancers, including some types of squamous cell carcinoma. However, the participation of RAP1 in cervical carcinogenesis is unknown. We conducted a cross-sectional study of paraffin-embedded cervical biopsies to determine the association of RAP1 with cervical intraepithelial neoplasia (CIN). Standard and quantitative immunohistochemistry assessment of RAP1 expression in fixed tissue was performed on 183 paraffin-embedded cervical biopsies that were classified as normal or non-dysplastic mucosa (NDM) (n = 33); CIN grade 1 (n = 84) and CIN grade 2/3 (n = 66). A gradual increase in RAP1 expression in NDM < CIN 1 < CIN 2/3 (p<0.001) specimens was observed and was in agreement with the histopathologic diagnosis. A progressive increase in the RAP1 expression levels increased the risk of CIN 1 [odds ratio (OR) = 3.50; 95% confidence interval (CI) 1.30-10.64] 3.5 fold and the risk of CIN 2/3 (OR = 19.86, 95% CI 6.40-70.79) nearly 20 fold when compared to NDM. In addition, stereotype ordinal regression analysis showed that this progressive increase in RAP1 expression more strongly impacted CIN 2/3 than CIN 1. Our findings suggest that RAP1 may be a useful biomarker for the diagnosis of CIN.

Potential biomarkers for the clinical prognosis of severe dengue.

Currently, several assays can confirm acute dengue infection at the point-of-care. However, none of these assays can predict the severity of the disease symptoms. A prognosis test that predicts the likelihood of a dengue patient to develop a severe form of the disease could permit more efficient patient triage and treatment. We hypothesise that mRNA expression of apoptosis and innate immune response-related genes will be differentially regulated during the early stages of dengue and might predict the clinical outcome. Aiming to identify biomarkers for dengue prognosis, we extracted mRNA from the peripheral blood mononuclear cells of mild and severe dengue patients during the febrile stage of the disease to measure the expression levels of selected genes by quantitative polymerase chain reaction. The selected candidate biomarkers were previously identified by our group as differentially expressed in microarray studies. We verified that the mRNA coding for CFD, MAGED1, PSMB9, PRDX4 and FCGR3B were differentially expressed between patients who developed clinical symptoms associated with the mild type of dengue and patients who showed clinical symptoms associated with severe dengue. We suggest that this gene expression panel could putatively serve as biomarkers for the clinical prognosis of dengue haemorrhagic fever.

Potential biomarkers for the clinical prognosis of severe dengue

Currently, several assays can confirm acute dengue infection at the point-of-care. However, none of these assays can predict the severity of the disease symptoms. A prognosis test that predicts the likelihood of a dengue patient to develop a severe form of the disease could permit more efficient patient triage and treatment. We hypothesise that mRNA expression of apoptosis and innate immune response-related genes will be differentially regulated during the early stages of dengue and might predict the clinical outcome. Aiming to identify biomarkers for dengue prognosis, we extracted mRNA from the peripheral blood mononuclear cells of mild and severe dengue patients during the febrile stage of the disease to measure the expression levels of selected genes by quantitative polymerase chain reaction. The selected candidate biomarkers were previously identified by our group as differentially expressed in microarray studies. We verified that the mRNA coding for CFD, MAGED1, PSMB9, PRDX4 and FCGR3B were differentially expressed between patients who developed clinical symptoms associated with the mild type of dengue and patients who showed clinical symptoms associated with severe dengue. We suggest that this gene expression panel could putatively serve as biomarkers for the clinical prognosis of dengue haemorrhagic fever.

Modeling the zing finger protein SmZF1 from Schistosoma mansoni: Insights into DNA binding and gene regulation.

Zinc finger proteins are widely found in eukaryotes, representing an important class of DNA-binding proteins frequently involved in transcriptional regulation. Zinc finger motifs are composed by two antiparallel ß-strands and one α-helix, stabilized by a zinc ion coordinated by conserved histidine and cysteine residues. In Schistosoma mansoni, these regulatory proteins are known to modulate morphological and physiological changes, having crucial roles in parasite development. A previously described C(2)H(2) zinc finger protein, SmZF1, was shown to be present in cell nuclei of different life stages of S. mansoni and to activate gene transcription in a heterologous system. A high-quality SmZF1 tridimensional structure was generated using comparative modeling. Molecular dynamics simulations of the obtained structure revealed stability of the zinc fingers motifs and high flexibility on the terminals, comparable to the profile observed on the template X-ray structure based on thermal b-factors. Based on the protein tridimensional features and amino acid composition, we were able to characterize four C(2)H(2) zinc finger motifs, the first involved in protein-protein interactions while the three others involved in DNA binding. We defined a consensus DNA binding sequence using three distinct algorithms and further carried out docking calculations, which revealed the interaction of fingers 2-4 with the predicted DNA. A search for S. mansoni genes presenting putative SmZF1 binding sites revealed 415 genes hypothetically under SmZF1 control. Using an automatic annotation and GO assignment approach, we found that the majority of those genes code for proteins involved in developmental processes. Taken together, these results present a consistent base to the structural and functional characterization of SmZF1.