A ligand motif enables differential vascular targeting of endothelial junctions between brain and retina.

Endothelial heterogeneity has important implications in health and disease. Molecular markers selectively expressed in the vasculature of different organs and tissues are currently being explored in targeted therapies with promising results in preclinical and clinical studies. Noteworthy is the role that combinatorial approaches such as phage display have had in identifying such markers by using phage as nanoparticles and surrogates for billions of different peptides, screening noninvasively the vascular lumen for binding sites. Here, we show that a new peptide motif that emerged from such combinatorial screening of the vasculature binds selectively to blood vessels in the brain in vivo but not to vessels in other organs. Peptides containing a conserved motif in which amino acids Phenylalanine-Arginine-Tryptophan (FRW) predominate could be visualized by transmission electron microscopy bound to the junctions between endothelial cells in all areas of the brain, including the optic nerve, but not in other barrier-containing tissues, such as intestines and testis. Remarkably, peptides containing the motif do not bind to vessels in the retina, implying an important molecular difference between these two vascular barriers. Furthermore, the peptide allows for in vivo imaging, demonstrating that new tools for studying and imaging the brain are likely to emerge from this motif.

Anti-Trypanosoma cruzi activity of costic acid isolated from Nectandra barbellata (Lauraceae) is associated with alterations in plasma membrane electric and mitochondrial membrane potentials.

As part of our continuous studies on prospecting metabolites from Brazilian plant species with pharmacologic activity against Trypanosoma cruzi, the n-hexane extract from twigs of Nectandra barbellata (Lauraceae) was subjected to a bioactivity-guided fractionation to afford the sesquiterpene costic acid. As results, costic acid induced a trypanocidal effect with IC50 of 37.8 and 7.9 µM to trypomastigotes and intracellular amastigotes, respectively. When tested in L929 cells, no cytotoxicity was detected in the highest tested concentration (CC50 > 200 µM), resulting in SI values >5 and >25 to trypomastigotes and amastigotes, respectively. Based on these promising results against T. cruzi, a mechanistic study of the parasite death was investigated. The flow cytometry analysis of costic acid-treated parasites showed depolarization of the plasma membrane electric potential. Spectrofluorimetrical analysis and transmission electron microscopy showed no evidence of plasma membrane permeability alteration of trypomastigotes, but strong ultrastructural damage, evidenced by large vacuoles. Although Ca2+ and reactive oxygen species (ROS) levels were unaltered after short time incubation with costic acid, it rapidly affected the mitochondria, leading to a depolarized potential of the membrane, reducing the ATP levels. In silico studies of costic acid showed good predictions for drug-likeness, with adherence to Lipinskís rules of five (RO5), good ADMET properties and no alerts for Pan-Assay Interference Compounds (PAINS). Therefore, costic acid demonstrated promising activity against T. cruzi parasites, with high selectivity to intracellular amastigotes. Considering the lethal action of costic acid in affecting a vital and unique organelle as the mitochondria, it could be considered a new hit compound for future drug design studies for Chagas disease.

Exploring Peripheral Blood-Derived Extracellular Vesicles as Biomarkers: Implications for Chronic Chagas Disease with Viral Infection or Transplantation.

Extracellular vesicles (EVs) are lipid bilayer envelopes that encapsulate cell-specific cargo, rendering them promising biomarkers for diverse diseases. Chagas disease, caused by the parasite Trypanosoma cruzi, poses a significant global health burden, transcending its initial epicenter in Latin America to affect individuals in Europe, Asia, and North America. In this study, we aimed to characterize circulating EVs derived from patients with chronic Chagas disease (CCD) experiencing a reactivation of acute symptoms. Blood samples collected in EDTA were processed to isolate plasma and subsequently subjected to ultracentrifugation for particle isolation and purification. The EVs were characterized using a nanoparticle tracking analysis and enzyme-linked immunosorbent assay (ELISA). Our findings revealed distinctive differences in the size, concentration, and composition of EVs between immunosuppressed patients and those with CCD. Importantly, these EVs play a critical role in the pathophysiology of Chagas disease and demonstrate significant potential as biomarkers in the chronic phase of the disease. Overall, our findings support the potential utility of the CL-ELISA assay as a specific sensitive tool for detecting circulating EVs in chronic Chagasic patients, particularly those with recurrent infection following an immunosuppressive treatment or with concurrent HIV and Chagas disease. Further investigations are warranted to identify and validate the specific antigens or biomarkers responsible for the observed reactivity in these patient groups, which may have implications for diagnosis, the monitoring of treatment, and prognosis.

Immunocytochemical expression of p16INK4A and Ki-67 in cytologically negative and equivocal pap smears positive for oncogenic human papillomavirus.

This study was designed to analyze the cross-sectional comparison of the p16 and Ki-67 immunocytochemical expression in negative and equivocal (atypical squamous cells of undetermined significance (ASC-US)) liquid-based cytology (LBC) samples testing positive for high-risk human papillomavirus (HPV) types with HC2 assay or polymerase-chain reaction (PCR). A series of 199 consecutive LBC specimens derived from the same number of women participating in the ongoing Latin American Screening Study at Leonor Mendes de Barros Hospital, Sao Paulo, were analyzed using immunocytochemistry for expression of p16 and Ki-67 in negative and equivocal LBC samples testing positive for high-risk HPV types with hybrid capture II test (HC2) or PCR. All patients with at least one test positive (cytology, PCR, and/or HC2) were followed each 6 months for 3 years. The follow-up procedure consisted of visual examination, colposcopic inspection, cytology, and HC2 assay. Among the negative cytologic samples, 101 were HPV-positive and 55 HPV-negative. Of the HPV-positive group, 59 of 101 cases (58.4%) were positive for both p16 and Ki67 immunostaining, and 17 of 101 (16.8%) were negative for both. The proportion of Ki-67-positivity increased almost in parallel with the increasing grade of p16-positivity (p = 0.0001 for linear trend). In the HPV-negative group, both markers were negative in 41 of 55 cases (74.5%), and no statistical relationship was observed between the two markers (Pearson, p = 0.595). HPV-positive ASC-US samples demonstrated a simultaneous positive immunoreaction for p16 and Ki67 in 11 of 16 cases (68.7%), whereas 3 (18.7%) were concurrently negative. The relationship between the two markers was of borderline significance (Pearson, p = 0.053), but no linear relationship was found between the graded p16 and Ki-67 expression (p = 0.065 for linear trend). In the HPV-negative ASC-US group, there was no statistical association between the graded p16 and Ki-67 positivity (Pearson, p = 0.281). After 36 months of follow-up of the ASC-US patients, 6 women still displayed ASC-US smear, of which 4 of 6 were HPV-positive and expressed both p16 and Ki-67 markers. Two of 43 ASC-US smears had high-grade squamous intraepithelial lesions diagnosed (4.6%), and 1 had low-grade squamous intraepithelial lesion (2.3%). All of those were positive for HPV, p16 and Ki-67. Patients with ASC-US diagnosis and positive high-risk HPV status and positive for p16 Ki67 should be carefully observed to exclude occurrence of a squamous intraepithelial lesion. The combination of these two markers can be a useful implement for management of women with equivocal cytology.