Biology subjects

Albuquerque, P.

Publications and source records attributed to Albuquerque, P..

10 recordsLinked to original sources

bioRxiv2023

Isolation of Biologically Active Extracellular Vesicles-Associated AAVs for Gene Delivery to the Brain by Size Exclusion Chromatography

Extracellular vesicles-associated adeno-associated viral vectors (EV-AAVs) emerged as a new opportunity for non-invasive gene therapy targeting the central nervous system (CNS). However, in previous reports, only AAV serotypes with known ability to cross the blood-brain barrier (BBB) have been used for EV-AAV production and testing through non-invasive strategies. In this work, we aimed at optimizing a size exclusion chromatography (SEC) protocol for the production and isolation of natural and biologically active brain-targeting EV-AAVs, that could be applied to any AAV serotype and further used for non-invasive gene delivery to the CNS. We performed a comparison between SEC and differential ultracentrifugation (UC) isolation protocols in terms of yield, contaminants, and transgene expression efficiency. We found that SEC allows a higher recovery of EV-AAVs, free of cell contaminating proteins and with less solo AAVs than UC. Remarkably, SEC-purified EV-AAVs also showed to be more potent at transgene expression than solo AAVs in neuronal cell lines. EV-AAVs exhibited the ability to cross the BBB in neonatal mice upon intravenous administration. In conclusion, SEC-purified brain-targeting EV-AAVs show to be a promising gene delivery vector for therapy of brain disorders. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/542901v1_ufig1.gif" ALT="Figure 1"> View larger version (70K): org.highwire.dtl.DTLVardef@7016b8org.highwire.dtl.DTLVardef@e3a5f3org.highwire.dtl.DTLVardef@1a43286org.highwire.dtl.DTLVardef@f110c5_HPS_FORMAT_FIGEXP M_FIG C_FIG During the production of AAV vectors, a small percentage of AAVs is secreted in association with extracellular vesicles, named "EV-AAVs". EV-AAVs can be efficiently isolated by size exclusion chromatography (SEC). When intravenously injected in mice, brain targeting EV-AAVs can cross the blood brain barrier (BBB) and transduce neuronal cells.

molecular biology↗

bioRxiv2021

Hinge Influences in Murine IgG Binding to Cryptococcus neoformans Capsule

Decades of studies on antibody structure led to the tenet that the V region binds antigens while the C region interacts with immune effectors. In some antibodies, however, the C region affects affinity and/or specificity for the antigen. One such case is that of the 3E5 antibodies, a family of monoclonal murine IgGs in which the mIgG3 isotype has different fine specificity to the Cryptococcus neoformans capsule polysaccharide than the other mIgG isotypes. Our group serendipitously found another pair of mIgG1/mIgG3 antibodies based on the 2H1 hybridoma to the C. neoformans capsule that recapitulated the differences observed with 3E5. In this work, we report the molecular basis of the constant domain effects on antigen binding using recombinant antibodies. As with 3E5, immunofluorescence experiments show a punctate pattern for 2H1-mIgG3 and an annular pattern for 2H1-mIgG1. Also as observed with 3E5, 2H1-mIgG3 bound on ELISA to both acetylated and non-acetylated capsular polysaccharide, whereas 2H1-mIgG1 only bound well to the acetylated form, consistent with differences in fine specificity. In engineering hybrid mIgG1/mIgG3 antibodies, we found that switching the 2H1-mIgG3 hinge for its mIgG1 counterpart changed the immunofluorescence pattern to annular, but a 2H1-mIgG1 antibody with a mIgG3 hinge still had an annular pattern. The hinge is thus necessary but not sufficient for these changes in binding to the antigen. This important role for the constant region in binding of antibodies to the antigen could affect the design of therapeutic antibodies and our understanding of their function in immunity. Key pointsO_LIKey point 1- 2H1 antibodies recapitulate differences between mIgG isotypes observed with 3E5. C_LIO_LIKey point 2 - The hinge region is necessary but not sufficient for these differences. C_LIO_LIKey point 3 - The antibody constant region can also play a role in mIgG binding to antigen. C_LI

immunology↗

bioRxiv2020

Molecular mechanisms of LC3-associated phagocytosis in the macrophage response to Paracoccidioides spp.

Paracoccidiomycosis is a systemic fungal infection that is endemic in Latin America. The etiologic agents are thermodimorphic fungi from the Paracoccidiodes genus, which are facultative intracellular parasites of macrophages. LC3-associated phagocytosis (LAP), a noncanonical form of autophagy, is important in the immune response to similar pathogens, so we sought to determine the role LAP plays in the macrophage response to Paracoccidioides spp. By immunofluorescence, we found that LC3 was recruited to phagosomes containing Paracoccidioides spp. in both RAW264.7 and J774.16 cell lines and in bone marrow-derived macrophages. Interference with autophagy using RNAi against ATG5 reduced the antifungal activity of J774.16 cells, showing that LC3 recrutiment is important for proper control of the fungus by macrophages. Finally, we used pharmacological Syk kinase and NAPH oxidase inhibitors, which inhibit signalling pathways necessary for macrophage LAP against Aspergillus fumigatus and Candida albicans, to dissect part of the signaling pathways that trigger LAP agains Paracoccidioides spp. Interestingly, these inhibitors did not decrease LAP against P. brasiliensis, possibly due to differences in the fungal cell surface compositions. These observations suggest a potential role for autophagy as target for host-directed paracoccidioidomycosis therapies.

immunology↗

bioRxiv2020

Paracoccidioides HSP90 can be found in the cell surface and is a target for antibodies with therapeutic potential

Paracoccidioidomycosis (PCM) is one of the most frequent systemic mycoses in Latin America. It affects mainly male rural workers in impoverished regions, and the therapy can last up to two years or use drugs that are very toxic. Given the need for novel safe and effective approaches to treat PCM, we have been developing monoclonal antibodies (mAbs) that could be used not only to block specific fungal targets, but also modulate the hosts antifungal immunity. In this work we show the generation of and promising results with a mAb against HSP90, a molecular chaperone that is an important virulence factor in fungi. Using recombinant Paracoccidioides lutzii (Pb01) and P. brasiliensis (Pb18) HSP90 proteins produced in E. coli, we immunized mice and generated polyclonal antibodies and an IgG1 hybridoma mAb. The proteins were very immunogenic and both the polyclonal serum and mAb were used in immunofluorescence experiments, which showed binding of antibodies to the yeast cell surface. The mAb successfully opsonized P. lutzii and P. brasiliensis cells in co-incubations with J774.16 macrophage-like cells. Our results suggest that this mAb could serve as the basis for new immunotherapy regimens for PCM. Author summaryParacoccidioidomycosis (PCM) is a severe disease caused by fungi, common in Latin America. It is treatable, but some of the drugs that are available are very toxic or not very effective, and the treatment can take as long as two years to clear the infection. To address the need for improved therapeutic alternatives, we have been developing drug candidates based on antibody technologies against Paracoccidioides brasiliensis and P. lutzii, which cause PCM. In this work, we produced monoclonal antibodies (mAbs) that bind to the fungal protein HSP90, which is essential for fungal cells to survive. One mAb, 4D11, recognized the HSP90 target on the surface of fungal cells. These antibody-covered cells were ingested more efficiently by immune cells called macrophages, suggesting they could improve the host resistance to infection by Paracoccidioides. Future improvements on these antibodies could thus lead to more effective and safer PCM treatments.

microbiology↗

bioRxiv2020

Faster Cryptococcus melanization increases virulence in experimental and human cryptococcosis

Cryptococcus spp. are important human pathogens responsible for about 180,000 deaths per year. Studying their virulence attributes can lead to better cryptococcosis prevention and treatment. In this work, we systematically investigated virulence attributes of Cryptococcus spp. clinical isolates and correlated them with patient data. We collected 66 C. neoformans and 19 C. gattii isolates from Brazilian patients and analyzed multiple phenotypes related with their capsule, production of laccase, melanin and extracellular vesicles. We also tested their virulence in Galleria mellonella and ability to evade macrophage LC3-associated phagocytosis (LAP). All phenotypes analyzed varied widely among the isolates, but C. neoformans isolates tended to melanize faster and more intensely and produce thinner capsules in comparison with C. gattii. We also observed correlations that match previous studies, such as that between secreted laccase - but not total melanin production - and disease outcome in patients. The most striking results, though, came from our measurements of Cryptococcus colony melanization kinetics, which followed a sigmoidal curve for most isolates. Faster melanization correlated positively with LAP evasion, virulence in G. mellonella and worse prognosis in humans. These results suggest that the speed of melanization, more than the total amount of melanin Cryptococcus spp. produces, is crucial for virulence. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=162 SRC="FIGDIR/small/222794v2_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@41c48borg.highwire.dtl.DTLVardef@1ce8e1eorg.highwire.dtl.DTLVardef@1d4568corg.highwire.dtl.DTLVardef@17e2003_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2019

A hidden battle in the dirt: soil amoebae interactions with Paracoccidioides spp

Paracoccidioides spp. are thermodimorphic pathogenic fungi endemic to Latin America. Predation is believed to drive the evolution of virulence for soil saprophytes. We evaluated the presence of environmental amoeboid predators in soil from armadillo burrows where Paracoccidioides had been previously detected and tested if interaction of Paracoccidioides with amoebae increased fungal virulence. Nematodes, ciliates and amoebae - all potential predators of fungi - grew in cultures from soil samples. Microscopical observation and ITS sequencing identified the amoebae as Acanthamoeba spp, Allovahlkampfia spelaea and Vermamoeba vermiformis. These three amoebae efficiently ingested, killed and digested Paracoccidioides spp. yeast cells, as did laboratory-adapted axenic Acanthamoeba castellanii. Sequential co-cultivation of Paracoccidioides with A. castellanii selected for phenotypical traits related to survival of the fungus within a natural predator as well as in murine macrophages and in vivo (Galleria mellonella and mice). This increase in virulence is linked to the accumulation of cell wall alpha-glucans, polysaccharides that masks recognition of fungal molecular patterns by host pattern recognition receptors. Altogether, our results indicate that Paracoccidioides inhabits a complex environment with multiple amoeboid predators that can exert selective pressure to guide the evolution of virulence traits.

microbiology↗

bioRxiv2019

Cryptococcus neoformans secretes small molecules that inhibit IL-1β inflammasome-dependent secretion

Cryptococcus neoformans is an encapsulated yeast that causes disease mainly in immunosuppressed hosts. It is considered a facultative intracellular pathogen because of its capacity to survive and replicate inside phagocytes, especially macrophages. This capacity is heavily dependent on various virulence factors, particularly the glucuronoxylomannan (GXM) component of the polysaccharide capsule, that render the non- or poorly-activated macrophage ineffective against phagocytosed yeast. Strategies utilized by macrophages to prevent this scenario include pyroptosis (a rapid highly inflammatory cell death) and vomocytosis (the expulsion of the pathogen from the intracellular environment without lysis). Inflammasome activation in phagocytes is usually protective against fungal infections, including cryptococcosis. Nevertheless, recognition of C. neoformans by inflammasome receptors requires specific changes in morphology or the opsonization of the yeast, impairing a proper inflammasome function. In this context, we analyzed the impact of molecules secreted by C. neoformans B3501 strain and its acapsular mutant{Delta} cap67 in an inflammasome activation in vitro model. Our results showed that conditioned media derived from B3501 was capable of inhibiting inflammasome dependent events (i. e. IL-1{beta} secretion and LDH release via pyroptosis) more strongly than conditioned media from{Delta} cap67, regardless of GXM presence. We also demonstrated that macrophages treated with conditioned media were less responsive against infection with the virulent strain H99, exhibiting lower rates of phagocytosis, increased fungal burdens and enhanced vomocytosis. Moreover, we showed that the aromatic metabolite DL-Indole-3-lactic acid (ILA) was present in B3501s conditioned media and that this fungal metabolite is involved in the regulation of inflammasome activation by C. neoformans. Overall, the results presented show that conditioned media from a wild-type strain can inhibit an important recognition pathway and subsequent fungicidal functions of macrophages, contributing to fungal survival in vitro and suggesting that this serves as an important role for secreted molecules during cryptococcal infections. Authors SummaryCryptococcus neoformans is the agent of cryptococcal meningitis, a disease that can be life-threatening in immunocompromised hosts such as those infected with HIV. The infection thrives in hosts that poorly activate their immune system, mainly because of the yeasts ability to survive inside macrophages and migrate towards the central nervous system. Emerging data indicate that cryptococci modulate the host immune response, but the underlying mechanisms remain largely uncharacterized. Here we show that secreted molecules from a wild-type strain of C. neoformans impair inflammatory responses driven by inflammasome activation, which in turn impact the macrophage antifungal activity. We further show that this inhibition does not involve GXM, the main constituent of the fungal capsule, but rather is partially dependent on DL-Indole-3-lactic acid (ILA), a metabolite not previously implicated in fungal virulence.

immunology↗

bioRxiv2018

Genotyping and epidemiological metadata provides new insights into population structure of Xanthomonas isolated from walnut trees

Xanthomonas arboricola pv. juglandis (Xaj) is the etiological agent of walnut diseases affecting leaves, fruits, branches and trunks. Although this phytopathogen is widely spread in walnut producing regions and has a considerable genetic diversity, there is still a poor understanding of its epidemic behaviour. To shed some light on the epidemiology of these bacteria, 131 Xanthomonas isolates obtained from 64 walnut trees were included in this study considering epidemiological metadata such as year of isolation, bioclimatic regions, walnut cultivars, production regimes, host walnut specimen and plant organs. Genetic diversity was assessed by multilocus sequence analysis (MLSA) and dot blot hybridization patterns obtained with nine Xaj-specific DNA markers (XAJ1 - XAJ9). The results showed that Xanthomonas isolates grouped in ten distinct MLSA clusters and in 18 hybridization patterns (HP). The majority of isolates (112 out of 131) were closely related with X. arboricola strains of pathovar juglandis as revealed by MLSA (clusters I to VI) and hybridize with more than five Xaj-specific markers. Nineteen isolates clustered in four MLSA groups (clusters VII to X) which do not include Xaj strains, and hybridize to less than five markers. Taking this data together, was possible to distinguish 17 lineages of Xaj, three lineages of X. arboricola and 11 lineages of Xanthomonas sp. Some Xaj lineages appeared to be widely distributed and prevalent across the different bioclimatic regions and apparently not constrained by the other features considered. Assessment of type III effector genes and pathogenicity tests revealed that representative lineages of MLSA clusters VII to X were nonpathogenic on walnut, with exception for strain CPBF 424, making this bacterium particularly appealing to address Xanthomonas pathoadaptations to walnut.\n\nIMPORTANCEXanthomonas arboricola pv. juglandis is one of the most serious threats of walnut trees. New disease epidemics caused by this phytopathogen has been a big concern causing high economic losses on walnut production worldwide. Using a comprehensive sampling methodology to disclose the diversity of walnut infective Xanthomonas, we were able to identify a genetic diversity higher than previously reported and generally independent of bioclimatic regions and the other epidemiological features studied. Furthermore, co-colonization of the same plant sample by distinct Xanthomonas strains were frequent and suggested a sympatric lifestyle. The extensive sampling carried out resulted in a set of non-arboricola Xanthomonas sp. strains, including a pathogenic strain, therefore diverging from the nonpathogenic phenotype that have been associated to these atypical strains, generally considered to be commensal. This new strain might be particularly informative to elucidate novel pathogenicity traits and unveil pathogenesis evolution within walnut infective xanthomonads. Beyond extending the present knowledge about walnut infective xanthomonads, this study might contribute to provide a methodological framework for phytopathogen epidemiological studies, still largely disregarded.

microbiology↗

bioRxiv2018

Opsonin-free, real-time imaging of Cryptococcus neoformans capsule during budding

Cryptococcus neoformans is a unicellular fungal pathogen that causes meningoencephalitis, killing hundreds of thousands of patients each year. Its most distinctive characteristic is a polysaccharide capsule that envelops the whole cell. It is the major virulence attribute and the antigen for serologic diagnosis. We have developed a method for easy observation of the capsule and its growth dynamics using the cell-separation reagent Percoll and differential interference contrast (DIC) microscopy. Percoll suspension is far less disruptive of cell physiology than methods relying on antibody binding to the capsule, and measurements made with it are equivalent with India ink. Time-lapse microscopy observations using this method suggest that during budding, a dividing cell can regulate whether the capsule polysaccharide it produces is deposited on the capsule of the bud or on its own. This observation has important implications for our understanding of the C. neoformans capsule induction process during budding.\n\nList of abbreviations and acronyms

microbiology↗

bioRxiv2017

Integrin alpha 4/beta 1 (CD49d/CD29) is a component of the murine IgG3 receptor

Antibodies exert several of their effector functions by binding to cell surface receptors. For murine IgG3 (mIgG3) the identity of its receptors (and the very existence of a receptor) is still under debate, as not all mIgG3 functions can be explained by interaction with Fc{gamma}-receptor I (Fc{gamma}RI). This implies the existence of an alternate receptor, whose identity we sought to pinpoint. We found that blockage of the alpha4/beta1 integrin (Itga4/Itgb1) selectively hampered binding of mIgG3 to macrophages and mIgG3-mediated phagocytosis. Manganese, an integrin activator, increased mIgG3 binding to macrophages. Blockage of Fc{gamma}RI or Itgb1 inhibited binding of different mIgG3 antibodies to variable extents. Our results indicate an integrin component in the mIgG3 receptor. Given the more ancient origin of integrins in comparison with Fc{gamma}R, this observation could have far ranging implications for our understanding of the evolution of antibody-mediated immunity, as well as in immunity to microorganisms, pathogenesis of autoimmune diseases and antibody engineering.

immunology↗