Comprehensive characterization of extracellular vesicles produced by environmental (Neff) and clinical (T4) strains of Acanthamoeba castellanii.

We conducted a comprehensive comparative analysis of extracellular vesicles (EVs) from two Acanthamoeba castellanii strains, Neff (environmental) and T4 (clinical). Morphological analysis via transmission electron microscopy revealed slightly larger Neff EVs (average = 194.5 nm) compared to more polydisperse T4 EVs (average = 168.4 nm). Nanoparticle tracking analysis (NTA) and dynamic light scattering validated these differences. Proteomic analysis of the EVs identified 1,352 proteins, with 1,107 common, 161 exclusive in Neff, and 84 exclusively in T4 EVs. Gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) mapping revealed distinct molecular functions and biological processes and notably, the T4 EVs enrichment in serine proteases, aligned with its pathogenicity. Lipidomic analysis revealed a prevalence of unsaturated lipid species in Neff EVs, particularly triacylglycerols, phosphatidylethanolamines (PEs), and phosphatidylserine, while T4 EVs were enriched in diacylglycerols and diacylglyceryl trimethylhomoserine, phosphatidylcholine and less unsaturated PEs, suggesting differences in lipid metabolism and membrane permeability. Metabolomic analysis indicated Neff EVs enrichment in glycerolipid metabolism, glycolysis, and nucleotide synthesis, while T4 EVs, methionine metabolism. Furthermore, RNA-seq of EVs revealed differential transcript between the strains, with Neff EVs enriched in transcripts related to gluconeogenesis and translation, suggesting gene regulation and metabolic shift, while in the T4 EVs transcripts were associated with signal transduction and protein kinase activity, indicating rapid responses to environmental changes. In this novel study, data integration highlighted the differences in enzyme profiles, metabolic processes, and potential origins of EVs in the two strains shedding light on the diversity and complexity of A. castellanii EVs and having implications for understanding host-pathogen interactions and developing targeted interventions for Acanthamoeba-related diseases.IMPORTANCEA comprehensive and fully comparative analysis of extracellular vesicles (EVs) from two Acanthamoeba castellanii strains of distinct virulence, a Neff (environmental) and T4 (clinical), revealed striking differences in their morphology and protein, lipid, metabolites, and transcripts levels. Data integration highlighted the differences in enzyme profiles, metabolic processes, and potential distinct origin of EVs from both strains, shedding light on the diversity and complexity of A. castellanii EVs, with direct implications for understanding host-pathogen interactions, disease mechanisms, and developing new therapies for the clinical intervention of Acanthamoeba-related diseases.

Antibody Isolation in C. neoformans.

The importance of humoral immunity to fungal infections remains to be elucidated. In cryptococcosis, patients that fail to generate antibodies against antigens of the fungus Cryptococcus neoformans are more susceptible to the disease, demonstrating the importance of these molecules to the antifungal immune response. Historically, antibodies against C. neoformans have been applied in diagnosis, therapeutics, and as important research tools to elucidate fungal biology. Throughout the process of generating monoclonal antibodies (mAbs) from a single B-cell clone and targeting a single epitope, several immunization steps might be required for the detection of responsive antibodies to the antigen of interest in the serum. This complex mixture of antibodies comprises the polyclonal antibodies. To obtain mAbs, B-lymphocytes are harvested (from spleen or peripheral blood) and fused with tumor myeloma cells, to generate hybridomas that are individually cloned and specifically screened for mAb production. In this chapter, we describe all the necessary steps, from the immunization to polyclonal antibody harvesting, hybridoma generation, and mAb production and purification. Additionally, we discuss new cutting-edge approaches for generating interspecies mAbs, such as humanized mAbs, or for similar species in distinct host backgrounds.

ß-1,3-Glucan recognition by Acanthamoeba castellanii as a putative mechanism of amoeba-fungal interactions.

In this study, we conducted an in-depth analysis to characterize potential Acanthamoeba castellanii (Ac) proteins capable of recognizing fungal ß-1,3-glucans. Ac specifically anchors curdlan or laminarin, indicating the presence of surface ß-1,3-glucan-binding molecules. Using optical tweezers, strong adhesion of laminarin- or curdlan-coated beads to Ac was observed, highlighting their adhesive properties compared to controls (characteristic time τ of 46.9 and 43.9 s, respectively). Furthermore, Histoplasma capsulatum (Hc) G217B, possessing a ß-1,3-glucan outer layer, showed significant adhesion to Ac compared to a Hc G186 strain with an α-1,3-glucan outer layer (τ of 5.3 s vs τ 83.6 s). The addition of soluble ß-1,3-glucan substantially inhibited this adhesion, indicating the involvement of ß-1,3-glucan recognition. Biotinylated ß-1,3-glucan-binding proteins from Ac exhibited higher binding to Hc G217B, suggesting distinct recognition mechanisms for laminarin and curdlan, akin to macrophages. These observations hinted at the ß-1,3-glucan recognition pathway's role in fungal entrance and survival within phagocytes, supported by decreased fungal viability upon laminarin or curdlan addition in both phagocytes. Proteomic analysis identified several Ac proteins capable of binding ß-1,3-glucans, including those with lectin/glucanase superfamily domains, carbohydrate-binding domains, and glycosyl transferase and glycosyl hydrolase domains. Notably, some identified proteins were overexpressed upon curdlan/laminarin challenge and also demonstrated high affinity to ß-1,3-glucans. These findings underscore the complexity of binding via ß-1,3-glucan and suggest the existence of alternative fungal recognition pathways in Ac.IMPORTANCEAcanthamoeba castellanii (Ac) and macrophages both exhibit the remarkable ability to phagocytose various extracellular microorganisms in their respective environments. While substantial knowledge exists on this phenomenon for macrophages, the understanding of Ac's phagocytic mechanisms remains elusive. Recently, our group identified mannose-binding receptors on the surface of Ac that exhibit the capacity to bind/recognize fungi. However, the process was not entirely inhibited by soluble mannose, suggesting the possibility of other interactions. Herein, we describe the mechanism of ß-1,3-glucan binding by A. castellanii and its role in fungal phagocytosis and survival within trophozoites, also using macrophages as a model for comparison, as they possess a well-established mechanism involving the Dectin-1 receptor for ß-1,3-glucan recognition. These shed light on a potential parallel evolution of pathways involved in the recognition of fungal surface polysaccharides.

In vitro and in vivo efficacies of Dectin-1-Fc(IgG)(s) fusion proteins against invasive fungal infections.

Fungal infections have increased in the last years, particularly associated to an increment in the number of immunocompromised individuals and the emergence of known or new resistant species, despite the difficulties in the often time-consuming diagnosis. The controversial efficacy of the currently available strategies for their clinical management, apart from their high toxicity and severe side effects, has renewed the interest in the research and development of new broad antifungal alternatives. These encompass vaccines and passive immunization strategies with monoclonal antibodies (mAbs), recognizing ubiquitous fungal targets, such as fungal cell wall ß-1,3-glucan polysaccharides, which could be used in early therapeutic intervention without the need for the diagnosis at species level. As additional alternatives, based on the Dectin-1 great affinity to ß-1,3-glucan, our group developed broad antibody-like Dectin1-Fc(IgG)(s) from distinct subclasses (IgG2a and IgG2b) and compared their antifungal in vitro and passive immunizations in vivo performances. Dectin1-Fc(IgG2a) and Dectin1-Fc(IgG2b) demonstrated high affinity to laminarin and the fungal cell wall by ELISA, flow cytometry, and microscopy. Both Dectin-1-Fc(IgG)(s) inhibited Histoplasma capsulatum and Cryptococcus neoformans growth in a dose-dependent fashion. For Candida albicans, such inhibitory effect was observed with concentrations as low as 0.098 and 0.049 µg/ml, respectively, which correlated with the impairment of the kinetics and lengths of germ tubes in comparison to controls. Previous opsonization with Dectin-1-Fc(IgG)(s) enhanced considerably the macrophage antifungal effector functions, increasing the fungi macrophages interactions and significantly reducing the intraphagosome fungal survival, as lower CFUs were observed. The administration of both Dectin1-Fc(IgG)(s) reduced the fungal burden and mortality in murine histoplasmosis and candidiasis models, in accordance with previous evaluations in aspergillosis model. These results altogether strongly suggested that therapeutic interventions with Dectin-1-Fc(IgG)(s) fusion proteins could directly impact the innate immunity and disease outcome in favor of the host, by direct neutralization, opsonization, phagocytosis, and fungal elimination, providing interesting information on the potential of these new strategies for the control of invasive fungal infections. LAY SUMMARY: Mycoses have increased worldwide, and new efficient therapeutics are needed. Passive immunizations targeting universally the fungal cell would allow early interventions without the species-level diagnosis. Lectins with affinity to carbohydrates could be used to engineer 'antibody-like' strategies.

Recognition of Cell Wall Mannosylated Components as a Conserved Feature for Fungal Entrance, Adaptation and Survival Within Trophozoites of Acanthamoeba castellanii and Murine Macrophages.

Acanthamoeba castellanii (Ac) is a species of free-living amoebae (FLAs) that has been widely applied as a model for the study of host-parasite interactions and characterization of environmental symbionts. The sharing of niches between Ac and potential pathogens, such as fungi, favors associations between these organisms. Through predatory behavior, Ac enhances fungal survival, dissemination, and virulence in their intracellular milieu, training these pathogens and granting subsequent success in events of infections to more evolved hosts. In recent studies, our group characterized the amoeboid mannose binding proteins (MBPs) as one of the main fungal recognition pathways. Similarly, mannose-binding lectins play a key role in activating antifungal responses by immune cells. Even in the face of similarities, the distinct impacts and degrees of affinity of fungal recognition for mannose receptors in amoeboid and animal hosts are poorly understood. In this work, we have identified high-affinity ligands for mannosylated fungal cell wall residues expressed on the surface of amoebas and macrophages and determined the relative importance of these pathways in the antifungal responses comparing both phagocytic models. Mannose-purified surface proteins (MPPs) from both phagocytes showed binding to isolated mannose/mannans and mannosylated fungal cell wall targets. Although macrophage MPPs had more intense binding when compared to the amoeba receptors, the inhibition of this pathway affects fungal internalization and survival in both phagocytes. Mass spectrometry identified several MPPs in both models, and in silico alignment showed highly conserved regions between spotted amoeboid receptors (MBP and MBP1) and immune receptors (Mrc1 and Mrc2) and potential molecular mimicry, pointing to a possible convergent evolution of pathogen recognition mechanisms.

Complex and Controversial Roles of Eicosanoids in Fungal Pathogenesis.

The prevalence of fungal infections has increased in immunocompromised patients, leading to millions of deaths annually. Arachidonic acid (AA) metabolites, such as eicosanoids, play important roles in regulating innate and adaptative immune function, particularly since they can function as virulence factors enhancing fungal colonization and are produced by mammalian and lower eukaryotes, such as yeasts and other fungi (Candida albicans, Histoplasma capsulatum and Cryptococcus neoformans). C. albicans produces prostaglandins (PG), Leukotrienes (LT) and Resolvins (Rvs), whereas the first two have been well documented in Cryptococcus sp. and H. capsulatum. In this review, we cover the eicosanoids produced by the host and fungi during fungal infections. These fungal-derived PGs have immunomodulatory functions analogous to their mammalian counterparts. Prostaglandin E2 (PGE2) protects C. albicans and C. parapsilosis cells from the phagocytic and killing activity of macrophages. H. capsulatum PGs augment the fungal burden and host mortality rates in histoplasmosis. However, PGD2 potentiates the effects and production of LTB4, which is a very potent neutrophil chemoattractant that enhances host responses. Altogether, these data suggest that eicosanoids, mainly PGE2, may serve as a new potential target to combat diverse fungal infections.

Protective Efficacy of Lectin-Fc(IgG) Fusion Proteins In Vitro and in a Pulmonary Aspergillosis In Vivo Model.

Aspergillosis cases by Aspergillus fumigatus have increased, along with fungal resistance to antifungals, urging the development of new therapies. Passive immunization targeting common fungal antigens, such as chitin and ß-glucans, are promising and would eliminate the need of species-level diagnosis, thereby expediting the therapeutic intervention. However, these polysaccharides are poorly immunogenic. To overcome this drawback, we developed the lectin-Fc(IgG) fusion proteins, Dectin1-Fc(IgG2a), Dectin1-Fc(IgG2b) and wheat germ agglutinin (WGA)-Fc(IgG2a), based on their affinity to ß-1,3-glucan and chitooligomers, respectively. The WGA-Fc(IgG2a) previously demonstrated antifungal activity against Histoplasma capsulatum, Cryptococcus neoformans and Candida albicans. In the present work, we evaluated the antifungal properties of these lectin-Fc(s) against A. fumigatus. Lectin-Fc(IgG)(s) bound in a dose-dependent manner to germinating conidia and this binding increased upon conidia germination. Both lectin-Fc(IgG)(s) displayed in vitro antifungal effects, such as inhibition of conidia germination, a reduced length of germ tubes and a diminished biofilm formation. Lectin-Fc(IgG)(s) also enhanced complement deposition on conidia and macrophage effector functions, such as increased phagocytosis and killing of fungi. Finally, administration of the Dectin-1-Fc(IgG2b) and WGA-Fc(IgG2a) protected mice infected with A. fumigatus, with a 20% survival and a doubled life-span of the infected mice, which was correlated to a fungal burden reduction in lungs and brains of treated animals. These results confirm the potential of lectin-Fc(IgGs)(s) as a broad-spectrum antifungal therapeutic.

Acute Kidney Failure in a Young African American Male.

Retroperitoneal fibrosis (RPF) is a condition characterized by chronic inflammatory and fibrotic changes in the retroperitoneum that can lead to serious complications including kidney failure, mesenteric and limb ischemia, and deep venous thrombosis among others. Affected individuals may present with nonspecific symptomology that would require a high clinical index of suspicion for prompt diagnosis. We herein discuss a case of a young African-American man with recurrent deep venous thrombosis who presents with a 4-week history of constant aching pain of abdomen and back and kidney failure. Initial noncontrast computed tomogram (CT) only revealed mild bilateral hydroureteronephrosis with inflammatory changes but without obvious mass or lymphadenopathy. At the insistence of the renal consulting team to rule out RPF, a CT-urogram was performed which revealed an infiltrative mass encasing the aorta, inferior vena cava, and common iliac vessels. Laparoscopic biopsy revealed dense fibroadipose tissue, lymphocytic aggregates, focal scattered IgG4-positive plasma cells, and fibrin deposition. Patient underwent bilateral nephrostomy placement and empirical corticosteroid therapy with resolution of kidney failure. Our case illustrates a classic presentation of RPF with relatively benign findings on noncontrast CT that could have been missed if clinicians did not keep a high index of suspicion for the condition.

Effect of two glycyrrhizinic acid nanoparticle carriers on MARC-145 cells actin filaments.

The development of technologies that combine the advantages of nanomedicine with natural medicine represents a versatile approach to improve the safety and efficacy of drugs. Glycyrrhizinic acid (GA) is a natural compound that has a wide range of biological activities for the treatment of diseases. To establish a safe nanotransport system for this drug, two different nanoparticles with glycyrrhizinic acid, solid lipid nanoparticles (SLN-GA) and polymeric nanoparticles (PNPS-GA) were elaborated to obtain nanostructure sizes between 200 and 300 nm. The nanoparticles were evaluated at concentrations of 1.25-100 µl/ml using the MARC-145 cell line to determine the effects on cell morphology, cellular structure (actin filaments) and cell viability (mitochondrial and lysosomal) at 24 and 72 h post-exposure. The safety range of the nanoparticles was 50 µl/ml, to determine that PNPs-GA had an optimal safety profile and no cytotoxic effects, as there was no evidence of changes in morphology, internal cellular structures (stress fibers and the cell cortex formed by actin filaments) or viability under the experimental concentrations and conditions employed.

Uso de palivizumab en el control de un brote de VRS en una unidad de cuidado intensivo neonatal / Use of palivizumab in the control of an outbreak of respiratory syncytial virus in a neonatal intensive care unit

El virus respiratorio sincicial (VRS) es una causa importante de morbilidad en pediatría, especialmente en los menores de 2 años. Los grupo de mayor riesgo de presentar una infección grave son los recién nacidos prematuros (RNPrT) menores de 35 semanas de edad gestacional, los pacientes portadores de enfermedad pulmonar crónica y los pacientes portadores de cardiopatías congénitas congestivas. La presencia de un brote de VRS en la unidad de cuidados intensivo neonatal (UCIN) se asocia a resultados desfavorables, incluyendo la muerte. Palivizumab es un anticuerpo monoclonal que se usa para la prevención de la infección por VRS en pacientes de riesgo durante la época epidémica estacional y cuyo uso no está formalmente indicado para el control de brotes. Se describe un brote de VRS en época epidémica en una UCIN y les medidas tomadas para su control. El uso de palivizumab asociado al uso óptimo de medidas de aislamiento para el control de infecciones virales respiratorias pareciera ser altamente efectivo en el control de un brote de VRS en la UCIN.

Respiratory syncytial virus (RSV) infection is the leading cause of morbidity in infants. High risk group presents serious infections, particularly preterm birth. An RSV outbreak occurred in the neonatal intensive care and we described the control measurements taken, including the use of palivizumab

Streptococcus pneumoniae resists intracellular killing by olfactory ensheathing cells but not by microglia.

Olfactory ensheathing cells (OECs) are a type of specialized glial cell currently considered as having a double function in the nervous system: one regenerative, and another immune. Streptococcus pneumoniae is a major agent of severe infections in humans, including meningitis. It is commonly found in the nasopharynx of asymptomatic carriers, and, under certain still unknown conditions, can invade the brain. We evaluated whether pneumococcal cells recovered from lysed OECs and microglia are able to survive by manipulating the host cell activation. An intracellular-survival assay of S. pneumoniae in OECs showed a significant number of bacterial CFU recovered after 3 h of infection. In contrast, microglia assays resulted in a reduced number of CFU. Electron-microscopy analysis revealed a large number of pneumococci with apparently intact morphology. However, microglia cells showed endocytic vesicles containing only bacterial cell debris. Infection of OEC cultures resulted in continuous NF-κB activation. The IFN-γ-induced increase of iNOS expression was reversed in infected OECs. OECs are susceptible to S. pneumoniae infection, which can suppress their cytotoxic mechanisms in order to survive. We suggest that, in contrast to microglia, OECs might serve as safe targets for pneumococci, providing a more stable environment for evasion of the immune system.

Genetic and antigenic changes in porcine rubulavirus.

Blue eye disease, caused by a porcine rubulavirus (PoRV), is an emergent viral swine disease that has been endemic in Mexico since 1980. Atypical outbreaks were detected in 1990 and 2003. Growing and adult pigs presented neurological signs, mild neurological signs were observed in piglets, and severe reproductive problems were observed in adults. Amino acid sequence comparisons and phylogenetic analysis of the hemagglutinin-neuraminidase (HN) protein revealed genetically different lineages. We used cross-neutralization assays, with homologous and heterologous antisera, to determine the antigenic relatedness values for the PoRV isolates. We found antigenic changes among several strains and identified a highly divergent one, making up a new serogroup. It seems that genetically and antigenically different PoRV strains are circulating simultaneously in the swine population in the geographical region studied. The cross neutralization studies suggest that the HN is not the only antigenic determinant participating in the antigenic changes among the different PoRV strains.

Antigenic differentiation of classical swine fever vaccinal strain PAV-250 from other strains, including field strains from Mexico.

Twenty-nine classical swine fever virus (CSFv) strains were grown in the PK15 or SK6 cell lines. Antigenic differentiation studies were performed using monoclonal antibodies (McAbs), produced at Lelystad (CDI-DLO), The Netherlands. The monoclonals which were classified numerically as monoclonals 2-13. Epitope map patterns that resulted from the reactivity with McAbs were found to be unrelated to the pathogenicity of the viruses studied. Antigenic determinants were recognized by McAbs 5 and 8, were not detected in some Mexican strains; however, sites for McAb 6 were absent in all strains. The PAV-250 vaccine strain was recognized by all MAbs, except by MAb 6. Furthermore, the Chinese C-S vaccine strain was found to be very similar to the GPE(-) vaccine. None of the studied Mexican vaccines or field strains was found to be similar to the PAV-250 vaccine strain.

Allelic loss at 10q26 in osteosarcoma in the region of the BUB3 and FGFR2 genes.

Loss of heterozygosity at 10q26 was mapped using microsatellite markers in 20 osteosarcomas. A four-megabase region centered on marker D10S587 was affected by allelic loss in 60 percent of osteosarcomas. The most frequently lost marker was D10S1723. Around 15 known genes are found in this region. The gene immediately adjacent to D10S1723 encodes BUB3, an element of the spindle assembly mitotic checkpoint. Loss of BUB3 function could contribute to chromosomal instability. The fibroblast growth factor receptor 2 (FGFR2) gene is located 2 Mb from the BUB3 gene and has the potential for a role in cancer. Inherited mutations of the FGFR2 gene result in skeletal dysplasias. FGFR2 alterations have also been implicated in gastric cancer. Human genome project data were used to design primers for amplifying FGFR2 in 18 genomic segments and BUB3 in 7 genomic segments. In each case, the segments encompassed coding exons and flanking intron sequences. The primers were used to search for mutations by polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP). Several shifted bands were detected in the BUB3 exon 3 fragment. Sequencing resolved the BUB3 exon 3 fragment shifts into polymorphisms in intron 2. No mutations of BUB3 or FGFR2 were detected. It remains possible that BUB3 or FGFR2 hemizygosity alone contributes to osteosarcoma, or that one of the genes is cryptically inactivated by a higher-order modification or mutation outside the coding region. There may also be a yet undiscovered tumor suppressor gene in this region.

Prevención de ceguera por glaucoma: resultados y aspectos operativos de catorce campañas / Prevention of blindness by glaucoma: results and operative aspects of 14 campaigns

El Glaucoma ocasiona aproximadamente el 20% de los casos de ceguera en el mundo, constituyendo la tercera causa de ceguera en nuestro país. Siendo el tipo de ceguera evitable por excelencia, el examen en masa y la educación de la Comunidad desempeñan un papel muy importante en la identificación precoz de los enfermos y en el seguimiento de los casos sospechosos. En este reporte describimos la metodología empleada y los resultados de 14 Campañas de Detección de Glaucoma desde 1983 hasta 1986, que 36 Centros Detectores y 13,428 personas examinadas, destacando la importancia del nivel socio-económico de las diversas poblaciones que habitan Lima Metropolitana. Dicha muestra fue comparada con otra de extracción rural efectuada durante los Programas de la Organización Peruana de Lucha contra la Ceguera en las zonas rurales del país, y que comprendió 2,482 personas. Los resultados muestran una incidencia en la población general de 1.48% de casos de Glaucoma no diagnosticados, 0,43% de casos antiguos y 5.06% de sospechosos de padecer la enfermedad. En la población de Lima, las zonas de nivel socio-económico medio-altos mostró una incidencia de 1.16% y las zonas medio-bajas 2.58%. Por otra parte, el grupo de las zonas rurales representativa de población hospitalaria mostró 2.56% de casos nuevos. En casi todos los grupos estudiados existe similitud en la incidencia de sospechosos, con un promedio de 6.2%. Señalamos las estrategias recomendadas para prevenir la ceguera por Glaucoma y la importancia de una acción sistemática para reducir la incidencia de los casos de ceguera por este mal