Identification of protein and mannoprotein antigens of Candida albicans of relevance for the serodiagnosis of invasive candidiasis

Antigens from Candida albicans blastoconidia and germ tubes were identified by two-dimensional electrophoresis and Western blotting and characterized by microsequencing, reactivity with concanavalin A, and a panel of human sera. Antigens identified included a polydispersed area in the acidic high-molecular-mass regions of blastoconidium and germ-tube extracts, and 16 antigens varying in molecular masses and isoelectric points (pIs). The majority of the detected antigens, especially those in the polydispersed region, showed mannosyl groups, as determined by concanavalin A reactivity. Antibodies present in sera from patients with invasive candidiasis showed high reactivity with a number of antigens not detected with sera from blood donors. Eight of the 16 antigens could be identified by reactivity with monoclonal antibodies or by microsequencing. Five antigens showed homology with five enzymes previously described as antigens in C. albicans: enolase, phosphoglycerate kinase, malate dehydrogenase, and two isoforms of the fructose biphosphate aldolase. However, to our knowledge, this is the first report of the immunogenic activity of a kexin precursor, a mitochondrial complex I chaperone, and a diacylglycerol kinase catalytic domain from C. albicans. Antigens described in this study may be of potential interest for the serodiagnosis of invasive candidiasis. [Int Microbiol 2007; 10(2):103-108

The role of osmolarity adjusting agents in the regulation of encapsulated cell behavior to provide a safer and more predictable delivery of therapeutics

[Abstract] Transplantation of cells within alginate microspheres has been extensively studied for sustained drug delivery. However, the lack of control over cell behavior represents a major concern regarding the efficacy and the safety of the therapy. Here, we demonstrated that when formulating the biosystem, an adequate selection of osmolarity adjusting agents significantly contributes to the regulation of cell responses. Our data showed that these agents interact in the capsule formation process, influencing the alginate crosslinking degree. Therefore, when selecting inert or electrolyte-based osmolarity adjusting agents to encapsulate D1 multipotent mesenchymal stromal cells (MSCs), alginate microcapsules with differing mechanical properties were obtained. Since mechanical forces acting on cells influence their behavior, contrasting cell responses were observed both, in vitro and in vivo. When employing mannitol as an inert osmolarity adjusting agent, microcapsules presented a more permissive matrix, allowing a tumoral-like behavior. This resulted in the formation of enormous cell-aggregates that presented necrotic cores and protruding peripheral cells, rendering the therapy unpredictable, dysfunctional, and unsafe. Conversely, the use of electrolyte osmolarity adjusting agents, including calcium or sodium, provided the capsule with a suitable crosslinking degree that established a tight control over cell proliferation and enabled an adequate therapeutic regimen in vivo. The crucial impact of these agents was confirmed when gene expression studies reported pivotal divergences not only in proliferative pathways, but also in genes involved in survival, migration, and differentiation. Altogether, our results prove osmolarity adjusting agents as an effective tool to regulate cell behavior and obtain safer and more predictable therapies.Gobierno Vasco; IT-907-16Universidad del País Vasco; UFI11/3

Fumagilina mikotoxinak aspergilosi inbaditzailearen garapenean duen rola aztertzeko SPE-UHPLC-DAD metodo analitikoa

Invasive aspergillosis caused by Aspergillus fumigatus is a threat for immunocompromised patients. According to recent studies, fumagillin, a mycotoxin produced by this fungus, has been associated with the propagation of the disease. Therefore, this molecule might help to understand the mechanisms of this disease and to study the use of fumagillin as a potential biomarker of invasive aspergillosis. In spite of the relevance of fumagillin analysis in microbiological research, no quanti-tative method has been developed so far for its determination in cell culture media. Here, we present the first validated method for the quantitative analysis of fumagillin in RPMI-1640. The sample treatment consists of a mixed-mode anion exchange Solid Phase Extraction that effectively removes potential interferences and offered a recovery of 83 ± 7%. The analysis was carried out by Ultra High Performance Liquid Chromatography coupled to Diode Array Detection at 336 nm. The method fulfilled all the validation criteria established by EMA (European Medicine Agency) and FDA (Food and Drug Administration) guidelines for bioanalysis. Finally, the method was satisfactorily applied to the quantification of the fumagillin produced by different strains of Aspergillus fumigatus and it was observed that they had a different micotoxin production capacity.; Aspergillus fumigatus onddoak sortutako aspergilosi inbaditzailea mehatxua da immunoeskasia duten gai-xoentzat. Azkeneko ikerketa batzuen arabera, fumagilinak, onddoak sortutako mikotoxinak, gaixotasunaren hedapenarekin zerikusia duela ikusi da. Hori dela eta, konposatu honen determinazioa lagungarria izan daiteke bai gaixotasunaren mekanismoak hobeto ulertzeko eta baita aspergilosi inbaditzailearen biomarkatzaile gisa erabili ahal izateko ere. Ikerketa mikro-biologikoetan fumagilinaren analisiak garrantzia izan arren, oraindik ez da haren determinaziorako metodo kuantitatiborik garatu zelula-hazkuntzako inguruneetan. Beraz, lan honetan fumagilinaren determinazio kuantitatiborako lehenengo metodo analitikoa balidatu da RPMI-1640 zelula-hazkuntzako ingurunean. Laginaren tratamendua fase solidoko erauzketarekin egin da, anioi trukatzaile sendoak diren modu mistoko kartutxoak erabiliz. Horrela, egon daitezkeen interferentziak modu eraginkorrean ezabatu dira, eta % 83 ± 7ko berreskurapena lortu da. Analisia fotodiodo detektagailuari akoplaturiko bereizmen oso altuko likido kromatografia erabiliz egin da 336 nm-ko uhin-luzeran. Horrela, metodoak EMA (Europako Medikamentuen Agentzia) eta FDA (Elikagai eta Sendagaien Administrazioa) agentziek balidazio bioanalitikoetarako zehazten dituzten parametro guztien onartze-irizpideak betetzen dituela egiaztatu da. Gero, metodoa A. fumigatus-en lau andui analizatzeko aplikatu da, eta bakoitzak mikotoxinaren kantitate desberdina ekoizteko gaitasuna daukala ikusi da

Mikroorganismoek minbizia eragin dezakete?

Many studies have analyzed relationships between microorganisms and cancer, demonstrating that microorganisms are able to prevent the onset of cancer and, others to provoke it. Specifically, more and more scientific articles are publishing on microorganisms, linking them to the creation, implementation and dispersion of cancer. In fact, it is estimated that microorganisms cause 17.8% of all cancers. The cancer-causing viral capability is the most studied and, in consequence, many different viral mechanisms that can cause cancer have been described. The International Cancer Re-search Institute has categorized eight viruses for the first time as "carcinogenic to hu-mans", including a human papillomavirus, two herpesvirus and two hepatitis viruses. Regarding bacteria, among cancerous agents, Helicobacter pylori is the most studied in relation to stomach cancer. In addition, many other bacteria, such as Salmonella typhi, Chlamydia pneumoniae and Streptococcus bovis, have been directly related to cancer. Although relatively little research on the effect of fungi on cancer has been investi-gated, some of the toxins produced by these microorganisms have been shown to cause cancer. In addition, some mechanism for the generation and spread of cancer have been described in Candida albicans. Studies to date have shown the influence of microor-ganisms on the development and promotion of cancer. For this reason, to face cancer in the next future, deepen into the relationship between cancer and microorganisms will be essential; Ikerketa askok mikroorganismoen eta minbizien arteko erlazioak aztertu dituzte, eta erakutsi dute mikroorganismo batzuek minbiziaren agerpena saihesten dutela eta beste batzuek, aldiz, minbizia eragin dezaketela. Hain zuzen ere, gero eta artikulu zientifiko gehiago argitaratzen ari dira mikroorganismoak minbiziaren sortzearekin, ezarpenarekin eta sakabanaketarekin erlazionatuz. Izan ere, mikroorganismoek minbizi guztien % 17,8 eragiten dutela estimatu da. Minbizia sortzeko birusen gaitasuna da gehien ikertu dena eta, ondorioz, minbizia sor dezaketen mekanismo desberdin asko deskribatu dira. Minbizia Ikertzeko Nazioarteko Agentziak zortzi birus 1. mailako "gizakiontzat kartzinogeno"-tzat sailkatu ditu; haien artean, giza papiloma birusa, bi herpesbirus eta bi hepatitisaren birus aurkitzen dira. Bakterioei dagokienez, minbizi-eragileen artean, Helicobacter pylori da gehien ikertu dena urdaileko minbiziarekin erlazionatuta. Baina honetaz gain, beste hainbat bakterio, hala nola Salmonella typhi, Chlamydia pneumoniae eta Streptococcus bovis minbiziarekin zuzenki erlazionatu dira. Onddoek daukaten minbiziarekiko erlazioa oso gutxi ikertu den arren, mikroorganismo hauek sortutako toxina batzuek minbizia eragin dezaketela frogatu da. Horrez gain, Candida albicans onddoak minbiziaren sorrera eta hedapena eragin dezakeen hainbat mekanismo deskribatu dira. Orain arte egindako ikerketek mikroorganismoek minbiziaren garapenean eta sustapenean daukaten eragina agerrarazi dute. Hori dela eta, etorkizunean minbiziari aurre egiteko, minbiziaren eta mikroorganismoen arteko erlazioan sakontzea ezinbestekoa da

Study of Humoral Responses against Lomentospora/Scedosporium spp. and Aspergillus fumigatus to Identify L. prolificans Antigens of Interest for Diagnosis and Treatment

The high mortality rates of Lomentospora prolificans infections are due, above all, to the tendency of the fungus to infect weakened hosts, late diagnosis and a lack of effective therapeutic treatments. To identify proteins of significance for diagnosis, therapy or prophylaxis, immunoproteomics-based studies are especially important. Consequently, in this study murine disseminated infections were carried out using L. prolificans, Scedosporium aurantiacum, Scedosporium boydii and Aspergillus fumigatus, and their sera used to identify the most immunoreactive proteins of L. prolificans total extract and secreted proteins. The results showed that L. prolificans was the most virulent species and its infections were characterized by a high fungal load in several organs, including the brain. The proteomics study showed a high cross-reactivity between Scedosporium/Lomentospora species, but not with A. fumigatus. Among the antigens identified were, proteasomal ubiquitin receptor, carboxypeptidase, Vps28, HAD-like hydrolase, GH16, cerato-platanin and a protein of unknown function that showed no or low homology with humans. Finally, Hsp70 deserves a special mention as it was the main antigen recognized by Scedosporium/Lomentospora species in both secretome and total extract. In conclusion, this study identifies antigens of L. prolificans that can be considered as potential candidates for use in diagnosis and as therapeutic targets and the production of vaccines.This research was funded by the Basque Government, grant number IT1362-19. I.B., L.M.S. and L.A.F. received a predoctoral fellowship from the Basque Government