Generation and characterisation of a semi-synthetic siderophore-immunogen conjugate and a derivative recombinant triacetylfusarinine C-specific monoclonal antibody with fungal diagnostic application.

Invasive pulmonary aspergillosis (IPA) is a severe life-threatening condition. Diagnosis of fungal disease in general, and especially that caused by Aspergillus fumigatus is problematic. A. fumigatus secretes siderophores to acquire iron during infection, which are also essential for virulence. We describe the chemoacetylation of ferrated fusarinine C to diacetylated fusarinine C (DAFC), followed by protein conjugation, which facilitated triacetylfusarinine C (TAFC)-specific monoclonal antibody production with specific recognition of the ferrated form of TAFC. A single monoclonal antibody sequence was ultimately elucidated by a combinatorial strategy involving protein LC-MS/MS, cDNA sequencing and RNAseq. The resultant murine IgG2a monoclonal antibody was secreted in, and purified from, mammalian cell culture (5 mg) and demonstrated to be highly specific for TAFC detection by competitive ELISA (detection limit: 15 nM) and in a lateral flow test system (detection limit: 3 ng), using gold nanoparticle conjugated- DAFC-bovine serum albumin for competition. Overall, this work reveals for the first time a recombinant TAFC-specific monoclonal antibody with diagnostic potential for IPA diagnosis in traditional and emerging patient groups (e.g., COVID-19) and presents a useful strategy for murine Ig sequence determination, and expression in HEK293 cells, to overcome unexpected limitations associated with aberrant or deficient murine monoclonal antibody production.

Identidade do homem resiliente no contexto de adoecer por câncer de próstata: uma perspectiva cultural / Identity of the resilient man in the context of ill with prostate cancer: a cultural perspective / Identidad del hombre resiliente en el contexto al enfermar por cáncer de próstata: una perspectiva cultural

O estudo objetivou conhecer o contexto do homem resiliente ao adoecer por câncer de próstata. Trata-se de um estudo de caso etnográfico realizado com dois homens sobreviventes ao câncer de próstata, com alto grau de resiliência. Os dados foram coletados no domicílio, no período de abril e maio de 2012, por meio da entrevista semiestruturada em profundidade, de observação participante e do ecomapa. Pela análise dos dados construíram-se duas unidades de sentido: "Identidade do homem resiliente: contextualizando os informantes" e "O homem resiliente descobrindo-se doente". Apreende-se que a identidade de ser homem resiliente, para estes informantes, foi marcada pela diferença histórica e cultural que permeou as suas ações, no processo de adoecimento por câncer de próstata. Considera-se importante que os enfermeiros atentem para os aspectos culturais da saúde do homem, para que este possa sentir-se parte integrante do processo de cura, tornando-se sujeito ativo frente à própria saúde.

The study aimed to understand the context of resilient man when ill with prostate cancer. This is an ethnographic case study conducted with two prostate cancer survival men with a high degree of resilience. The data was collected on their places, in 2012 April and May, using semi-structured in-depth interviews, participant observation and ecomap. For the data analysis, it was built two units of meaning: "Identity of the resilient man: contextualizing the informants" and "The resilient man finding himself ill". It was noticed that the identity of being a resilient man, to these informants, was marked by historical and cultural difference which permeated their actions in the process of being ill with prostate cancer. It is important that nurses pay attention to the cultural aspects of human health, so that they can feel part of the healing process, becoming an active subject facing their own health.

El estudio enfocó conocer el contexto del hombre resiliente al enfermar por cáncer de próstata. Se trata de un estudio de caso etnográfico realizado con dos hombres sobrevivientes al cáncer de próstata con alto grado de resiliencia. Los datos fueron recogidos en el domicilio, en el período de abril y mayo de 2012, por medio de entrevista semiestructurada en profundidad, observación participante y ecomapa. Por el análisis de los datos, se construyeron dos unidades de sentido: "Identidad del hombre resiliente: contextualizando a los informantes" y "El hombre resiliente descubriéndose enfermo". Se comprende que la identidad de ser hombre resiliente, para estos informantes, fue marcada por la diferencia histórica y cultural que hicieron permeables sus acciones en el proceso de enfermar por cáncer de próstata. Se considera importante que los enfermeros estén atentos a los aspectos culturales de la salud del hombre, para que este se pueda sentir parte integrante del proceso de cura, tornándose sujeto activo frente a la propia salud.

Advances in vaccination against avian pathogenic Escherichia coli respiratory disease: potentials and limitations.

Avian pathogenic Escherichia coli (APEC) is one of the most economically devastating pathogens affecting the poultry industry. This group of extra-intestinal E. coli causes a variety of clinical conditions including airsacculitis and cellulitis. The economic impact of APEC is mainly due to mortality, slower growth rates, and carcass downgrading. In commercial broiler operations, APEC infections are controlled indirectly by vaccination against other respiratory diseases and minimizing stress conditions, and directly by administration of antimicrobial agents to suppress the infection in already infected flocks. The fact that most APEC strains possess some common virulence factors suggests that an effective vaccine against APEC is a viable option. The most important virulence factors that have been investigated over the years include type I and P fimbriae, aerobactin iron-acquisition system, and serum resistance traits. Despite the potential for developing an efficacious vaccine to combat this economically important poultry disease, several obstacles hinder such efforts. Those obstacles include the cost, vaccine delivery method and timing of vaccination as the birds should be immune to APEC by 21 days of age. Herein, we review the various attempts to develop an effective vaccine against the respiratory form of APEC diseases in poultry. We also discuss in-depth the potentials and limitations of such vaccines.

Neutrophil apoptosis is associated with loss of signal regulatory protein alpha (SIRPα) from the cell surface.

Cells of the innate immune system, including monocytes, macrophages, and neutrophils, play a major role in the development of inflammatory diseases. During inflammation, large numbers of neutrophils are recruited from the blood and subsequently undergo apoptosis, which involves changes in the cell surface expression of a number of receptors. Neutrophils express the Ig superfamily member, SIRPα, which is a receptor involved in regulating cell adhesion and migration. As apoptotic neutrophils down-regulate their capacity for adhesion and migration, we here investigated whether neutrophil expression of SIRPα was affected during apoptosis. We found that apoptotic neutrophils lost SIRPα from their cell surface with kinetics similar to the loss of CD16. The majority of neutrophils with reduced SIRPα also expressed PS on their surface, and the loss of the receptor was reduced proportional to the reduction of apoptosis by caspase inhibitors during Fas-induced apoptosis but less so during spontaneous apoptosis. Neutrophil loss of SIRPα or CD16 was inhibited by the protease inhibitor TAPI-2, as well as specific inhibitors of MMP3 or -8, suggesting that proteolytic mechanisms were involved. Finally, SIRPα was also found on smaller membrane vesicles released from the cells during apoptosis. Our data suggest that neutrophils reduce their SIRPα expression during apoptosis, which may be part of the functional down-regulation seen in apoptotic neutrophils.

Inhibition of cell-mediated immunity by the histone deacetylase inhibitor vorinostat: implications for therapy of cutaneous T-cell lymphoma.

Several histone deacetylase inhibitors (HDACi), including vorinostat, have been approved for the therapy of cutaneous T-cell lymphoma (CTCL). Emerging data suggest that HDACi may exert immune suppressive effects which would be disadvantageous for therapy of CTCL. We describe a patient with Sezary syndrome who was monitored for drug-induced immunosuppression while undergoing treatment with vorinostat. Analysis of the patient's natural killer cell function before and after initiation of treatment confirmed inhibition of this important cell-mediated immune function. In addition, the in vitro effects of vorinostat on the immunity of healthy volunteers confirmed that this class of drug can profoundly suppress multiple arms of the cellular immune response. These findings raise concerns of increased susceptibility to infection in this high-risk population.

Hydroxamic acids derived from 2-hydroxy-2H-1,4-benzoxazin-3(4H)-one: key defense chemicals of cereals.

Many cereals accumulate hydroxamic acids derived from 2-hydroxy-2H-1,4-benzoxazin-3(4H)-one. These benzoxazinoid hydroxamic acids are involved in defense of maize against various lepidopteran pests, most notably the European corn borer, in defense of cereals against various aphid species, and in allelopathy affecting the growth of weeds associated with rye and wheat crops. The role of benzoxazinoid hydroxamic acids in defense against fungal infection is less clear and seems to depend on the nature of the interactions at the plant-fungus interface. Efficient use of benzoxazinoid hydroxamic acids as resistance factors has been limited by the inability to selectively increase their levels at the plant growth stage and the plant tissues where they are mostly needed for a given pest. Although the biosynthesis of benzoxazinoid hydroxamic acids has been elucidated, the genes and mechanisms controlling their differential expression in different plant tissues and along plant ontogeny remain to be unraveled.

Effects of the Aspergillus fumigatus siderophore systems on the regulation of macrophage immune effector pathways and iron homeostasis.

The saprophytic fungus Aspergillus fumigatus is the most prevalent airborne fungal pathogen, which is responsible for invasive aspergillosis in immunocompromised patients. Iron plays an essential role for the growth and proliferation of A. fumigatus. This fungus synthesizes three major siderophores. It excretes triacetylfusarinine C to capture iron, while it accumulates ferricrocin and hydroxyferricrocin for hyphal and conidial iron storage, respectively. Herein, we investigated the role of the siderophore system of A. fumigatus in the modulation of immune effector pathways and iron homeostasis in macrophages. We set up a co-culture system consisting of the murine macrophage cell line RAW264.7 and either A. fumigatus wild type or a siderophore-deficient mutant (DeltasidA). We used real-time quantitative RT-PCR and Western blot analyses to study the expression of macrophage iron metabolism and innate immune response genes in response to pathogen challenge. Infection of macrophages with A. fumigatus wild type, but not with the DeltasidA mutant, induced expression of TNF and phagocyte oxidase subunit 47 at the transcriptional level. Moreover, infection with A. fumigatus wild type, but not with the DeltasidA mutant, compromised macrophage iron homeostasis. Infection with wild-type A. fumigatus decreased expression of the two cellular iron importers, the divalent metal transporter-1 and the transferrin receptor, and the only known iron exporter ferroportin. At the same time, it increased macrophage iron retention and ferritin synthesis. These data indicate that A. fumigatus affects the regulation of macrophage iron homeostasis and innate immune effector pathways via its siderophore system. The changes in immune response may be a consequence of macrophage iron restriction.

Hydroxamate siderophores of Histoplasma capsulatum.

The zoopathogenic fungus Histoplasma capsulatum, like other eukaryotic aerobic microorganisms, requires iron for growth. Under conditions of low iron availability, the fungus secretes hydroxamates that function as siderophores (iron chelators). The experiments to be reported were designed to gather further information on the hydroxamate siderophores of H. capsulatum. The fungus was grown in a synthetic medium deferrated with the cationic exchange resin Chelex 100. Siderophores were detected after 4 days of incubation at 37 degrees C in media containing 0.3 to 1.0 microM iron. The secretion was suppressed by 10 microM iron. The hydroxamates were purified by reverse-phase and size-exclusion chromatography. On the basis of ions observed during electrospray mass spectroscopy, five hydroxamate siderophores were tentatively identified: dimerum acid, acetyl dimerum acid, coprogen B, methyl coprogen B, and fusarinine (monomeric). A polyclonal antibody to dimerum acid was generated. This reagent cross-reacted with coprogen B and fusarinine. Thus, the antibody detects hydroxamates in all three families of siderophores excreted by H. capsulatum.

Results of passive and active immunization directed against ferric aerobactin in experimental enterobacterial infections in mice and chickens.

Production of aerobactin has been reported to be a virulence factor in members of the family Enterobacteriaceae. To investigate the protection afforded by humoral immunity directed towards aerobactin in infectious diseases caused by aerobactin-producing strains, we tested the efficacy of mAbAERO1, a murine monoclonal antibody directed to ferric aerobactin, which, in vitro, was found to impair the growth of aerobactin-dependent strains of Enterobacteriaceae under iron-limited conditions. The mortality of mice experimentally infected with the aerobactin-producing strains Escherichia coli V2019 (LD50 = 3.5 x 10(5) CFU/mice) or Klebsiella pneumoniae Caroli (LD50 = 1.3 CFU/mice) was not reduced when 1 mg of mAbAERO1 was injected intravenously 1 h before or 1 h after bacterial challenge. Nor was mortality reduced after challenge with either E. coli V2019 or K. pneumoniae Caroli, even though the active immunization of mice with purified FeAero (ferric aerobactin) conjugated with thyroglobulin as followed by a rise in systemic anti-FeAero antibodies. Lastly, chicks born of hens immunized with FeAero showed evidence of antibody transmission towards FeAero, but were not protected when challenged with E. coli MT78, an aerobactin-producing strain highly virulent for chickens. Therefore, under the experimental conditions tested, humoral immunity against aerobactin appeared to play only a minor role in protection against infections caused by aerobactin-producing members of the family Enterobacteriaceae. However, other experimental models should be tested to confirm these observations.

Activity and specificity of a mouse monoclonal antibody to ferric aerobactin.

We isolated a monoclonal antibody directed against the ferric complex of aerobactin purified from Escherichia coli KH576. This antibody, which we designated MAb AERO1, was identified as an immunoglobulin G, subtype 2. A competitive enzyme-linked immunosorbent assay with MAb AERO1 had a limit of 10 nM for the detection of purified ferric aerobactin and allowed detection of the crude aerobactin produced by various members of the family Enterobacteriaceae isolated from cancer patients with bacteremia. The only two other structurally related siderophores recognized by MAb AERO1 were ferric arthrobactin and ferrioxamine B. These results suggest that the epitope recognized by MAb AERO1 was the lysyl moiety of ferric aerobactin. We also showed that MAb AERO1 reduced the growth of an aerobactin-producing strain of E. coli in newborn calf serum, which indicates that it might be effective in reducing the severity of infections caused by bacteria for which the production of aerobactin is an important virulence factor.

A simple colorimetric method for the measurement of phagocytosis of crystals of low molecular weight compound by macrophages.

Sodium salt of 9-oxo-10-acridineacetohydroxamic acid (HCA), a new synthetic compound, forms small crystals in aqueous solutions. These crystals were easily phagocytable by the mouse bone marrow-derived macrophages. The ingested HCA crystals were visible under light microscope as dark granules. The counting of cells with the granules allowed the calculation of number of the phagocytic cells in the preparation. It was also possible to measure the amount of ingested HCA by the cells using colorimetric method. The kinetics of phagocytosis of HCA showed that the process was rapid. Quantitative measurements of ingestion of HCA reflected the phagocytic activity of the cultured cells.