81 research outputs found
Exploiting the potential of Insects for in vivo pathogenicity testing of Microbial pathogen
Conventional assays for quantifying the virulence of microbial pathogens and
mutants have traditionally relied upon the use of a range of mammalian species. A
number of workers have demonstrated that insects can be used for evaluating microbial
pathogenicity and provide results comparable to those that can be obtained with
mammals since one component of the vertebrate immune system, the innate immune
response, remains similar to that found in insects. Larvae of the Greater Wax Moth Galleria mellonella have been used to evaluate the virulence of a range of bacterial and
fungal pathogens and a correlation with the virulence of these microbes in mice has been established. This review highlights the similarities of the vertebrate and insect innate immune responses to infection and identifies the potential use of insects for the in vivo evaluation of the microbial pathogenicity
Exploiting the potential of Insects for in vivo pathogenicity testing of Microbial pathogen
Conventional assays for quantifying the virulence of microbial pathogens and
mutants have traditionally relied upon the use of a range of mammalian species. A
number of workers have demonstrated that insects can be used for evaluating microbial
pathogenicity and provide results comparable to those that can be obtained with
mammals since one component of the vertebrate immune system, the innate immune
response, remains similar to that found in insects. Larvae of the Greater Wax Moth Galleria mellonella have been used to evaluate the virulence of a range of bacterial and
fungal pathogens and a correlation with the virulence of these microbes in mice has been established. This review highlights the similarities of the vertebrate and insect innate immune responses to infection and identifies the potential use of insects for the in vivo evaluation of the microbial pathogenicity
Inhibition of neutrophil function following exposure to the Aspergillus fumigatus toxin fumagillin
The filamentous fungus Aspergillus fumigatus produces a variety of enzymes and toxins that may
facilitate fungal colonization of tissue and evasion of the host immune response. One such toxin,
fumagillin, was investigated for its ability to inhibit the action of neutrophils, which are a central
component of the innate immune response to microbial infection. Neutrophils exposed to 2 mg
fumagillin ml”1 for 25 min showed a significantly reduced ability to kill yeast cells (P,0.02), to
phagocytose conidia of A. fumigatus (P,0.023) and to consume oxygen (P,0.032). The ability of
neutrophils to generate superoxide is dependent upon the action of a functional NADPH oxidase
complex which is composed of cytosolic (p40phox, p47phox, p67phox, Rac2) and membrane
(gp91phox) proteins. Exposure of neutrophils to fumagillin inhibited the formation of the NADPH
oxidase complex by blocking the translocation of p47phox from the cytosolic to the membrane
fraction (P50.02). In addition to the production of superoxide, neutrophils also undergo
degranulation, which leads to the release of proteolytic enzymes that contribute to the
microbicidal activity of the cell. Fumagillin-treated neutrophils showed reduced degranulation as
evidenced by lower myeloperoxidase activity (P,0.019). Fumagillin-treated cells demonstrated
reduced levels of F-actin, thus indicating that retarding the formation of F-actin may contribute to
the inhibition of the structural rearrangements required in the activated neutrophil. This work
indicates that fumagillin may contribute to reducing the local immune response by altering the
activity of neutrophils and thus facilitate the continued persistence and growth of A. fumigatus in
the host
The Aspergillus fumigatus toxin fumagillin suppresses the immune response of Galleria mellonella larvae by inhibiting the action of haemocytes
Larvae of Galleria mellonella are widely used to evaluate microbial virulence and to assess the in
vivo efficacy of antimicrobial agents. The aim of this work was to examine the ability of an
Aspergillus fumigatus toxin, fumagillin, to suppress the immune response of larvae. Administration
of fumagillin to larvae increased their susceptibility to subsequent infection with A. fumigatus
conidia (P50.0052). It was demonstrated that a dose of 2 mg fumagillin ml”1 reduced the ability
of insect immune cells (haemocytes) to kill opsonized cells of Candida albicans (P50.039) and to
phagocytose A. fumigatus conidia (P50.016). Fumagillin reduced the oxygen uptake of
haemocytes and decreased the translocation of a p47 protein which is homologous to p47phox, a
protein essential for the formation of a functional NADPH oxidase complex required for superoxide
production. In addition, toxin-treated haemocytes showed reduced levels of degranulation as
measured by the release of a protein showing reactivity to an anti-myeloperoxidase antibody
(P,0.049) that was subsequently identified by liquid chromatography-MS analysis as
prophenoloxidase. This work demonstrates that fumagillin suppresses the immune response of
G. mellonella larvae by inhibiting the action of haemocytes and thus renders the larvae susceptible
to infection. During growth of the fungus in the larvae, this toxin, along with others, may facilitate
growth by suppressing the cellular immune response
Inhibition of neutrophil function following exposure to the Aspergillus fumigatus toxin fumagillin
The filamentous fungus Aspergillus fumigatus produces a variety of enzymes and toxins that may
facilitate fungal colonization of tissue and evasion of the host immune response. One such toxin,
fumagillin, was investigated for its ability to inhibit the action of neutrophils, which are a central
component of the innate immune response to microbial infection. Neutrophils exposed to 2 mg
fumagillin ml”1 for 25 min showed a significantly reduced ability to kill yeast cells (P,0.02), to
phagocytose conidia of A. fumigatus (P,0.023) and to consume oxygen (P,0.032). The ability of
neutrophils to generate superoxide is dependent upon the action of a functional NADPH oxidase
complex which is composed of cytosolic (p40phox, p47phox, p67phox, Rac2) and membrane
(gp91phox) proteins. Exposure of neutrophils to fumagillin inhibited the formation of the NADPH
oxidase complex by blocking the translocation of p47phox from the cytosolic to the membrane
fraction (P50.02). In addition to the production of superoxide, neutrophils also undergo
degranulation, which leads to the release of proteolytic enzymes that contribute to the
microbicidal activity of the cell. Fumagillin-treated neutrophils showed reduced degranulation as
evidenced by lower myeloperoxidase activity (P,0.019). Fumagillin-treated cells demonstrated
reduced levels of F-actin, thus indicating that retarding the formation of F-actin may contribute to
the inhibition of the structural rearrangements required in the activated neutrophil. This work
indicates that fumagillin may contribute to reducing the local immune response by altering the
activity of neutrophils and thus facilitate the continued persistence and growth of A. fumigatus in
the host
Amphotericin B enhances the synthesis and release of the immunosuppressive agent gliotoxin from the pulmonary pathogen Aspergillus fumigatus
Exposure of the pulmonary pathogen Aspergillus fumigatus to amphotericin B alters membrane
permeability as indicated by the escape of amino acids and protein from the mycelium. Amphotericin
B exposure for periods of 2-4 h also leads to increased release of the immunosuppressive agent
gliotoxin into the surrounding culture medium. Examination of the intracellular gliotoxin concentration
following exposure to amphotericin B indicated elevated levels within the hyphae as well as in the
culture medium - an effect which was also evident upon exposure of A. fumigatus to DMSO. These
results indicate that in parallel with the ability of amphotericinBto act as a fungistatic agent it can also
induce the synthesis of gliotoxin and facilitate its release by increasing the permeability of the fungal
cell membrane. Increased synthesis of gliotoxin may result from the commencement of secondary
metabolism in the presence of amphotericin B. The ability of amphotericin B to enhance the
synthesis and release of gliotoxin may exacerbate the effects of the toxin and facilitate fungal invasion
of pulmonary tissue
Translocation of proteins homologous to human neutrophil p47phox and p67phox to the cell membrane in activated hemocytes of Galleria mellonella
Activation of the superoxide forming respiratory burst oxidase of human neutrophils, crucial in host defence, requires the cytosolic proteins p47phox and p67phox which translocate to the plasma membrane upon cell stimulation and activate flavocytochrome b558, the redox centre of this enzyme system. We have previously demonstrated the presence of proteins (67 and 47 kDa) in hemocytes of the insect Galleria mellonella homologous to proteins of the superoxide-forming NADPH oxidase complex of neutrophils. The work presented here illustrates for the first time translocation of homologous hemocyte proteins, 67 and 47 kDa from the cytosol to the plasma membrane upon phorbol 12-myristate 13 acetate (PMA)activation. In hemocytes, gliotoxin (GT), the fungal secondary metabolite significantly suppressed PMA-induced superoxide generation in a concentration dependent manner and reduced translocation to basel nonstimulated levels.
Primarily these results correlate translocation of hemocyte 47 and 67 kDa proteins with PMA induced oxidase activity.
Collectively results presented here, demonstrate further cellular and functional similarities between phagocytes of insects and mammals and further justify the use of insects in place of mammals for modelling the innate immune response to microbial pathogens
Hypertonic Saline in Treatment of Pulmonary Disease in Cystic Fibrosis
The pathogenesis of lung disease in cystic fibrosis is characterised by decreased airway surface liquid volume and subsequent failure of normal mucociliary clearance. Mucus within the cystic fibrosis airways is enriched in negatively charged matrices composed of DNA released from colonizing bacteria or inflammatory cells, as well as F-actin and elevated concentrations of anionic glycosaminoglycans. Therapies acting against airway mucus in cystic fibrosis include aerosolized hypertonic saline. It has been shown that hypertonic saline possesses mucolytic properties and aids mucociliary clearance by restoring the liquid layer lining the airways. However, recent clinical and bench-top studies are beginning to broaden our view on the beneficial effects of hypertonic saline, which now extend to include anti-infective as well as anti-inflammatory properties. This review aims to discuss the described therapeutic benefits of hypertonic saline and specifically to identify novel models of hypertonic saline action independent of airway hydration
New Research on the Importance of Cystic Fibrosis Transmembrane Conductance Regulator Function for Optimal Neutrophil Activity
Despite tremendous recent advances in our understanding of the molecular and cellular basis of cystic fibrosis (CF), there remains a paradox of why recruited neutrophils fail to eradicate bacterial infections in the airways of individuals with CF. The focus of this chapter is on new research authenticating the CF neutrophil as a key player in disease pathogenesis. Studies specifying intrinsic abnormalities due to a lack of cystic fibrosis transmembrane conductance regulator (CFTR) function, along with reports indicating reprogrammed cell activity secondary to chronic bacterial infection and inflammation, will be discussed
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