Globally, there are several million individuals with life-threatening invasive fungal diseases such as candidiasis, aspergillosis, cryptococcosis, Pneumocystis pneumonia (PCP), and mucormycosis. The ...mortality rate for these diseases generally exceeds 40%. Annual medical costs to treat these invasive fungal diseases in the United States exceed several billion dollars. In addition to AIDS patients, the risks of invasive mycoses are increasingly found in immune-impaired individuals or in immunosuppressed patients following stem cell or organ transplant or implantation of medical devices. Current antifungal drug therapies are not meeting the challenge, because (1) at safe doses, they do not provide sufficient fungal clearance to prevent reemergence of infection; (2) most become toxic with extended use; (3) drug-resistant fungal isolates are emerging; and (4) only one new class of antifungal drugs has been approved for clinical use in the last 2 decades. DectiSomes represent a novel design of drug delivery to drastically increase drug efficacy. Antifungals packaged in liposomes are targeted specifically to where the pathogen is, through binding to the fungal cell walls or exopolysaccharide matrices using the carbohydrate recognition domains of pathogen receptors. Relative to untargeted liposomal drug, DectiSomes show order of magnitude increases in the binding to and killing of Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus in vitro and similarly improved efficacy in mouse models of pulmonary aspergillosis. DectiSomes have the potential to usher in a new antifungal drug treatment paradigm.
Purpose
Obstructive sleep apnea (OSA) results in systemic intermittent hypoxia. By one model, hypoxic stress signaling in OSA patients alters the levels of inflammatory soluble cytokines TNF and IL6, ...damages the blood brain barrier, and activates microglial targeting of neuronal cell death to increase the risk of neurodegenerative disorders and other diseases. However, it is not yet clear if OSA significantly alters the levels of the soluble isoforms of TNF receptors TNFR1 and TNFR2 and IL6 receptor (IL6R) and co-receptor gp130, which have the potential to modulate TNF and IL6 signaling.
Methods
Picogram per milliliter levels of the soluble isoforms of these four cytokine receptors were estimated in OSA patients, in OSA patients receiving airways therapy, and in healthy control subjects. Triplicate samples were examined using Bio-Plex fluorescent bead microfluidic technology. The statistical significance of cytokine data was estimated using the nonparametric Wilcoxon rank-sum test. The clustering of these high-dimensional data was visualized using
t
-distributed stochastic neighbor embedding (t-SNE).
Results
OSA patients had significant twofold to sevenfold reductions in the soluble serum isoforms of all four cytokine receptors, gp130, IL6R, TNFR1, and TNFR2, as compared with control individuals (
p
= 1.8 × 10
−13
to 4 × 10
−8
). Relative to untreated OSA patients, airways therapy of OSA patients had significantly higher levels of gp130 (
p
= 2.8 × 10
−13
), IL6R (
p
= 1.1 × 10
−9
), TNFR1 (
p
= 2.5 × 10
−10
), and TNFR2 (
p
= 5.7 × 10
−9
), levels indistinguishable from controls (
p
= 0.29 to 0.95). The data for most airway-treated patients clustered with healthy controls, but the data for a few airway-treated patients clustered with apneic patients.
Conclusions
Patients with OSA have aberrantly low levels of four soluble cytokine receptors associated with neurodegenerative disease, gp130, IL6R, TNFR1, and TNFR2. Most OSA patients receiving airways therapy have receptor levels indistinguishable from healthy controls, suggesting a chronic intermittent hypoxia may be one of the factors contributing to low receptor levels in untreated OSA patients.
,
, and
cause life-threatening candidiasis, cryptococcosis, and aspergillosis, resulting in several hundred thousand deaths annually. The patients at the greatest risk of developing these ...life-threatening invasive fungal infections have weakened immune systems. The vulnerable population is increasing due to rising numbers of immunocompromised individuals as a result of HIV infection or immunosuppressed individuals receiving anticancer therapies and/or stem cell or organ transplants. While patients are treated with antifungals such as amphotericin B, all antifungals have serious limitations due to lack of sufficient fungicidal effect and/or host toxicity. Even with treatment, 1-year survival rates are low. We explored methods of increasing drug effectiveness by designing fungicide-loaded liposomes specifically targeted to fungal cells. Most pathogenic fungi are encased in cell walls and exopolysaccharide matrices rich in mannans. Dectin-2 is a mammalian innate immune membrane receptor that binds as a dimer to mannans and signals fungal infection. We coated amphotericin-loaded liposomes with monomers of Dectin-2's mannan-binding domain, sDectin-2. sDectin monomers were free to float in the lipid membrane and form dimers that bind mannan substrates. sDectin-2-coated liposomes bound orders of magnitude more efficiently to the extracellular matrices of several developmental stages of
,
, and
than untargeted control liposomes. Dectin-2-coated amphotericin B-loaded liposomes reduced the growth and viability of all three species more than an order of magnitude more efficiently than untargeted control liposomes and dramatically decreased the effective dose. Future efforts focus on examining pan-antifungal targeted liposomal drugs in animal models of fungal diseases.
Invasive fungal diseases caused by
,
, and
have mortality rates ranging from 10 to 95%. Individual patient costs may exceed $100,000 in the United States. All antifungals in current use have serious limitations due to host toxicity and/or insufficient fungal cell killing that results in recurrent infections. Few new antifungal drugs have been introduced in the last 2 decades. Hence, there is a critical need for improved antifungal therapeutics. By targeting antifungal-loaded liposomes to α-mannans in the extracellular matrices secreted by these fungi, we dramatically reduced the effective dose of drug. Dectin-2-coated liposomes loaded with amphotericin B bound 50- to 150-fold more strongly to
,
, and
than untargeted liposomes and killed these fungi more than an order of magnitude more efficiently. Targeting drug-loaded liposomes specifically to fungal cells has the potential to greatly enhance the efficacy of most antifungal drugs.
Invasive fungal diseases cause millions of deaths each year. There are currently approximately 300,000 acute cases of aspergillosis, most of which result from a pulmonary infection of ...immunocompromised patients by the common soil organism and opportunistic pathogen
Patients are treated with antifungal drugs, such as amphotericin B (AmB). However, AmB has serious limitations due to human organ toxicity. AmB is slightly less toxic if loaded in liposomes, such as AmBisome or AmB-loaded liposomes (AmB-LLs). Even with antifungal therapy, recurrent infections are common, and 1-year fatality rates may exceed 50%. We have previously shown that coating AmB-LLs with the extracellular oligomannan-binding domain of the C-type lectin receptor Dectin-2 (DEC2-AmB-LLs) effectively targets DEC2-AmB-LLs to cell walls, exopolysaccharide matrices, and biofilms of fungal pathogens
, DEC2-AmB-LLs reduce the effective dose of AmB for 95% inhibition and killing of
10-fold compared to that of untargeted AmB-LLs. Herein we tested the antifungal activity of DEC2-AmB-LLs relative to that of untargeted AmB-LLs in immunosuppressed mice with pulmonary aspergillosis. Remarkably, DEC2-AmB-LLs bound 30-fold more efficiently to
at sites of infection in the lungs. Furthermore, Dectin-2-targeted liposomes delivering AmB at a dose of 0.2 mg/kg of body weight significantly reduced the fungal burden in lungs compared to results with untargeted AmB-LLs at 0.2 mg/kg and micellar voriconazole at 20 mg/kg and prolonged mouse survival. By dramatically increasing the efficacy of antifungal drugs at low doses, targeted liposomes have the potential to create a new clinical paradigm to treat diverse fungal diseases.
Invasive aspergillosis (IA) generally results from a pulmonary infection of immunocompromised patients by the common soil organism and opportunistic pathogen
The susceptible population has expanded rapidly due to the increased number of cancer patients with immunocompromising chemotherapy and transplant patients taking immunosuppressants. Patients are treated with antifungals, such as liposomal amphotericin B, with per-patient costs exceeding $50,000 in the United States. However, AmB has serious side effects due to host toxicity, which limits its usage and contributes to the lack of fungal clearance in patients at safe doses. Fifty percent of IA patients die within a year. Herein, we employed liposomal amphotericin B coated with the innate immune receptor Dectin-2 to direct antifungals specifically to the fungal pathogen. Using two mouse models of pulmonary aspergillosis, we demonstrate that Dectin-2-targeted delivery of amphotericin B to
resulted in remarkably higher efficacy than that of the untargeted antifungal formulations.
species cause pulmonary invasive aspergillosis resulting in nearly 100,000 deaths each year. Patients at the greatest risk of developing life-threatening aspergillosis have weakened immune systems ...and/or various lung disorders. Patients are treated with antifungals such as amphotericin B (AmB), caspofungin acetate, or triazoles (itraconazole, voriconazole, etc.), but these antifungal agents have serious limitations due to lack of sufficient fungicidal effect and human toxicity. Liposomes with AmB intercalated into the lipid membrane (AmB-LLs; available commercially as AmBisome) have severalfold-reduced toxicity compared to that of detergent-solubilized drug. However, even with the current antifungal therapies, 1-year survival among patients is only 25 to 60%. Hence, there is a critical need for improved antifungal therapeutics. Dectin-1 is a mammalian innate immune receptor in the membrane of some leukocytes that binds as a dimer to beta-glucans found in fungal cell walls, signaling fungal infection. Using a novel protocol, we coated AmB-LLs with Dectin-1's beta-glucan binding domain to make DEC-AmB-LLs. DEC-AmB-LLs bound rapidly, efficiently, and with great strength to
and to
and
, highly divergent fungal pathogens of global importance. In contrast, untargeted AmB-LLs and bovine serum albumin (BSA)-coated BSA-AmB-LLs showed 200-fold-lower affinity for fungal cells. DEC-AmB-LLs reduced the growth and viability of
an order of magnitude more efficiently than untargeted control liposomes delivering the same concentrations of AmB, in essence decreasing the effective dose of AmB. Future efforts will focus on examining pan-antifungal targeted liposomal drugs in animal models of disease.
The fungus
causes pulmonary invasive aspergillosis resulting in nearly 100,000 deaths each year. Patients are often treated with antifungal drugs such as amphotericin B (AmB) loaded into liposomes (AmB-LLs), but all antifungal drugs, including AmB-LLs, have serious limitations due to human toxicity and insufficient fungal cell killing. Even with the best current therapies, 1-year survival among patients with invasive aspergillosis is only 25 to 60%. Hence, there is a critical need for improved antifungal therapeutics. Dectin-1 is a mammalian protein that binds to beta-glucan polysaccharides found in nearly all fungal cell walls. We coated AmB-LLs with Dectin-1 to make DEC-AmB-LLs. DEC-AmB-LLs bound strongly to fungal cells, while AmB-LLs had little affinity. DEC-AmB-LLs killed or inhibited
10 times more efficiently than untargeted liposomes, decreasing the effective dose of AmB. Dectin-1-coated drug-loaded liposomes targeting fungal pathogens have the potential to greatly enhance antifungal therapeutics.
Cry3Bb toxin from
is an important insecticidal protein due to its potency against coleopteran pests, especially rootworms. Cadherin, a protein in the insect midgut epithelium, is a receptor of Cry ...toxins; in some insect species toxin-binding domains of cadherins-synergized Cry toxicity. Previously, we reported that the DvCad1-CR8-10 fragment of
cadherin-like protein (GenBank Accession #EF531715) enhanced Cry3Bb toxicity to the Colorado Potato Beetle (CPB),
(
). We report that individual CR domains of the DvCad1-CR8-10 fragment were found to have strong binding affinities to α-chymotrypsin-treated Cry3Bb. The dissociation constant (
) of Cry3Bb binding to the CR8, CR9, and CR10 domain was 4.9 nM, 28.2 nM, and 4.6 nM, respectively. CR8 and CR10, but not CR9, enhanced Cry3Bb toxicity against
and the lesser mealworm
neonates. In-frame deletions of the DvCad1-CR10 open reading frame defined a high-affinity binding and synergistic site to a motif in residues I1226-D1278. A 26 amino acid peptide from the high affinity Cry3Bb-binding region of CR10 functioned as a Cry3Bb synergist against coleopteran larvae.
Mucormycosis (a.k.a. zygomycosis) is an often-life-threatening disease caused by fungi from the ancient fungal division Mucoromycota. Globally, there are nearly a million people with the disease.
...spp., and
(
) in particular, are responsible for most of the diagnosed cases. Pulmonary, rhino-orbito-cerebral, and invasive mucormycosis are most effectively treated with amphotericin B (AmB) and particularly with liposomal formulations (e.g., AmBisome
). However, even after antifungal therapy, there is still a 50% mortality rate. Hence, there is a critical need to improve therapeutics for mucormycosis. Targeting AmB-loaded liposomes (AmB-LLs) with the pathogen receptor Dectin-1 (DEC1-AmB-LLs) to the beta-glucans expressed on the surface of
and
lowers the effective dose required to kill cells relative to untargeted AmB-LLs. Because Dectin-1 is an immune receptor for
infections and may bind it directly, we explored the Dectin-1-mediated delivery of liposomal AmB to
. DEC1-AmB-LLs bound 100- to 1000-fold more efficiently to the exopolysaccharide matrix of
germlings and mature hyphae relative to AmB-LLs. DEC1-AmB-LLs delivering sub-micromolar concentrations of AmB were an order of magnitude more efficient at inhibiting and/or killing
than AmB-LLs. Targeted antifungal drug-loaded liposomes have the potential to improve the treatment of mucormycosis.
Life-threatening invasive fungal infections are treated with antifungal drugs such as Amphotericin B (AmB) loaded liposomes. Our goal herein was to show that targeting liposomal AmB to fungal cells ...with the C-type lectin pathogen recognition receptor DC-SIGN improves antifungal activity. DC-SIGN binds variously crosslinked mannose-rich and fucosylated glycans and lipomannans that are expressed by helminth, protist, fungal, bacterial and viral pathogens including three of the most life-threatening fungi, Aspergillus fumigatus, Candida albicans and Cryptococcus neoformans. Ligand recognition by human DC-SIGN is provided by a carbohydrate recognition domain (CRD) linked to the membrane transit and signaling sequences. Different combinations of the eight neck repeats (NR1 to NR8) expressed in different protein isoforms may alter the orientation of the CRD to enhance its binding to different glycans.
We prepared two recombinant isoforms combining the CRD with NR1 and NR2 in isoform DCS12 and with NR7 and NR8 in isoform DCS78 and coupled them to a lipid carrier. These constructs were inserted into the membrane of pegylated AmB loaded liposomes AmB-LLs to produce DCS12-AmB-LLs and DCS78-AmB-LLs. Relative to AmB-LLs and Bovine Serum Albumin coated BSA-AmB-LLs, DCS12-AmB-LLs and DCS78-AmB-LLs bound more efficiently to the exopolysaccharide matrices produced by A. fumigatus, C. albicans and C. neoformans in vitro, with DCS12-AmB-LLs performing better than DCS78-AmB-LLs. DCS12-AmB-LLs inhibited and/or killed all three species in vitro significantly better than AmB-LLs or BSA-AmB-LLs. In mouse models of invasive candidiasis and pulmonary aspergillosis, one low dose of DCS12-AmB-LLs significantly reduced the fungal burden in the kidneys and lungs, respectively, several-fold relative to AmB-LLs.
DC-SIGN's CRD specifically targeted antifungal liposomes to three highly evolutionarily diverse pathogenic fungi and enhanced the antifungal efficacy of liposomal AmB both in vitro and in vivo. Targeting significantly reduced the effective dose of antifungal drug, which may reduce drug toxicity, be effective in overcoming dose dependent drug resistance, and more effectively kill persister cells. In addition to fungi, DC-SIGN targeting of liposomal packaged anti-infectives have the potential to alter treatment paradigms for a wide variety of pathogens from different kingdoms including protozoans, helminths, bacteria, and viruses which express its cognate ligands.
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