A dissemination-prone morphotype enhances extrapulmonary organ entry by Cryptococcus neoformans.

Environmental pathogens move from ecological niches to mammalian hosts, requiring adaptation to dramatically different environments. Microbes that disseminate farther, including the fungal meningitis pathogen Cryptococcus neoformans, require additional adaptation to diverse tissues. We demonstrate that the formation of a small C. neoformans morphotype-called "seed" cells due to their colonizing ability-is critical for extrapulmonary organ entry. Seed cells exhibit changes in fungal cell size and surface expression that result in an enhanced macrophage update. Seed cell formation is triggered by environmental factors, including C. neoformans' environmental niche, and pigeon guano with phosphate plays a central role. Seed cells show the enhanced expression of phosphate acquisition genes, and mutants unable to acquire phosphate fail to adopt the seed cell morphotype. Additionally, phosphate can be released by tissue damage, potentially establishing a feed-forward loop of seed cell formation and dissemination. Thus, C. neoformans' size variation represent inducible morphotypes that change host interactions to facilitate microbe spread.

Synergistic and antagonistic drug interactions in the treatment of systemic fungal infections.

Invasive fungal infections cause 1.6 million deaths annually, primarily in immunocompromised individuals. Mortality rates are as high as 90% due to limited treatments. The azole class antifungal, fluconazole, is widely available and has multi-species activity but only inhibits growth instead of killing fungal cells, necessitating long treatments. To improve treatment, we used our novel high-throughput method, the overlap2 method (O2M) to identify drugs that interact with fluconazole, either increasing or decreasing efficacy. We identified 40 molecules that act synergistically (amplify activity) and 19 molecules that act antagonistically (decrease efficacy) when combined with fluconazole. We found that critical frontline beta-lactam antibiotics antagonize fluconazole activity. A promising fluconazole-synergizing anticholinergic drug, dicyclomine, increases fungal cell permeability and inhibits nutrient intake when combined with fluconazole. In vivo, this combination doubled the time-to-endpoint of mice with Cryptococcus neoformans meningitis. Thus, our ability to rapidly identify synergistic and antagonistic drug interactions can potentially alter the patient outcomes.

Individuals with weakened immune systems ­ such as recipients of organ transplants ­ can fall prey to illnesses caused by fungi that are harmless to most people. These infections are difficult to manage because few treatments exist to fight fungi, and many have severe side effects. Antifungal drugs usually slow the growth of fungi cells rather than kill them, which means that patients must remain under treatment for a long time, or even for life. One way to boost efficiency and combat resistant infections is to combine antifungal treatments with drugs that work in complementary ways: the drugs strengthen each other's actions, and together they can potentially kill the fungus rather than slow its progression. However, not all drug combinations are helpful. In fact, certain drugs may interact in ways that make treatment less effective. This is particularly concerning because people with weakened immune systems often take many types of medications. Here, Wambaugh et al. harnessed a new high-throughput system to screen how 2,000 drugs (many of which already approved to treat other conditions) affected the efficiency of a common antifungal called fluconazole. This highlighted 19 drugs that made fluconazole less effective, some being antibiotics routinely used to treat patients with weakened immune systems. On the other hand, 40 drugs boosted the efficiency of fluconazole, including dicyclomine, a compound currently used to treat inflammatory bowel syndrome. In fact, pairing dicyclomine and fluconazole more than doubled the survival rate of mice with severe fungal infections. The combined treatment could target many species of harmful fungi, even those that had become resistant to fluconazole alone. The results by Wambaugh et al. point towards better treatments for individuals with serious fungal infections. Drugs already in circulation for other conditions could be used to boost the efficiency of fluconazole, while antibiotics that do not decrease the efficiency of this medication should be selected to treat at-risk patients.

Effect of Feeding Hay on Nonesterified Fatty Acids in Appetite-Suppressed Pregnant New Zealand White Rabbits.

Pregnant rabbits are a common nonrodent model for reproductive safety evaluation in preclinical drug development. During reproductive toxicology studies, rabbits are prone to decreased food consumption and anorexia. When persistent or severe, this condition can lead to hepatic lipidosis and pregnancy toxemia, which may confound the interpretation of study results. Non-Esterified Fatty Acids (NEFAs) have been used in veterinary production medicine to evaluate the impact of diet on the energy balance of pregnant animals. In the current study, sustained-release buprenorphine was used to suppress the appetite of pregnant New Zealand white rabbits, mimicking the clinical presentation of animals in reproductive toxicology studies. Sequential NEFA evaluations during gestation, along with other clinical endpoints, such as the necessity and duration of veterinary intervention, were used to evaluate the effects of feeding hay and a pelleted diet as compared with a pelleted diet alone. Elevated NEFA levels were directly correlated to litter size, the number of viable fetuses and the number of days on veterinary consult due to severely decreased consumption of pelleted diet. Animals with hay as part of their diet did not require additional diet supplementation as determined by qualitative evaluation of hay intake and adequate fecal output. These data suggest that including hay as a portion of the standard diet benefits pregnant rabbits in laboratory or production settings.

How Environmental Fungi Cause a Range of Clinical Outcomes in Susceptible Hosts.

Environmental fungi are globally ubiquitous and human exposure is near universal. However, relatively few fungal species are capable of infecting humans, and among fungi, few exposure events lead to severe systemic infections. Systemic infections have mortality rates of up to 90%, cost the US healthcare system $7.2 billion annually, and are typically associated with immunocompromised patients. Despite this reputation, exposure to environmental fungi results in a range of outcomes, from asymptomatic latent infections to severe systemic infection. Here we discuss different exposure outcomes for five major fungal pathogens: Aspergillus, Blastomyces, Coccidioides, Cryptococcus, and Histoplasma species. These fungi include a mold, a budding yeast, and thermal dimorphic fungi. All of these species must adapt to dramatically changing environments over the course of disease. These dynamic environments include the human lung, which is the first exposure site for these organisms. Fungi must defend themselves against host immune cells while germinating and growing, which risks further exposing microbe-associated molecular patterns to the host. We discuss immune evasion strategies during early infection, from disruption of host immune cells to major changes in fungal cell morphology.

Mechanisms of Pulmonary Escape and Dissemination by Cryptococcus neoformans.

Cryptococcus neoformans is a common environmental saprophyte and human fungal pathogen that primarily causes disease in immunocompromised individuals. Similar to many environmentally acquired human fungal pathogens, C. neoformans initiates infection in the lungs. However, the main driver of mortality is invasive cryptococcosis leading to fungal meningitis. After C. neoformans gains a foothold in the lungs, a critical early step in invasion is transversal of the respiratory epithelium. In this review, we summarize current knowledge relating to pulmonary escape. We focus on fungal factors that allow C. neoformans to disseminate from the lungs via intracellular and extracellular routes.

Regulated Release of Cryptococcal Polysaccharide Drives Virulence and Suppresses Immune Cell Infiltration into the Central Nervous System.

Cryptococcus neoformans is a common environmental yeast and opportunistic pathogen responsible for 15% of AIDS-related deaths worldwide. Mortality primarily results from meningoencephalitis, which occurs when fungal cells disseminate to the brain from the initial pulmonary infection site. A key C. neoformans virulence trait is the polysaccharide capsule. Capsule shields C. neoformans from immune-mediated recognition and destruction. The main capsule component, glucuronoxylomannan (GXM), is found both attached to the cell surface and free in the extracellular space (as exo-GXM). Exo-GXM accumulates in patient serum and cerebrospinal fluid at microgram/milliliter concentrations, has well-documented immunosuppressive properties, and correlates with poor patient outcomes. However, it is poorly understood whether exo-GXM release is regulated or the result of shedding during normal capsule turnover. We demonstrate that exo-GXM release is regulated by environmental cues and inversely correlates with surface capsule levels. We identified genes specifically involved in exo-GXM release that do not alter surface capsule thickness. The first mutant, the liv7Δ strain, released less GXM than wild-type cells when capsule was not induced. The second mutant, the cnag_00658Δ strain, released more exo-GXM under capsule-inducing conditions. Exo-GXM release observed in vitro correlated with polystyrene adherence, virulence, and fungal burden during murine infection. Additionally, we found that exo-GXM reduced cell size and capsule thickness under capsule-inducing conditions, potentially influencing dissemination. Finally, we demonstrated that exo-GXM prevents immune cell infiltration into the brain during disseminated infection and highly inflammatory intracranial infection. Our data suggest that exo-GXM performs a distinct role from capsule GXM during infection, altering cell size and suppressing inflammation.

A Translational Model for Diet-related Atherosclerosis: Effect of Statins on Hypercholesterolemia and Atherosclerosis in a Minipig.

Models of atherosclerosis are used in preclinical studies but often fail to translate to humans. A model that better reflects human atherosclerosis is necessary. We recently engineered the ExeGen™ low-density lipoprotein receptor (LDLR) miniswine, in which the LDL receptor gene is modified to drive hypercholesterolemia and atherosclerosis, and showed diet-related exacerbation of these phenotypes. Five groups of animals, either wild type (+/+) or heterozygous (+/-), were fed either a normal or high-fat diet for 6 months. One group of heterozygous pigs fed a high-fat diet was also administered atorvastatin at 3 mg/kg/day. Clinical chemistry and anatomic pathology parameters were measured biweekly and at termination. The high-fat diet resulted in increased adiposity and interspersion of adipocytes within the salivary glands. The heterozygous pigs on the high-fat diet gained more weight and had significant increases in total cholesterol, high-density lipoprotein, and LDL compared to wild-type animals or heterozygous animals fed a normal diet. Atorvastatin attenuated these parameters, indicating the statin had a beneficial effect, even in a high-fat diet scenario. Atorvastatin treatment also reduced the intensity of Oil Red O staining in pigs on high-fat diet. Atorvastatin-related amelioration of several indices of cardiovascular pathophysiology in this model underscores its utility for drug discovery.

Two Novel Bioassays to Assess the Effects of Pyrethroid-Treated Netting on Knockdown-Susceptible Versus Resistant Strains of Aedes aegypti.

We describe 2 new mosquito bioassays for use with insecticide-treated netting or other textiles. The 1st is a cylinder bioassay in which a mosquito is forced to contact treated material regardless of where it lands within the bioassay construct. The 2nd is a repellency/irritancy and biting-inhibition bioassay (RIBB) in which human arms and breath are used as attractants. Mosquitoes have the choice to pass through holes cut in untreated or treated netting to move from a center release chamber into side chambers to reach arms and potentially bite. Trials were conducted with pyrethroid-susceptible (New Orleans), moderately resistant (Hunucmá), and highly resistant (Vergel) strains of Aedes aegypti. Tests with netting treated with different pyrethroids demonstrated the utility of the cylinder bioassay to quantify knockdown and mortality following exposure to treated netting, and of the RIBB to quantify spatial repellency/contact irritancy of the treated netting and biting inhibition after females land on and then pass through holes in the treated netting. Both tested brands of pyrethroid-treated mosquitocidal netting (DuraNet® and NetProtect®) were effective against New Orleans but ineffective against Vergel strains. Mortality in the cylinder bioassay was 100% for New Orleans for all tested brands of treated netting, but only 10-14% for Vergel. Rates of passage through treated netting to reach a human arm in the RIBB were 10-15% for New Orleans versus 24-37% for Vergel. The reduction in biting after passage through treated netting, compared with untreated netting in the same trial replicates, was 12-39% for New Orleans versus ≤9% for Vergel.

Local evolution of pyrethroid resistance offsets gene flow among Aedes aegypti collections in Yucatan State, Mexico.

The mosquito Aedes aegypti is the major vector of the four serotypes of dengue virus (DENV1-4). Previous studies have shown that Ae. aegypti in Mexico have a high effective migration rate and that gene flow occurs among populations that are up to 150 km apart. Since 2000, pyrethroids have been widely used for suppression of Ae. aegypti in cities in Mexico. In Yucatan State in particular, pyrethroids have been applied in and around dengue case households creating an opportunity for local selection and evolution of resistance. Herein, we test for evidence of local adaptation by comparing patterns of variation among 27 Ae. aegypti collections at 13 single nucleotide polymorphisms (SNPs): two in the voltage-gated sodium channel gene para known to confer knockdown resistance, three in detoxification genes previously associated with pyrethroid resistance, and eight in putatively neutral loci. The SNPs in para varied greatly in frequency among collections, whereas SNPs at the remaining 11 loci showed little variation supporting previous evidence for extensive local gene flow. Among Ae. aegypti in Yucatan State, Mexico, local adaptation to pyrethroids appears to offset the homogenizing effects of gene flow.

Survey of retail milk composition as affected by label claims regarding farm-management practices.

A trend in food labeling is to make claims related to agricultural management, and this is occurring with dairy labels. A survey study was conducted to compare retail milk for quality (antibiotics and bacterial counts), nutritional value (fat, protein, and solids-not-fat), and hormonal composition (somatotropin, insulin-like growth factor-1 [IGF-1], estradiol, and progesterone) as affected by three label claims related to dairy-cow management: conventional, recombinant bovine somatotropin (rbST)-free (processor-certified not from cows supplemented with rbST), or organic (follows US Department of Agriculture organic practices). Retail milk samples (n=334) from 48 states were collected. Based on a statistical analysis that reflected the sampling schema and distributions appropriate to the various response variables, minor differences were observed for conventional, rbST-free, and organic milk labels. Conventionally labeled milk had the lowest (P<0.05) bacterial counts compared to either milk labeled rbST-free or organic; however, these differences were not biologically meaningful. In addition, conventionally labeled milk had significantly less (P<0.05) estradiol and progesterone than organic milk (4.97 vs 6.40 pg/mL and 12.0 vs 13.9 ng/mL, respectively). Milk labeled rbST-free had similar concentrations of progesterone vs conventional milk and similar concentrations of estradiol vs organic milk. Concentrations of IGF-1 in milk were similar between conventional milk and milk labeled rbST-free. Organic milk had less (P<0.05) IGF-1 than either conventional or rbST-free milk (2.73 ng/mL vs 3.12 and 3.04 ng/mL, respectively). The macronutrient profiles of the different milks were similar, except for a slight increase in protein in organic milk (about 0.1% greater for organic compared to other milks). Label claims were not related to any meaningful differences in the milk compositional variables measured. It is important for food and nutrition professionals to know that conventional, rbST-free, and organic milk are compositionally similar so they can serve as a key resource to consumers who are making milk purchase (and consumption) decisions in a marketplace where there are misleading milk label claims.