Sepsis, a systemic inflammatory response to infection, commonly progresses to acute lung injury (ALI), an inflammatory lung disease with high morbidity. We postulated that sepsis-associated ALI is initiated by degradation of the pulmonary endothelial glycocalyx, leading to neutrophil adherence and inflammation. Using intravital microscopy, we found that endotoxemia in mice rapidly induced pulmonary microvascular glycocalyx degradation via tumor necrosis factor-α (TNF-α)-dependent mechanisms. Glycocalyx degradation involved the specific loss of heparan sulfate and coincided with activation of endothelial heparanase, a TNF-α–responsive, heparan sulfate–specific glucuronidase. Glycocalyx degradation increased the availability of endothelial surface adhesion molecules to circulating microspheres and contributed to neutrophil adhesion. Heparanase inhibition prevented endotoxemia-associated glycocalyx loss and neutrophil adhesion and, accordingly, attenuated sepsis-induced ALI and mortality in mice. These findings are potentially relevant to human disease, as sepsis-associated respiratory failure in humans was associated with higher plasma heparan sulfate degradation activity; moreover, heparanase content was higher in human lung biopsies showing diffuse alveolar damage than in normal human lung tissue.

Rtp801, a stress – related protein triggered by adverse environmental conditions, inhibits mTOR and enhances oxidative stress – dependent cell death. We postulated that Rtp801 acts as potential amplifying switch in the development of cigarette smoke – induced lung injury, leading to emphysema. Rtp801 was overexpressed in human emphysematous lungs and in lungs of mice exposed to cigarette smoke. The upregulation of Rtp801 expression by cigarette smoke in the lung relied on oxidative stress – dependent activation of the CCAAT response element. Rtp801 was necessary and sufficient for NF – κ B activation in cultured cells and, when forcefully expressed in mouse lungs, it promoted NF – kB activation, alveolar inflammation, oxidative stress, and apoptosis of alveolar septal cells. On the other hand, Rtp801 − / − mice were markedly protected against acute cigarette smoke – induced lung injury, partly via increased mTOR signaling, and, when exposed chronically, against emphysema. Our data support the notion that Rtp801 may represent an important molecular sensor and mediator of lung injury to cigarette smoke.

No abstract

Aspergillus fumigatus is the most prevalent airborne filamentous fungal pathogen in humans, causing severe and often fatal invasive infections in immunocompromised patients. Currently available antifungal drugs to treat invasive aspergillosis have limited modes of action, and few are safe and effective. To identify and prioritize antifungal drug targets, we have developed a conditional promoter replacement (CPR) strategy using the nitrogen-regulated A. fumigatus NiiA promoter (pNiiA). The gene essentiality for 35 A. fumigatus genes was directly demonstrated by this pNiiA-CPR strategy from a set of 54 genes representing broad biological functions whose orthologs are confirmed to be essential for growth in Candida albicans and Saccharomyces cerevisiae. Extending this approach, we show that the ERG11 gene family (ERG11A and ERG11B) is essential in A. fumigatus despite neither member being essential individually. In addition, we demonstrate the pNiiA-CPR strategy is suitable for in vivo phenotypic analyses, as a number of conditional mutants, including an ERG11 double mutant (erg11BΔ, pNiiA-ERG11A), failed to establish a terminal infection in an immunocompromised mouse model of systemic aspergillosis. Collectively, the pNiiA-CPR strategy enables a rapid and reliable means to directly identify, phenotypically characterize, and facilitate target-based whole cell assays to screen A. fumigatus essential genes for cognate antifungal inhibitors.

Rhizopus oryzae is the most common cause of zygomycosis, a life-threatening infection that usually occurs in patients with diabetic ketoacidosis. Despite standard therapy, the overall rate of mortality from zygomycosis remains >50%, and new strategies for treatment are urgently needed. The activities of caspofungin acetate (CAS) and other echinocandins (antifungal inhibitors of the synthesis of 1,3-␤-D-glucan synthase [GS]) against the agents of zygomycosis have remained relatively unexplored, especially in animal models of infection. We found that R. oryzae has both an FKS gene, which in other fungi encodes a subunit of the GS synthesis complex, and CAS-susceptible, membrane-associated GS activity. Low-dose but not high-dose CAS improved the survival of mice with diabetic ketoacidosis infected with a small inoculum but not a large inoculum of R. oryzae. Fungal burden, assessed by a novel quantitative PCR assay, correlated with increasing inocula and progression of disease, particularly later in the infection, when CFU counts did not. CAS decreased the brain burden of R. oryzae when it was given prophylactically but not when therapy was started after infection. These results indicate that CAS has significant but limited activity against R. oryzae in vivo and demonstrates an inverse dose-response effect. The potential for CAS to play a role in combination therapy against zygomycosis merits further investigation.

Caspofungin acetate (MK-0991) is an antifungal antibiotic that inhibits the synthesis of 1,3-␤-D-glucan, an essential component of the cell wall of several pathogenic fungi. Caspofungin acetate was recently approved for the treatment of invasive aspergillosis in patients who are refractory to or intolerant of other therapies. The activity of 1,3-␤-D-glucan synthesis inhibitors against Aspergillus fumigatus has been evaluated in animal models of pulmonary or disseminated disease by using prolongation of survival or reduction in tissue CFU as assay endpoints. Because these methods suffer from limited sensitivity or poor correlation with fungal growth, we have developed a quantitative PCR-based (qPCR) (TaqMan) assay to monitor disease progression and measure drug efficacy. A. fumigatus added to naïve, uninfected kidneys as either ungerminated conidia or small germlings yielded a linear qPCR response over at least 4 orders of magnitude. In a murine model of disseminated aspergillosis, a burden of A. fumigatus was detected in each of five different organs at 4 days postinfection by the qPCR assay, and the mean fungal load in these organs was 1.2 to 3.5 log 10 units greater than mean values determined by CFU measurement. When used to monitor disease progression in infected mice, the qPCR assay detected an increase of nearly 4 log 10 conidial equivalents/g of kidney between days 1 and 4 following infection, with a peak fungal burden that coincided with the onset of significant mortality. Traditional CFU methodology detected only a marginal increase in fungal load in the same tissues. In contrast, when mice were infected with Candida albicans, which does not form true mycelia in tissues, quantitation of kidney burden by both qPCR and CFU assays was strongly correlated as the infection progressed. Finally, treatment of mice with induced disseminated aspergillosis with either caspofungin or amphotericin B reduced the A. fumigatus burden in infected kidneys to the limit of detection for the qPCR assay. Because of its much larger dynamic range, the qPCR assay is superior to traditional CFU determination for monitoring the progression of disseminated aspergillosis and evaluating the activity of antifungal antibiotics against A. fumigatus.Life-threatening fungal infections have become more prevalent as the population of immunocompromised patients has increased (6, 7). The value of existing therapies is tempered by such factors as a lack of sufficient spectrum, toxic side effects, or emerging resistance (13, 31, 37). Currently there are several antifungal agents in clinical development, including novel triazoles and members of a new class of compounds that inhibit the synthesis of 1,3-␤-D-glucan, an essential polysaccharide of the fungal cell wall. Caspofungin acetate (MK-0991) is the first compound of this class to be approved for therapeutic use. The development of caspofungin for treatment of aspergillosis has been supported by results from animal models of disseminated or pulmonary disease. The efficacy of caspofungin in t...

Protein kinase C (PKC) activation in the heart has been linked to a hypertrophic phenotype and to processes that influence contractile function. To establish whether PKC activation is sufficient to induce an abnormal phenotype, PKC ␤ was conditionally expressed in cardiomyocytes of transgenic mice. Transgene expression in adults caused mild and progressive ventricular hypertrophy associated with impaired diastolic relaxation, whereas expression in newborns caused sudden death associated with marked abnormalities in the regulation of intracellular calcium. Thus, the PKC signaling pathway in cardiocytes has different effects depending on the timing of expression and, in the adult, is sufficient to induce pathologic hypertrophy.

Mechanism-of-action (MOA) studies of bioactive compounds are fundamental to drug discovery. However, in vitro studies alone may not recapitulate a compound's MOA in whole cells. Here, we apply a chemogenomics approach in Candida albicans to evaluate compounds affecting purine metabolism. They include the IMP dehydrogenase inhibitors mycophenolic acid and mizoribine and the previously reported GMP synthase inhibitors acivicin and 6-diazo-5-oxo-L-norleucine (DON). We report important aspects of their whole-cell activity, including their primary target, off-target activity, and drug metabolism. Further, we describe ECC1385, an inhibitor of GMP synthase, and provide biochemical and genetic evidence supporting its MOA to be distinct from acivicin or DON. Importantly, GMP synthase activity is conditionally essential in C. albicans and Aspergillus fumigatus and is required for virulence of both pathogens, thus constituting an unexpected antifungal target.