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We previously reported medicinal chemistry efforts that identified MK-5204, an orally efficacious β-1,3-glucan synthesis inhibitor derived from the natural product enfumafungin. Further extensive optimization of the C2 triazole substituent identified 4-pyridyl as the preferred replacement for the carboxamide of MK-5204, leading to improvements in antifungal activity in the presence of serum, and increased oral exposure. Reoptimizing the aminoether at C3 in the presence of this newly discovered C2 substituent, confirmed that the (R) t-butyl, methyl aminoether of MK-5204 provided the best balance of these two key parameters, culminating in the discovery of ibrexafungerp, which is currently in phase III clinical trials. Ibrexafungerp displayed significantly improved oral efficacy in murine infection models, making it a superior candidate for clinical development as an oral treatment for Candida and Aspergillus infections.

We describe our optimization efforts to improve the physicochemical properties, solubility, and off-target profile of 1, an inhibitor of TarO, an early stage enzyme in the biosynthetic pathway for wall teichoic acid (WTA) synthesis. Compound 1 displayed a TarO IC50 of 125 nM in an enzyme assay and possessed very high lipophilicity (clogP = 7.1) with no measurable solubility in PBS buffer. Structure–activity relationship (SAR) studies resulted in a series of compounds with improved lipophilic ligand efficiency (LLE) consistent with the reduction of clogP. From these efforts, analog 9 was selected for our initial in vivo study, which in combination with subefficacious dose of imipenem (IPM) robustly lowered the bacterial burden in a neutropenic Staphylococci murine infection model. Concurrent with our in vivo optimization effort using 9, we further improved LLE as exemplified by a much more druglike analog 26.

Tedizolid phosphate, the prodrug of the active antibiotic tedizolid, is an oxazolidinone for the treatment of acute bacterial skin and skin structure infections. Studies in a mouse thigh infection model demonstrated that tedizolid has improved potency and pharmacokinetics/pharmacodynamics (PK/PD) compared with those of linezolid. Subsequent studies showed that the efficacy of tedizolid was enhanced in immunocompetent (IC) mice compared with neutropenic (immunosuppressed [IS]) mice, with stasis at clinically relevant doses being achieved only in the presence of granulocytes. The tedizolid label therefore contains a warning about its use in neutropenic patients. This study reevaluated the PK/PD of tedizolid and linezolid in the mouse thigh infection model in IC and IS mice using a methicillin-resistant Staphylococcus aureus (MRSA) strain (ATCC 33591) and a methicillin-susceptible S. aureus (MSSA) strain (ATCC 29213). The antistaphylococcal effect of doses ranging from 1 to 150 mg/kg of body weight tedizolid (once daily) or linezolid (twice daily) was determined at 24, 48, and 72 h after initiating treatment. In IC mice, stasis was achieved in the absence of antibiotics, and both tedizolid and linezolid reduced the burden further beyond a static effect. In IS mice, tedizolid achieved stasis against MRSA ATCC 33591 and MSSA ATCC 29213 at 72 h at a human clinical dose of 200 mg, severalfold lower than that in earlier studies. Linezolid achieved a static effect against MRSA ATCC 33591 in IS mice at a dose lower than that used clinically. This study demonstrates that, with time, both tedizolid and linezolid at clinically relevant exposures achieve stasis in neutropenic mice with an MRSA or MSSA thigh infection.

The clinical success of the echinocandins, which can only be administered parentally, has validated β-1,3-glucan synthase (GS) as an antifungal target. Semi-synthetic modification of enfumafungin, a triterpene glycoside natural product, was performed with the aim of producing a new class of orally active GS inhibitors. Replacement of the C2 acetoxy moiety with various heterocycles did not improve GS or antifungal potency. However, replacement of the C3 glycoside with an aminoether moiety dramatically improved oral pharmacokinetic (PK) properties while maintaining GS and antifungal potency. Installing an aminotetrazole at C2 in conjunction with an N-alkylated aminoether at C3 produced derivatives with significantly improved GS and antifungal potency that exhibited robust oral efficacy in a murine model of disseminated candidiasis.

Current recommendations do not discourage pregnancy in stable, orthotopic heart transplant (OHT) recipients who are more than 1 year posttransplant, although a highly specialized level of care with a multidisciplinary team is recommended. These patients may incur significant risk to themselves, their allograft, and/or their fetuses. Recognition and treatment of posttransplant complications in pregnancy also may be difficult. Although the risk of recurrent pregnancies in patients with peripartum cardiomyopathy (PPCM) is well defined, the risk of pregnancy after a PPCM patient has undergone OHT is unknown. A case of severe allograft rejection in a woman with a history of PPCM who became pregnant nearly 3 years after OHT is presented and her subsequent management is described. The data available on pregnancy after transplant are based on case reports, registry data, and reviews. The decision to become pregnant should be preceded by an extensive discussion including the patient, her partner, her obstetrician, and the transplant team, reviewing potential risks to her health as well as to her allograft and the fetus. Providers should be clear that many questions pertaining to pregnancy after transplant remain unanswered, and the patient should be comfortable going forward in this situation.

Steadily increasing antifungal drug resistance and persistent high rates of fungal-associated mortality highlight the dire need for the development of novel antifungals. Characterization of inhibitors of one enzyme in the GPI anchor pathway, Gwt1, has generated interest in the exploration of targets in this pathway for further study. Utilizing a chemical genomics-based screening platform referred to as the Candida albicans fitness test (CaFT), we have identified novel inhibitors of Gwt1 and a second enzyme in the glycosylphosphatidylinositol (GPI) cell wall anchor pathway, Mcd4. We further validate these targets using the model fungal organism Saccharomyces cerevisiae and demonstrate the utility of using the facile toolbox that has been compiled in this species to further explore target specific biology. Using these compounds as probes, we demonstrate that inhibition of Mcd4 as well as Gwt1 blocks the growth of a broad spectrum of fungal pathogens and exposes key elicitors of pathogen recognition. Interestingly, a strong chemical synergy is also observed by combining Gwt1 and Mcd4 inhibitors, mirroring the demonstrated synthetic lethality of combining conditional mutants of GWT1 and MCD4. We further demonstrate that the Mcd4 inhibitor M720 is efficacious in a murine infection model of systemic candidiasis. Our results establish Mcd4 as a promising antifungal target and confirm the GPI cell wall anchor synthesis pathway as a promising antifungal target area by demonstrating that effects of inhibiting it are more general than previously recognized.

Purpose Cognitive impairment (CI) in heart failure pts. may affect performance of everyday activities that impact ability to manage chronic illness. Anticholinergic drugs are a risk factor for CI. Higher anticholinergic burden (ACB) can occur with specific medications with high anticholinergic activity or an accumulation of drugs with low, medium, and high anticholinergic burden. The ACB of drugs in HF pts. may be a factor contributing to a high prevalence of CI; however, no studies have compared ACB and CI based on severity of HF. This study aims to compare CI and ACB in pts. with Class II vs. Class III/IV HF. Methods 113 adult pts. were recruited from a HF/Transplant Program. Exclusion criteria included diagnosis of dementia, LVAD or total artificial heart. Demographic data were obtained using a self-report questionnaire; clinical data were retrieved using a medical record review. Cognitive function was measured with the MOS Cognitive Function Scale. ACB was calculated using the Anticholinergic Cognitive Burden Scoring Scale. Comorbidity was measured with the Charlson Index. Polypharmacy (>=5 drugs) was determined using a medication count. Data were analyzed using mean (SD) for continuous variables f (%) for categorical variables. Significant differences between groups were examined using Chi Square and independent t-tests. Results Participants (Class II=48, Class III/IV=65) were 62% male, 50% AA, and 56 years old. There were no sig, differences between groups based on EF (p=.116), comorbidity (p=.485), evaluation for transplant (p=.235), IV inotrope therapy (p=.132), or ACB (Class II=2.47 vs Class III/IV-2.67; p=.811). There were between group differences based on age (p=.006), listed for transplant (p=.001), evaluation for MCS (p=.007), ICD (p=.002), polypharmacy ( n=105; Class II=9.5 vs 10.03, p=.034), and CI (Class II=11.06 vs Class III/IV-16.3, p=.035). Conclusion Findings support significant CI and ACB in NYHA Class II-IV pts. with greater CI in Class III/IV pts. Pts. with HF have substantial polypharmacy that may lead to high anticholinergic drug exposure which has been correlated with increased risk for CI. Future studies should target factors influencing CI in patients with greater severity of HF.

Riboswitches are bacterial-specific, broadly conserved, non-coding RNA structural elements that control gene expression of numerous metabolic pathways and transport functions essential for cell growth. As such, riboswitch inhibitors represent a new class of potential antibacterial agents. Recently, we identified ribocil-C, a highly selective inhibitor of the flavin mononucleotide (FMN) riboswitch that controls expression of de novo riboflavin (RF, vitamin B2) biosynthesis in Escherichia coli. Here, we provide a mechanistic characterization of the antibacterial effects of ribocil-C as well as of roseoflavin (RoF), an antimetabolite analog of RF, among medically significant Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA) and Enterococcus faecalis. We provide genetic, biophysical, computational, biochemical, and pharmacological evidence that ribocil-C and RoF specifically inhibit dual FMN riboswitches, separately controlling RF biosynthesis and uptake processes essential for MRSA growth and pathogenesis. Such a dual-targeting mechanism is specifically required to develop broad-spectrum Gram-positive antibacterial agents targeting RF metabolism.

Clostridium difficile causes infections of the colon in susceptible patients. Specifically, gut dysbiosis induced by treatment with broad-spectrum antibiotics facilitates germination of ingested C. difficile spores, expansion of vegetative cells, and production of symptom-causing toxins TcdA and TcdB. The current standard of care for C. difficile infections (CDI) consists of administration of antibiotics such as vancomycin that target the bacterium but also perpetuate gut dysbiosis, often leading to disease recurrence. The monoclonal antitoxin antibodies actoxumab (anti-TcdA) and bezlotoxumab (anti-TcdB) are currently in development for the prevention of recurrent CDI. In this study, the effects of vancomycin or actoxumab/bezlotoxumab treatment on progression and resolution of CDI were assessed in mice and hamsters. Rodent models of CDI are characterized by an early severe phase of symptomatic disease, associated with high rates of morbidity and mortality; high intestinal C. difficile burden; and a disrupted intestinal microbiota. This is followed in surviving animals by gradual recovery of the gut microbiota, associated with clearance of C. difficile and resolution of disease symptoms over time. Treatment with vancomycin prevents disease initially by inhibiting outgrowth of C. difficile but also delays microbiota recovery, leading to disease relapse following discontinuation of therapy. In contrast, actoxumab/bezlotoxumab treatment does not impact the C. difficile burden but rather prevents the appearance of toxin-dependent symptoms during the early severe phase of disease, effectively preventing disease until the microbiota (the body's natural defense against C. difficile ) has fully recovered. These data provide insight into the mechanism of recurrence following vancomycin administration and into the mechanism of recurrence prevention observed clinically with actoxumab/bezlotoxumab.

The widespread emergence of methicillin-resistant Staphylococcus aureus (MRSA) has dramatically eroded the efficacy of current β-lactam antibiotics and created an urgent need for new treatment options. We report an S. aureus phenotypic screening strategy involving chemical suppression of the growth inhibitory consequences of depleting late-stage wall teichoic acid biosynthesis. This enabled us to identify early-stage pathway-specific inhibitors of wall teichoic acid biosynthesis predicted to be chemically synergistic with β-lactams. We demonstrated by genetic and biochemical means that each of the new chemical series discovered, herein named tarocin A and tarocin B, inhibited the first step in wall teichoic acid biosynthesis (TarO). Tarocins do not have intrinsic bioactivity but rather demonstrated potent bactericidal synergy in combination with broad-spectrum β-lactam antibiotics against diverse clinical isolates of methicillin-resistant staphylococci as well as robust efficacy in a murine infection model of MRSA. Tarocins and other inhibitors of wall teichoic acid biosynthesis may provide a rational strategy to develop Gram-positive bactericidal β-lactam combination agents active against methicillin-resistant staphylococci.

To assess cardiovascular risk in both clinical and basic research settings, it is imperative to be able to accurately measure plasma lipid levels. Here, methods commonly used to measure lipoproteins and lipids: ultracentrifugation (UC), fast protein liquid chromatography (FPLC), Roche auto-analyzer, and enzymatic assays were tested and compared. Plasma samples from 20 healthy humans and 22 cynomolgus monkeys were analyzed for their total cholesterol (TC), cholesterol in low density lipoproteins (LDL) and high density lipoproteins (HDL), and triglycerides (TG). Major lipid classes from UC and FPLC separated lipoprotein fractions from human plasma were further characterized by liquid chromatography-mass spectrometry analysis. All the tested methods showed acceptable performance with Roche analyzer among the best in approximate dilution linearity and recovery for most lipids as well as in repeatability between measurements of the same samples. TC, LDL, HDL, and TG values measured in human vs. monkey were-183.9 ± 35.5 (mean ± SD) vs. 105.6 ± 24.6 mg/dl, 106.0 ± 30.1 vs. 42.8 ± 13.0 mg/dl, 50.0 ± 11.4 vs. 53.4 ± 14.8 mg/dl, and 107.6 ± 50.7 vs. 58.0 ± 52.3 mg/dl. While no single method was uniformly the best, we recommend the Roche analyzer for routine measurements. UC or FPLC separation is needed for further functional characterization for specific lipid fraction. We have shown athero-protective profile in cynomolgus monkey compared with humans.

Apolipoprotein B100 (apoB100) and apolipoprotein A1 (apoA1) are the primary protein components of low density lipoprotein (LDL) and high density lipoprotein (HDL) particles, respectively, and plasma levels of these proteins are associated with risks of cardiovascular disease. Existing apoB100 quantitation methods for animal models have been limited to affinity capture techniques such as enzyme-linked immunosorbent assay (ELISA) and Western blot which require specialized reagents for each species and in many cases are not readily available. Here we demonstrate a single translatable ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) assay that is fast and robust and can be used to measure apolipoprotein concentrations in plasma for six species. When possible, peptide sequences that are conserved across species were identified for this assay. The sample preparation is limited and can be carried out in 96-well microtiter plates and thus allows for multiplexed preparation of samples for analysis of large numbers of samples in a short time frame when combined with UPLC/MS/MS. Separation and quantitation of the tryptic peptides is carried out at 700 μL/min using a 1.7 µm core shell C18 column (2.1 × 50 mm). The chromatography is designed for the analysis of over 100 samples per day, and the UPLC run is less than 10 min. This assay is capable of supporting cardiovascular research by providing a single assay to measure critical biomarkers across multiple species without the need for antibodies, and does so in a high-throughput manner.

We investigated the effects of chronic mineralocorticoid receptor blockade with eplerenone on the development and progression of hypertension and end organ damage in Dahl salt-sensitive rats. Eplerenone significantly attenuated the progressive rise in systolic blood pressure (SBP) (204 ± 3 vs. 179±3 mmHg, p < 0.05), reduced proteinuria (605.5 ± 29.6 vs. 479.7 ± 26.1 mg/24h, p < 0.05), improved injury scores of glomeruli, tubules, renal interstitium, and vasculature in Dahl salt-sensitive rats fed a high-salt diet. These results demonstrate that mineralocorticoid receptor antagonism provides target organ protection and attenuates the development of elevated blood pressure (BP) in a model of salt-sensitive hypertension.

Neonatal candidiasis is an increasingly common occurrence causing significant morbidity and mortality and a higher risk of dissemination to the central nervous system (CNS) than that seen with older patients. The current understanding of optimal antifungal therapy in this setting is limited. We have developed a model of disseminated candidiasis with CNS involvement in juvenile mice to assess the efficacy of the echinocandin caspofungin relative to amphotericin B (AmB). Juvenile mice were inoculated intravenously with 5.64 × 10 4 CFU of Candida albicans MY1055. Treatment with caspofungin at 1, 2, 4, and 8 mg/kg of body weight/day, AmB at 1 mg/kg/day, or a vehicle control (VC) was initiated 30 h after infection and continued for 7 days. Pharmacokinetic parameters for caspofungin were also determined. Culture and histology showed evidence of disseminated candidiasis with multifocal encephalitis at the start of antifungal therapy. Survival was 100% in all treated groups, while mortality was 100% in the VC by day 11 after infection. By day 5, all mice in the caspofungin treatment (four doses) groups showed reductions in kidney and brain burden relative to the VC, while AmB treatment reduced kidney burden but gave no reduction of brain fungal burden. Systemic levels of caspofungin were similar in infected and uninfected mice, while brain levels were higher in infected animals. In this juvenile mouse model, caspofungin demonstrated dose-dependent activity, equivalent to or better than that of AmB at 1 mg/kg, against disseminated candidiasis with CNS involvement.

Emerging evidence suggests apolipoprotein B (apoB) and apolipoprotein AI (apoAI) are strong risk predictors for atherosclerosis. Non-human primates (NHP), including rhesus monkeys, cynomolgus monkeys, and African green monkeys, are important preclinical species for studying dyslipidemia and atherosclerosis as they more closely resemble humans in lipid metabolism and disease physiology compared to lower species such as rodents. However, no commercial assays are currently available for measuring apoB and apoAI in NHP. We therefore evaluated analytical methods for routinely measuring apoB and apoAI in our NHP dyslipidemia and atherosclerosis research. Since NHP apoB and apoAI sequences are likely highly similar to human, we focused on the clinically validated and widely utilized human apoB and apoAI immunoturbidity assays. We carried out technical validation of these assays with NHP samples and leveraged orthogonal technical platforms including mass spectrometry, independent ELISA assay, and absolute quantitation via SDS-PAGE for further characterization. Analysis of purified lipoproteins demonstrated that the immunoturbidity assays detect NHP apoAI and apoB, with good dilution linearity and spike recovery from NHP plasma. Orthogonal studies showed apoAI correlated with protein concentration and apoB levels correlated with LC/MS and an independent ELISA. NHP samples from a drug treatment study were analyzed with the immunoturbidity assays and levels of apoB and apoAI fit our understanding of biology and expectations from literature. These studies serve as important technical and biological validation of the immunoturbidity assays for NHP samples, and demonstrate that these assays provide a high-throughput, fully automated analytical platform for NHP samples. Our studies pave the way for future translational research in NHP for developing therapies for treating dyslipidemia and atherosclerosis.

The echinocandin caspofungin is a potent inhibitor of the activity of 1,3-β- d -glucan synthase from Aspergillus flavus , Aspergillus terreus , and Aspergillus nidulans . In murine models of disseminated infection, caspofungin prolonged survival and reduced the kidney fungal burden. Caspofungin was at least as effective as amphotericin B against these filamentous fungi in vivo.

Induction immunosuppression utilizing lymphocytolytic agents in the early peri-operative period has a number of theoretical and practical advantages and disadvantages. However, the efficacy of cytolytic agents as induction therapy remains unproven.To assess the current impact of induction therapy in heart transplantation, we queried a multi-institutional database regarding the frequency of use, type of agent, duration of therapy and outcomes of 6,553 patients transplanted from 1990 to 2001. A study group of 5,897 patients were identified who survived the first 48 hours post-transplant and received either no induction therapy (n = 4,161) or induction with OKT3 or anti-thymocyte preparations (n = 1,736).By multivariate analysis, risk factors for rejection death were identified and then applied to a model of overall mortality. Among patients with a 1-year risk of rejection death at5%, induction therapy provided a survival advantage, but survival with induction was decreased when the risk of rejection death was2%. Specific patient sub-sets that received a survival benefit in the current era with induction included younger patients of black race with/=4 HLA mismatches and long-term (6 months) support on a ventricular assist device (VAD).Use and application of induction therapy continues to be controversial in heart transplantation. At present, this approach appears to be beneficial in selected patients who are at high risk for rejection death, but likely detrimental in patients who are at low risk for rejection death. Those with a combination of longer term VAD support, of black ethnicity, and having extensive HLA mismatching are most likely to benefit from cytolytic induction therapy.

Global University is an international school with a network of offices in 169 countries. The university operates on every continent with students from a vast array of backgrounds, cultures, and learning styles. As an exclusively distance education institution, Global University offers courses by independent study. The mission of the school involves evangelism, discipleship, and training. At any given time, the University has about 475,000 students enrolled in its courses and programs of instruction. The impact of Global University around the world has been enormous, and the future of the school is bright .

The exotoxins TcdA and TcdB are the major virulence factors of Clostridium difficile . Circulating neutralizing antitoxin antibodies are protective in C. difficile infection (CDI), as demonstrated, in part, by the protective effects of actoxumab and bezlotoxumab, which bind to and neutralize TcdA and TcdB, respectively. The question of how systemic IgG antibodies neutralize toxins in the gut lumen remains unresolved, although it has been suggested that the Fc receptor FcRn may be involved in active antibody transport across the gut epithelium. In this study, we demonstrated that genetic ablation of FcRn and excess irrelevant human IgG have no impact on actoxumab-bezlotoxumab-mediated protection in murine and hamster models of CDI, suggesting that Fc-dependent transport of antibodies across the gut wall is not required for efficacy. Tissue distribution studies in hamsters suggest, rather, that the transport of antibodies depends on toxin-induced damage to the gut lining. In an in vitro two-dimensional culture system that mimics the architecture of the intestinal mucosal epithelium, toxins on the apical side of epithelial cell monolayers are neutralized by basolateral antibodies, and antibody transport across the cell layer is dramatically increased upon addition of toxin to the apical side. Similar data were obtained with F(ab′) 2 fragments, which lack an Fc domain, consistent with FcRn-independent paracellular, rather than transcellular, transport of antibodies. Kinetic studies show that initial damage caused by apical toxin is required for efficient neutralization by basolateral antibodies. These data may represent a general mechanism of humoral response-mediated protection against enteric pathogens.

Clostridium difficile is the causative agent of C. difficile -associated diarrhea (CDAD), with increased risk in elderly populations. Kibdelomycin, a novel natural-product inhibitor of type II topoisomerase enzymes, was evaluated for activity against C. difficile and gastrointestinal anaerobic organisms. Toxigenic C. difficile isolates ( n = 168) from U.S. hospitals and anaerobic Gram-positive and Gram-negative organisms ( n = 598) from Chicago-area hospitals were tested. Kibdelomycin showed potent activity against toxigenic C. difficile (MIC 90 = 0.25 μg/ml) and most Gram-positive aerobic organisms but had little activity against Bacteroides species (MIC 50 > 32 μg/ml; n = 270). Potent anti- C. difficile activity was also observed in the hamster model of C. difficile colitis. Dosing at 1.6 mg/kg (twice-daily oral dose) resulted in protection from a lethal infection and a 2-log reduction in C. difficile cecal counts. A 6.25-mg/kg twice-daily oral dose completely eliminated detectable C. difficile counts in cecal contents. A single 6.25-mg/kg oral dose showed that cecal contents were exposed to the drug at >2 μM (eightfold higher than the MIC), with no significant plasma exposure. These findings support further exploration of kibdelomycin for development of an anti- C. difficile agent.

In a screening of natural products with antifungal activity derived from endophytic fungi, we detected a potent activity in a culture belonging to the form-genus Hormonema, isolated from leaves of Juniperus communis. The compound is a new triterpene glycoside, showing an antifungal activity highly potent in vitro against Candida and Aspergillus and with moderate efficacy in an in vivo mouse model of disseminated candidiasis. The agent is especially interesting since its antifungal spectrum and its effect on morphology of Aspergillus fumigatus is comparable to that of the glucan synthase inhibitor pneumocandin B,,, the natural precursor of the clinical candidate MK-0991 (caspofungin acetate). An additional search for other Hormonema isolates producing improved titers or derivatives resulted in the isolation of two more strains recovered from the same plant host showing identical activity. The producing isolates were compared with other non-producing Hormonema strains by DNA fingerprinting and sequencing of the rDNA internal transcribed spacers. Comparison of rDNA sequences with other fungal species suggests that the producing fungus could be an undetermined Kabatina species. Kabatina is a coelomycetous genus whose members are known to produce Hormonema-like states in culture.

The in vivo efficacy of the echinocandin antifungal caspofungin acetate (caspofungin; MK-0991) was evaluated in models of disseminated aspergillosis and candidiasis in mice with cyclophosphamide (CY)-induced immunosuppression. Caspofungin is a 1,3-β- d -glucan synthesis inhibitor efficacious against a number of clinically relevant fungi including Aspergillus and Candida species. Models of CY-induced transient or chronic leukopenia were used with once daily administration of therapy initiated 24 h after microbial challenge. Caspofungin was effective in treating disseminated aspergillosis in mice that were transiently leukopenic (significant prolongation of survival at doses of ≥0.125 mg/kg of body weight and a 50% protective dose [PD 50 ] of 0.245 mg/kg/day at 28 days after challenge) or chronically leukopenic (50 to 100% survival at doses of ≥0.5 mg/kg and PD 50 s ranging from 0.173 to 0.400 mg/kg/day). Caspofungin was effective in the treatment and sterilization of Candida infections in mice with transient leukopenia with a 99% effective dose based on reduction in log 10 CFU of Candida albicans /gram of kidneys of 0.119 mg/kg and 80 to 100% of the caspofungin-treated mice having sterile kidneys at caspofungin doses from 0.25 to 2.0 mg/kg. In Candida -infected mice with chronic leukopenia, caspofungin was effective at all dose levels tested (0.25 to 1.0 mg/kg), with the log 10 CFU of C. albicans /gram of kidneys of caspofungin-treated mice being significantly lower (>99% reduction) than that of sham-treated mice from day 4 to day 28 after challenge. Also, 70 to 100% of the caspofungin-treated, chronic leukopenic mice had sterile kidneys at caspofungin doses of 0.5 to 1.0 mg/kg from day 8 to 28 after challenge. Sterilization of Candida infections by caspofungin in the absence of host leukocytes provides compelling in vivo evidence for fungicidal activity against C. albicans . Further human clinical trials with caspofungin against serious fungal infections are in progress.

A novel oxazolidinone, AM 7359, was evaluated in two mouse models of Staphylococcus aureus infection. AM 7359 and linezolid were equally efficacious in a methicillin-susceptible S. aureus organ burden model and a methicillin-resistant S. aureus localized infection model. However, AM 7359 was eightfold more efficacious than linezolid against a linezolid- and methicillin-resistant S. aureus strain in this localized (thigh) infection model.

Pneumocandins and echinocandins are fungicidal antibiotics, currently in clinical development, that inhibit 1,3-beta-D-glucan synthase (GS) in several human fungal pathogens. We have identified a gene from the diploid organism Candida albicans that encodes a target of these inhibitors. A 2.1-kb portion of this gene, designated CaFKS1, has significant homology to the Saccharomyces cerevisiae FKS1 and FKS2 genes, which encode partially functionally redundant subunits of GS. To evaluate the role of CaFkslp in susceptibility to echinocandins, we disrupted CaFKS1 on one homolog each of the spontaneous pneumocandin-resistant C. albicans mutants CAI4R1, NR2, NR3, and NR4. These mutants had been selected previously on agar plates containing the pneumocandin L-733,560. The clones derived from this transformation were either resistant (Ech[r]) or fully sensitive (Ech[s]) to inhibition by L-733,560 in both liquid broth microdilution and in vitro GS assays. The site of plasmid insertion in the transformants was mapped by Southern blot analysis, using restriction site polymorphisms in the CaFKS1 gene to distinguish between the two alleles (designated CaFKS1h and CaFKS1b). For strains CAI4R1 and NR2, the CaFKS1b allele was disrupted in each Ech(r) transformant; for strain NR4, CaFKS1h was disrupted in each Ech(r) transformant. We conclude that (i) strains CAI4R1, NR2, and NR4 are heterozygous for a dominant or semidominant pneumocandin resistance mutation at CaFKS1, (ii) drug resistance mutations can occur in either CaFKS1 allele, and (iii) CaFks1p is a target of the echinocandins. For transformants of strain NR3, all the clones we analyzed were uniformly Ech(r), and only the CaFKS1h allele, either in disrupted or wild-type form, was detected on genomic Southern blots. We believe gene conversion at the CaFKS1 locus may have produced two Cafks1h alleles that each contain an Ech(r) mutation. Transformants derived from the mutants were analyzed for susceptibility to pneumocandin treatment in a mouse model of disseminated candidiasis. Strains heterozygous for the resistant allele (i.e., C. albicans CAI4R1, NR2, and NR4) were moderately resistant to treatment, while strains without a functional Ech(s) allele (i.e., strain NR3 and derivatives of strain CAI4R1 with the disruption plasmid integrated in the Ech[s] allele) displayed strong in vivo echinocandin resistance. Finally, we were unable to inactivate both alleles at CaFKS1 by two-step integrative disruption, suggesting that CaFks1p is likely to be an essential protein in C. albicans.

The echinocandin MK-0991, formerly L-743,872, is a water-soluble lipopeptide that has been demonstrated in preclinical studies to have potent activity against Candida spp., Aspergillus fumigatus, and Pneumocystis carinii. An extensive in vitro biological evaluation of MK-0991 was performed to better define the potential activities of this novel compound. Susceptibility testing with MK-0991 against approximately 200 clinical isolates of Candida, Cryptococcus neoformans, and Aspergillus isolates was conducted to determine MICs and minimum fungicidal concentrations MF(s). The MFC at which 90% of isolates are inhibited for 40 C. albicans clinical isolates was 0.5 microg/ml. Susceptibility testing with panels of antifungal agent-resistant species of Candida and C. neoformans isolates indicated that the MK-0991 MFCs for these isolates are comparable to those obtained for susceptible isolates. Growth kinetic studies of MK-0991 against Candida albicans and Candida tropicalis isolates showed that the compound exhibited fungicidal activity (i.e., a 99% reduction in viability) within 3 to 7 h at concentrations ranging from 0.06 to 1 microg/ml (0.25 to 4 times the MIC). Drug combination studies with MK-0991 plus amphotericin B found that this combination was not antagonistic against C. albicans, C. neoformans, or A. fumigatus in vitro. Studies with 0 to 50% pooled human or mouse serum established that fungal susceptibility to MK-0991 was not significantly influenced by the presence of human or mouse serum. Results from resistance induction studies suggested that the susceptibility of C. albicans was not altered by repeated exposure (40 passages) to MK-0991. Erythrocyte hemolysis studies with MK-0991 with washed and unwashed human or mouse erythrocytes indicated minimal hemolytic potential with this compound. These favorable results of preclinical studies support further studies with MK-0991 with humans.

Background: A new mouse model of Helicobacter felis infection, which mimics the human infection observed with H. pylori, has recently been developed utilizing polymerase chain reaction (PCR) based on the 16S rRNA gene sequence for detection of infection. Methods: We tested several therapeutic regimens in this model, including some currently utilized in the clinic and some shown ineffective in the clinic. Results: The therapeutic results obtained by PCR with this model are consistent with results observed in the published human H. pylori clinical trials and also with results obtained in another H. felis mouse model utilizing culture and histology. Conclusions: These results support further use of this new model in screening for new therapeutic regimens for the management of Helicobacter disease.

Modern medicine is founded on the discovery of penicillin and subsequent small molecules that inhibit bacterial peptidoglycan (PG) and cell wall synthesis. However, the discovery of new chemically and mechanistically distinct classes of PG inhibitors has become exceedingly rare, prompting speculation that intracellular enzymes involved in PG precursor synthesis are not 'druggable' targets. Here, we describe a β-lactam potentiation screen to identify small molecules that augment the activity of β-lactams against methicillin-resistant Staphylococcus aureus (MRSA) and mechanistically characterize a compound resulting from this screen, which we have named murgocil. We provide extensive genetic, biochemical, and structural modeling data demonstrating both in vitro and in whole cells that murgocil specifically inhibits the intracellular membrane-associated glycosyltransferase, MurG, which synthesizes the lipid II PG substrate that penicillin binding proteins (PBPs) polymerize and cross-link into the cell wall. Further, we demonstrate that the chemical synergy and cidality achieved between murgocil and the β-lactam imipenem is mediated through MurG dependent localization of PBP2 to the division septum. Collectively, these data validate our approach to rationally identify new target-specific bioactive β-lactam potentiation agents and demonstrate that murgocil now serves as a highly selective and potent chemical probe to assist our understanding of PG biosynthesis and cell wall biogenesis across Staphylococcal species.

The pneumocandins are potent antifungal agents of the echinocandin class which are under development for use as broad-spectrum antimycotic therapy. One important consideration for any new therapeutic class for treating serious fungal infections is the potential for drug resistance development. In this study we have isolated and characterized four independent spontaneous Candida albicans mutants resistant to the potent semisynthetic pneumocandin L-733,560. These mutants have many of the properties of FKS1/ETG1 echinocandin-resistant mutants of Saccharomyces cerevisiae, including (i) cross-resistance to other 1,3-beta-D-glucan synthase inhibitors, such as papulacandin and echinocandins, but no change in sensitivity to other antifungal agents; (ii) in vitro glucan synthase activity that is more resistant to pneumocandins than the wild-type parent enzyme; and (iii) semidominant drug resistance in spheroplast fusion strains. The mutants were compared with C. albicans echinocandin-resistant mutants isolated by mutagenesis by L. Beckford and D. Kerridge (mutant M-2) (abstr. PS3.11, in Proceedings of the XI Congress of the International Society for Human and Animal Mycology, Montreal, Canada, 1992) and by A. Cassone, R. E. Mason, and D. Kerridge (mutant CA-2) (Sabouraudia 19:97-110, 1981). All of the strains had resistant enzyme activity in vitro. M-2 grew poorly and had low levels of enzyme activity. In contrast, CA-2 and the spontaneous mutants grew as well as the parents and had normal levels of glucan synthase activity. These results suggest that these resistant mutants may have alterations in glucan synthase. CA-2 was unable to form germ tubes, an ability retained by the spontaneous mutants. The virulence of the spontaneous mutants was unimpaired in a mouse model of disseminated candidiasis, while M-2 and CA-2 were 2 orders of magnitude less virulent than their parent strains. Significantly, mice challenged with the spontaneous mutant CAI4R1 responded therapeutically to lower levels of L-733,560 than would he predicted by the increase in in vitro susceptibility.

Although many detection methods have been used to determine Helicobacter colonization in small animal models, the sensitivity and specificity of these detection methods are limited. To improve the Helicobacter felis conventional mouse model for accurate evaluation of therapeutic regimens, we developed a PCR for detection of, and a competitive PCR for quantitation of, H. felis in viral antibody-free (VAF) mice. The PCR was based on the H. felis 16S rRNA gene. An internal control DNA was used for competitive quantitation of the PCR. VAF conventional Swiss-Webster mice were infected with an H. felis culture by oral gavage. At various times after H. felis challenge and therapy, stomach mucosa was collected and evaluated by PCR. PCR detected approximately 50 to 100 H. felis cells per mouse stomach and showed no cross-reaction with other bacteria commonly found in mouse stomachs. Colonization of H. felis in the mouse stomach was confirmed by culture isolation from germfree mice and histological examination of VAF mice. Response to therapy in this H. felis model correlated well with results seen in human clinical trials with H. pylori. A model utilizing PCR detection which may be useful for discovering new antibiotics and/or vaccines against Helicobacter ulcer disease has been developed.

Summary Innovative strategies are needed to combat drug resistance associated with methicillin-resistant Staphylococcus aureus (MRSA). Here, we investigate the potential of wall teichoic acid (WTA) biosynthesis inhibitors as combination agents to restore β-lactam efficacy against MRSA. Performing a whole-cell pathway-based screen, we identified a series of WTA inhibitors (WTAIs) targeting the WTA transporter protein, TarG. Whole-genome sequencing of WTAI-resistant isolates across two methicillin-resistant Staphylococci spp. revealed TarG as their common target, as well as a broad assortment of drug-resistant bypass mutants mapping to earlier steps of WTA biosynthesis. Extensive in vitro microbiological analysis and animal infection studies provide strong genetic and pharmacological evidence of the potential effectiveness of WTAIs as anti-MRSA β-lactam combination agents. This work also highlights the emerging role of whole-genome sequencing in antibiotic mode-of-action and resistance studies.

Ilicicolin H is a polyketide-nonribosomal peptide synthase (NRPS)-natural product isolated from Gliocadium roseum, which exhibits potent and broad spectrum antifungal activity, with sub-μg/mL MICs against Candida spp., Aspergillus fumigatus, and Cryptococcus spp. It showed a novel mode of action, potent inhibition (IC50 = 2-3 ng/mL) of the mitochondrial cytochrome bc1 reductase, and over 1000-fold selectivity relative to rat liver cytochrome bc1 reductase. Ilicicolin H exhibited in vivo efficacy in murine models of Candida albicans and Cryptococcus neoformans infections, but efficacy may have been limited by high plasma protein binding. Systematic structural modification of ilicicolin H was undertaken to understand the structural requirement for the antifungal activity. The details of the biological activity of ilicicolin H and structural modification of some of the key parts of the molecule and resulting activity of the derivatives are discussed. These data suggest that the β-keto group is critical for the antifungal activity.

A reduction of peripheral CD4+ cell levels has been correlated with the onset of Pneumocystis carinii pneumonia in AIDS patients. Most in vivo drug discovery and development for P. carinii have been conducted in corticosteroid-treated rats. There is need for the development of new small animal models with more selective methods of immunosuppression. This study outlines a new mouse model in which specific depletion of the CD4+ T-lymphocyte population was achieved by subcutaneous injection of G.K1.5 hybridoma cells into C3HeB/FeJ mice. A significant reduction in splenic CD4+ cells was maintained over a 10-week period following a single injection of cells. Circulating anti-CD4+ antibody was detected throughout the 10-week period in hybridoma-injected mice, while circulating antibody was undetectable 4 weeks after repeated injection of purified monoclonal antibody. There was no significant increase in the CD8+ cell populations of the hybridoma-injected mice. P. carinii cysts increased in the lungs of CD4+ T-cell-depleted mice, with the number of cysts detected comparable to levels in dexamethasone-treated mice. High levels of cysts were detected when CD4+ cell populations in the spleen remained below 5% and decreased when CD4+ populations increased above the 5% level. In mice whose CD4+ population was not reduced below 5%, there was no significant increase in P. carinii cysts detected. This study presents a new mouse model with specific immunosuppression requiring a minimum of animal manipulation for use in discovery and development of potential new therapeutics for P. carinii pneumonia.

Apolipoprotein B100 (apoB100) and apolipoprotein A1 (apoA1) are the primary protein components of low density lipoprotein (LDL) and high density lipoprotein (HDL) particles, respectively, and plasma levels of these proteins are associated with risks of cardiovascular disease. Existing apoB100 quantitation methods for animal models have been limited to affinity capture techniques such as enzyme-linked immunosorbent assay (ELISA) and Western blot which require specialized reagents for each species and in many cases are not readily available. Here we demonstrate a single translatable ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) assay that is fast and robust and can be used to measure apolipoprotein concentrations in plasma for six species. When possible, peptide sequences that are conserved across species were identified for this assay. The sample preparation is limited and can be carried out in 96-well microtiter plates and thus allows for multiplexed preparation of samples for analysis of large numbers of samples in a short time frame when combined with UPLC/MS/MS. Separation and quantitation of the tryptic peptides is carried out at 700 μL/min using a 1.7 µm core shell C18 column (2.1 × 50 mm). The chromatography is designed for the analysis of over 100 samples per day, and the UPLC run is less than 10 min. This assay is capable of supporting cardiovascular research by providing a single assay to measure critical biomarkers across multiple species without the need for antibodies, and does so in a high-throughput manner.

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Infections are the major complication among patients on left ventricular device (LVAD) support. One mechanism that may explain the elevated incidence of infection is a deficiency in cellular immunity after LVAD placement. To test this hypothesis, we evaluated the in vitro T-cell-proliferative and cytokine response following challenge with staphylococcal enterotoxin B in 14 LVAD and 6 non-LVAD stable outpatients awaiting heart transplantation. Clinical outcome was followed for 12 months. Clinical outcome was poorer (P.05) for LVAD vs non-LVAD patients. T-cell-proliferative response was lower (P = .002) for LVAD vs non-LVAD patients. Th1 cytokine expression was lower (P.05) and Th2 cytokine expression was higher (P.05) for LVAD vs non-LVAD patients. We conclude that LVAD recipients are hyporesponsive to superantigen challenge, indicating a depressed cellular immunity that may contribute to their increased susceptibility to infections.

An association between reduced susceptibility to echinocandins and changes in the 1,3-β- d -glucan synthase (GS) subunit Fks1p was investigated. Specific mutations in fks1 genes from Saccharomyces cerevisiae and Candida albicans mutants are described that are necessary and sufficient for reduced susceptibility to the echinocandin drug caspofungin. One group of amino acid changes in ScFks1p, ScFks2p, and CaFks1p defines a conserved region (Phe 641 to Asp 648 of CaFks1p) in the Fks1 family of proteins. The relationship between several of these fks1 mutations and the phenotype of reduced caspofungin susceptibility was confirmed using site-directed mutagenesis or integrative transformation. Glucan synthase activity from these mutants was less susceptible to caspofungin inhibition, and heterozygous and homozygous Ca fks1 C. albicans mutants could be distinguished based on the shape of inhibition curves. The C. albicans mutants were less susceptible to caspofungin than wild-type strains in a murine model of disseminated candidiasis. Five Candida isolates with reduced susceptibility to caspofungin were recovered from three patients enrolled in a clinical trial. Four C. albicans strains showed amino acid changes at Ser 645 of CaFks1p, while a single Candida krusei isolate had a deduced R1361G substitution. The clinical C. albicans mutants were less susceptible to caspofungin in the disseminated candidiasis model, and GS inhibition profiles and DNA sequence analyses were consistent with a homozygous fks1 mutation. Our results indicate that substitutions in the Fks1p subunit of GS are sufficient to confer reduced susceptibility to echinocandins in S. cerevisiae and the pathogens C. albicans and C. krusei .

Background: The purposes of this study were to describe long-term (>1 year) cardiac transplant recipients' perceptions of barriers to health-promoting behaviors; ability to manage their health, health-promoting lifestyle, health status and quality of life (QOL); and determine predictors of QOL. Methods: Data were collected from 93 recipients who completed a self-report questionnaire incorporating: (1) patient characteristics; (2) barriers to health promotion, perceived health competence and health-promoting lifestyle; (3) perceived health status; and (4) QOL. All instruments used had acceptable reliability and validity. Objective measures of health status were retrieved from medical records. Data were analyzed using descriptive and comparative statistics, measures of association and multiple regression. Results: Recipients were uncertain about their ability to manage their health; felt that barriers sometimes interfered with engaging in health-promoting behaviors; and often included health-promoting behaviors in their lifestyle. They rated their health as good and were moderately satisfied with life. Predictors of better perceptions of QOL included less education, longer time since transplant, ischemic etiology of heart failure, fewer barriers, higher perceived health competence and a health-promoting lifestyle ( R 2 = 0.51; F = 14.77; p = 0.001). Conclusions: The present results provide information regarding factors that contribute to perceptions of quality of life after cardiac transplantation, have implications for identifying patients at risk for poor QOL, and sensitize clinicians to the need for ongoing patient education.

Deletion of the kexin gene (KEX2) in Candida albicans has a pleiotropic effect on phenotype and virulence due partly to a defect in the expression of two major virulence factors: the secretion of active aspartyl proteinases and the formation of hyphae. kex2/kex2 mutants are highly attenuated in a mouse systemic infection model and persist within cultured macrophages for at least 24 h without causing damage. Pathology is modest, with little disruption of kidney matrix. The infecting mutant cells are largely confined to glomeruli, and are aberrant in morphology. The complex phenotype of the deletion mutants reflects a role for kexin in a wide range of cellular processes. Taking advantage of the specificity of Kex2p cleavage, an algorithm we developed to scan the 9168 open reading frames in Assembly 6 of the C. albicans genome identified 147 potential substrates of Kex2p. These include all previously identified substrates, including eight secreted aspartyl proteinases, the exoglucanase Xog1p, the immunodominant antigen Mp65, and the adhesin Hwp1p. Other putative Kex2p substrates identified include several adhesins, cell wall proteins, and hydrolases previously not implicated in pathogenesis. Kexins also process fungal mating pheromones; a modification of the algorithm identified a putative mating pheromone with structural similarities to Saccharomyces cerevisiae alpha-factor.

Aims: Natural fungal products were screened for antifungal compounds. The mode of action of one of the hits found and the taxonomy of the producing organism were analysed. Methods and Results: An extract from a Trichoderma species showed a more potent activity in an agar-based assay against the null mutant fks1::HIS strain than against the wild-type strain, suggesting that it could contain a glucan synthesis inhibitor. The active component was identified as the known compound ergokonin A. The compound exhibited activity against Candida and Aspergillus species, but was inactive against Cryptococcus species. It induced alterations in the hyphal morphology of Aspergillus fumigatus. The identification of the producing isolate was confirmed by sequencing of the rDNA internal transcribed spacers and comparison with the sequences of other Trichoderma species. The analysis showed that the producing fungus had a high homology with other strains classified as Trichoderma longibrachiatum and its teleomorph Hypocrea schweinitzii. Conclusions: The antifungal activity spectrum of ergokonin A and the morphology alterations induced on A. fumigatus are consistent with glucan synthesis as the target for ergokonin A. The production of ergokonin A is not uncommon, but is probably restricted to Trichoderma species. Significance and Impact of the Study: The discovery that ergokonin A could be an inhibitor of glucan synthesis, having a structure very different to other inhibitors, increases the likelihood that orally active agents with this fungal-specific mode of action may be developed.

The increasing incidence of life-threatening fungal infections has driven the search for new, broad-spectrum fungicidal agents that can be used for treatment and prophylaxis in immunocompromised patients. Natural-product inhibitors of cell wall (1,3)-β- d -glucan synthase such as lipopeptide pneumocandins and echinocandins as well as the glycolipid papulacandins have been evaluated as potential therapeutics for the last two decades. As a result, MK-0991 (caspofungin acetate; Cancidas), a semisynthetic analogue of pneumocandin B o , is being developed as a broad-spectrum parenteral agent for the treatment of aspergillosis and candidiasis. This and other lipopeptide antifungal agents have limited oral bioavailability. Thus, we have sought new chemical structures with the mode of action of lipopeptide antifungal agents but with the potential for oral absorption. Results of natural-product screening by a series of newly developed methods has led to the identification of four acidic terpenoid (1,3)-β- d -glucan synthase inhibitors. Of the four compounds, the in vitro antifungal activity of one, enfumafungin, is comparable to that of L-733560, a close analogue of MK-0991. Like the lipopeptides, enfumafungin specifically inhibits glucan synthesis in whole cells and in (1,3)-β- d -glucan synthase assays, alters the morphologies of yeasts and molds, and produces a unique response in Saccharomyces cerevisiae strains with point mutations in FKS1 , the gene which encodes the large subunit of glucan synthase.