Faculty Bibliography

Antifungal efficacies of the echinocandin drugs caspofungin, micafungin, and anidulafungin were reduced significantly in the presence of 50% human serum, which yielded nearly equivalent MICs or minimum effective concentrations against diverse Candida spp. and Aspergillus spp. Consistent with a direct drug interaction, serum decreased the sensitivity of glucan synthase to echinocandin drugs.

Invasive fungal infections cause morbidity and mortality in severely ill patients, and limited drug classes restrict treatment choices. The echinocandin drugs are the first new class of antifungal compounds that target the fungal cell wall by blocking beta-1,3-d-glucan synthase. Elevated MIC values with occasional treatment failure have been reported for strains of Candida. Yet, an uncertain correlation exists between clinical failure and elevated MIC values for the echinocandin drugs. Fungi display several adaptive physiological mechanisms that result in elevated MIC values. However, resistance to echinocandin drugs among clinical isolates is associated with amino acid substitutions in two "hot-spot" regions of Fks1, the major subunit of glucan synthase. The mutations, yielding highly elevated MIC values, are genetically dominant and confer cross-resistance to all echinocandin drugs. Prominent Fks1 mutations decrease the sensitivity of glucan synthase for drug by 1000-fold or more, and strains harboring such mutations may require a concomitant increase in drug to reduce fungal organ burdens in animal infection models. The Fks1-mediated resistance mechanism is conserved in a wide variety of Candida spp. and can account for intrinsic reduced susceptibility of certain species. Fks1 mutations confer resistance in both yeasts and moulds suggesting that this mechanism is pervasive in the fungal kingdom.

An optimal system for monitoring in vivo corneal wound healing is inexpensive, has utility for wounding and imaging, and is able to provide previews before photography. We outline such an imaging system that takes advantage of a consumer digital camera and an LED-based light source for fluorescein excitation. Using FVB/NJ mice, 2mm diameter, circular, axial corneal epithelial defects were created using a crescent blade. The corneal wounds were imaged every four hours until healed using a Nikon Coolpix 5400 camera attached to a Nikon SMZ-10A stereomicroscope, using the illumination from a 16 LED 464nm flashlight. The wound area was calculated, and the linear regressions of the linear phase of wound healing were compared using the F-test. The slopes of the linear regressions for the 6 trials of 4 mice/trial had an average of -52.95microm/h (SEM=0.55microm/h) and were statistically equivalent (p>0.05). The mean of the R(2) values for the linear regressions was 0.9546 (SEM=0.0121). The equivalent linear regressions and R(2)>0.90 suggest that the imaging system could precisely monitor the wound healing of multiple trials and of animals within each trial, respectively. Using a consumer digital camera and LED-based illumination, we have established a system that is economical, is used in both wounding and imaging, is operated by a single person, and is able to provide real-time previews to monitor corneal wound healing precisely

Ras activation as a consequence of antigen receptor (T-cell receptor; TCR) engagement on T lymphocytes is required for T-cell development, selection and function. Lymphocyte function-associated antigen-1 (LFA-1) mediates lymphocyte adhesion, stabilization of the immune synapse and bidirectional signalling. Using a fluorescent biosensor we found that TCR activation with or without costimulation of CD28 led to activation of Ras only on the Golgi apparatus, whereas costimulation with LFA-1 induced Ras activation on both the Golgi and the plasma membrane. Ras activation on both compartments required RasGRP1, an exchange factor regulated by calcium and diacylglycerol (DAG), but phospholipase C (PLC) activity was required only for activation on the Golgi. Engagement of LFA-1 increased DAG levels at the plasma membrane by stimulating phospholipase D (PLD). PLD2 and phosphatidic acid phosphatase (PAP) were required for Ras activation on the plasma membrane. Thus, LFA-1 acts through PLD2 to reshape the pattern of Ras activation downstream of the TCR

The genetic pathways that partition the developing nervous system into functional systems are largely unknown. The engrailed (En) homeobox transcription factors are candidate regulators of this process in the dorsal midbrain (tectum) and anterior hindbrain (cerebellum). En1 mutants lack most of the tectum and cerebellum and die at birth, whereas En2 mutants are viable with a smaller cerebellum and foliation defects. Our previous studies indicated that the difference in phenotypes is due to the earlier expression of En1 as compared with En2, rather than differences in protein function, since knock-in mice expressing En2 in place of En1 have a normal brain. Here, we uncovered a wider spectrum of functions for the En genes by generating a series of En mutant mice. First, using a conditional allele we demonstrate that En1 is required for cerebellum development only before embryonic day 9, but plays a sustained role in forming the tectum. Second, by removing the endogenous En2 gene in the background of En1 knock-in alleles, we show that Drosophila en is not sufficient to sustain midbrain and cerebellum development in the absence of En2, whereas En2 is more potent than En1 in cerebellum development. Third, based on a differential sensitivity to the dose of En1/2, our studies reveal a genetic subdivision of the tectum into its two functional systems and the medial cerebellum into four regions that have distinct circuitry and molecular coding. Our study suggests that an ;engrailed code' is integral to partitioning the tectum and cerebellum into functional domains

BACKGROUND: B-type natriuretic peptide (BNP) released from cardiac myocytes plays an important role in cardiac homeostasis through cyclic guanosine monophosphate (cGMP) activation. BNP also reduces cardiac remodeling and fibrosis. The antifibrotic effects of BNP are mediated in part by blocking the effects of transforming growth factor beta, a profibrotic cytokine that plays a significant role in cutaneous wound healing. It is unclear if BNP plays any role in cutaneous wound healing. OBJECTIVES: To investigate if BNP levels would be elevated in thermally injured human skin and if human-derived fibroblasts would respond to BNP exposure by increasing levels of cGMP. METHODS: This was an in vitro analysis of human skin. Skin samples and cells were collected from patients with and without thermal injury. The authors stained three skin samples from normal skin (taken at the time of elective cosmetic surgery) with antibodies to BNP and compared these with three tissue samples obtained from burned human skin taken during tangential excision of deep burns. Normal human-derived fibroblasts and keratinocytes were exposed in triplicate to BNP in vitro, and cGMP accumulation was evaluated. Levels of cGMP were quantified and compared with analysis of variance. RESULTS: BNP was present in all specimens of thermally injured skin (especially around collagen, epithelial cells, and endothelial cells) but not in any uninjured skin samples (p = 0.05, single-tailed Fisher's exact test). In vitro grown fibroblasts showed significant increases of cGMP levels with increasing levels of BNP exposure (mean [+/-SD]: 0.6 [+/-0.3], 1.2 [+/-0.2], 4.6 [+/-0.1], and 5.0 [+/-0.9] pmol/mL with BNP concentrations of 0, 10, 500, and 1,000 nmol/L, respectively; p < 0.001). The effect of BNP on keratinocytes was minimal and below the level of quantification. CONCLUSIONS: These findings demonstrate proof of principle that human fibroblasts are responsive to the effects of BNP in vitro and that BNP is present in injured skin, suggesting that BNP may play a role in cutaneous wound healing.

Recent studies have demonstrated that the LIM homeodomain transcription factor Islet1 (Isl1) marks pluripotent cardiovascular progenitor cells and is required for proliferation, survival, and migration of recently defined second heart field progenitors. Factors that are upstream of Isl1 in cardiovascular progenitors have not yet been defined. Here we demonstrate that beta-catenin is required for Isl1 expression in cardiac progenitors, directly regulating the Isl1 promoter. Ablation of beta-catenin in Isl1-expressing progenitors disrupts multiple aspects of cardiogenesis, resulting in embryonic lethality at E13. beta-Catenin is also required upstream of a number of genes required for pharyngeal arch, outflow tract, and/or atrial septal morphogenesis, including Tbx2, Tbx3, Wnt11, Shh, and Pitx2. Our findings demonstrate that beta-catenin signaling regulates proliferation and survival of cardiac progenitors.

For more than 50 years, investigators have unsuccessfully tried to recreate in experimental animals the cardiovascular complications of diabetes seen in humans. In particular, accelerated atherosclerosis and dilated cardiomyopathy, the major causes of mortality in patients with diabetes, have been conspicuously absent in many mouse models of the disease. Under the auspices of the NIH, the Animal Models of Diabetic Complications Consortium has worked to address this issue. This effort has focused on the development of mouse models because of the high level of genomic information available and the many well-developed genetic manipulations that may be performed in mice. Importantly, the consortium has also worked to standardize many methods to assess metabolic and cardiovascular end points for measurement of the diabetic state and its macrovascular complications. Finally, for maximum benefits from these animal models in the study of atherosclerosis and of other diabetic complications, the consortium has created a system for sharing both the animal models and the accumulated phenotypic data with the greater scientific community.

The integrated stress response (ISR) is defined as a highly conserved response to several stresses that converge to the induction of the activating transcription factor 4 (ATF4). Because an uncontrolled response may have deleterious effects, cells have elaborated several negative feedback loops that attenuate the ISR. In the present study, we describe how induction of the human homolog of Drosophila tribbles (TRB3) attenuates the ISR by a negative feedback mechanism. To investigate the role of TRB3 in the control of the ISR, we used the regulation of gene expression by amino acid limitation as a model. The enhanced production of ATF4 upon amino acid starvation results in the induction of a large number of target genes like CHOP (CAAT/enhancer-binding protein-homologous protein), asparagine synthetase (ASNS), or TRB3. We demonstrate that TRB3 overexpression inhibits the transcriptional induction of CHOP and ASNS whereas TRB3 silencing induces the expression of these genes both under normal and stressed conditions. In addition, transcriptional profiling experiments show that TRB3 affects the expression of many ISR-regulated genes. Our results also suggest that TRB3 and ATF4 belong to the same protein complex bound to the sequence involved in the ATF4-dependent regulation of gene expression by amino acid limitation. Collectively, our data identify TRB3 as a negative feedback regulator of the ATF4-dependent transcription and participates to the fine regulation of the ISR.

STUDY DESIGN: In vitro assessment of the effects of recombinant human osteogenic protein-1 (rhOP-1) on the proteoglycan metabolism of human intervertebral disc cells. OBJECTIVES: To determine whether rhOP-1 is effective in stimulating the cell proliferation and proteoglycan metabolism of human intervertebral disc cells cultured in alginate beads. SUMMARY OF THE BACKGROUND DATA: OP-1 has been shown to stimulate the proteoglycan and collagen synthesis of rabbit intervertebral disc cells in vitro. In vivo, a single injection of rhOP-1 restored the disc height of a degenerated disc in the rabbit anular-puncture model. The effect of rhOP-1 on human intervertebral disc cells remains unknown. METHODS: Human nucleus pulposus and anulus fibrosus cells were isolated from the discs of 4 cadaveric spines and one surgical specimen. After preculture for 7 days, alginate beads containing nucleus pulposus and anulus fibrosus cells were cultured for 21 days in media containing 10% fetal bovine serum with 0, 100, or 200 ng/mL rhOP-1 and supplements. The synthesis and accumulation of proteoglycans and the DNA content were biochemically assessed. RESULTS: The addition of rhOP-1 to the media resulted in the prevention of a decreased cell number during culture. Treatment with rhOP-1, compared with the control condition (10% fetal bovine serum), significantly upregulated proteoglycan synthesis and accumulation in alginate beads in all cases tested. A longer exposure over 14 days to rhOP-1 resulted in a pronounced response. The retention of newly-synthesized proteoglycan was higher in the rhOP-1-treated cells than in the control. CONCLUSIONS: rhOP-1 was effective in stimulating the cell proliferation and proteoglycan metabolism of human intervertebral disc cells in vitro. The results supported the hypothesis that an in vivo injection of rhOP-1 may increase the metabolic activity of disc cells or prevent apoptosis of disc cells in a degenerated disc. However, the requirement for a long exposure to rhOP-1 for human cells may suggest the need for a prolonged supply of rhOP-1 by a drug delivery system or by repeated injections.