Faculty Bibliography

The adaptor protein SLP76 directs signaling downstream of the T cell receptor (TCR) and is essential for thymocyte development. SLP76 contains three N-terminal tyrosines that are critical for its function. To define the role of these residues in thymocyte development, we generated two lines of "knock-in" mice, one expressing a mutation in tyrosine 145 (Y145F) and a second harboring two point mutations at tyrosines 112 and 128 (Y112-128F). We show here that although thymocyte development requires both Y145- and Y112-128-generated signals, selection was more dependent upon Y145. Although several proximal TCR signaling events were defective in both mutant mice, phosphorylation of the guanine nucleotide exchange factor, Vav1, and activation of Itk-dependent pathways were differentially affected by mutations at Y112-128 and Y145, respectively. Analysis of mice expressing one Y145F and one Y112-128F allele revealed that these mutants could complement one another in trans, demonstrating cooperativity between two or more SLP76 molecules. Thus, the N-terminal tyrosines of SLP76 are required for thymocyte selection but can function on separate molecules to support TCR signaling.

Rapid and reliable detection of mutations at the genetic level is an integral part of modern molecular diagnostics. These mutations can range from dominant single nucleotide polymorphisms within specific loci to codominant heterozygotic insertions and they present considerable challenges to investigators in developing rapid nucleic acid-based amplification assays that can distinguish wild-type from mutant alleles. The recent improvements of real-time polymerase chain reaction (PCR) using self-reporting fluorescence probes have given researchers a powerful tool in developing assays for mutation detection that can be multiplexed for high-throughput screening of multiple mutations and cost effectiveness. Here we describe an application of a multiplexed real-time PCR assay using Molecular Beacon probes for the detection of mutations in codon 54 of the CYP51A gene in Aspergillus fumigatus conferring triazole resistance.

We identified three cases of C. tropicalis strains causing breakthrough fungemia in allogeneic stem cell recipients receiving caspofungin prophylaxis and treatment. Three genetically unrelated isolates with high echinocandin MICs were identified. Each strain carried a characteristic mutation conferring an amino acid substitution within Fks1p hot spot 1.

We have developed an experimental recombinant vesicular stomatitis virus (VSV) vectored plague vaccine expressing a secreted form of Yersinia pestis low calcium response protein V (LcrV) from the first position of the VSV genome. This vector, given intramuscularly in a single dose, induced high-level antibody titers to LcrV and gave 90-100% protection against pneumonic plague challenge in mice. This single-dose protection was significantly better than that generated by VSV expressing the non-secreted LcrV protein. Increased protection correlated with increased anti-LcrV antibody and a bias toward IgG2a and away from IgG1 isotypes. We also found that the depletion of CD4+ cells, but not CD8+ cells, at the time of challenge resulted in reduced vaccine protection, indicating a role for cellular immunity in protection.

The calcineurin pathway is a critical signal transduction pathway in fungi that mediates growth, morphology, stress responses, and pathogenicity. The importance of the calcineurin pathway in fungal physiology creates an opportunity for the development of new antifungal therapies that target this critical signaling pathway. In this study, we examined the role of the zinc finger transcription factor Crz1 homolog (CrzA) in the physiology and pathogenicity of the opportunistic human fungal pathogen Aspergillus fumigatus. Genetic replacement of the crzA locus in A. fumigatus resulted in a strain with significant defects in conidial germination, polarized hyphal growth, cell wall structure, and asexual development that are similar to but with differences from defects seen in the A. fumigatus DeltacnaA (calcineurin A) strain. Like the DeltacnaA strain, the DeltacrzA strain was incapable of causing disease in an experimental persistently neutropenic inhalational murine model of invasive pulmonary aspergillosis. Our results suggest that CrzA is an important downstream effector of calcineurin that controls morphology in A. fumigatus, but additional downstream effectors that mediate calcineurin signal transduction are likely present in this opportunistic fungal pathogen. In addition, the importance of CrzA to the production of disease is critical, and thus CrzA is an attractive fungus-specific antifungal target for the treatment of invasive aspergillosis.

Echinocandins target fungal beta-1,3 glucan synthesis and are used clinically to treat invasive aspergillosis. Although echinocandins do not completely inhibit in vitro growth of Aspergillus fumigatus, they do induce morphological changes in fungal hyphae. Because beta-1,3 glucans activate host antifungal pathways via the Dectin-1 receptor, we investigated the effect of echinocandins on inflammatory responses to A. fumigatus. Caspofungin- or micafungin-treated conidia and germlings induced less secretion of tumor necrosis factor (TNF) and CXCL2 by macrophages than did their untreated counterparts. Diminished secretion of TNF and CXCL2 correlated with diminished beta-glucan exposure on echinocandin-treated germ tubes. In contrast to treated conidia and germlings, echinocandin-treated hyphae stimulated increased release of TNF and CXCL2 by macrophages and demonstrated intense staining with a beta-glucan-specific antibody, particularly at hyphal tips. Our experiments demonstrate that echinocandin-induced morphological changes in A. fumigatus hyphae are accompanied by increased beta-glucan exposure, with consequent increases in Dectin-1-mediated inflammatory responses by macrophages.

BACKGROUND: Human metapneumovirus is a recently discovered RNA virus that typically causes respiratory disease in children. It has been linked to severe lower airway disease in hematopoietic stem cell and solid-organ transplant recipients. hMPV infection in a large population of patients with underlying cancer and varying degrees of immunosuppression has not been reported. We sought to characterize hMPV infection in patients with cancer. METHODS: Review of all cases of hMPV infection from two seasons (2005-6 and 2006-7) detected by DFA and/or real-time PCR at MSKCC, a tertiary cancer center in New York City. RESULTS: Among MSKCC patients with cancer, 51 (2.7%) of 1899 patients were positive for hMPV, including 3.2% with hematologic neoplasm and 1.7% with solid tumors. More children (4.5%) were positive than adults (2.2%). PCR detected twice as many cases as DFA. Cough and fever were common complaints. The longest shedding period was 80 days. 40 patients received radiographic evaluation; of these, 22 showed abnormalities including patchy (11), ground glass (5), and interstitial infiltrates (4). CONCLUSIONS: hMPV causes a nonspecific respiratory illness and was found in more than 2% of all tested persons with cancer. PCR detected substantially more cases than DFA. Unlike previous reports, we observed no fatalities due to hMPV, including 22 HSCT recipients with the infection.

Triazole resistance in Aspergillus fumigatus is an uncommon but rising phenomenon. Susceptibility testing is rarely performed and can take 48 h or longer, which is an impediment to effective therapy. Molecular diagnostic probing of well-defined resistance mechanisms, which serve as surrogate markers, provides an alternative approach to rapidly (within hours) and efficiently identify resistant strains. The mechanisms of triazole resistance in A. fumigatus are limited to amino acid substitutions in the drug target Cyp51A and include amino acid substitutions at the positions Gly 54, Gly 138, Met 220, and Leu 98, coupled with a tandem repetition in the gene promoter. We report the development of a real-time PCR assay utilizing molecular beacons to assess triazole resistance markers in A. fumigatus. When combined in a multiplex platform, the assay provides a comprehensive evaluation of drug resistance in A. fumigatus.

We describe a case of recurring Candida glabrata infection in a 68-year-old African-American female on caspofungin therapy. The initial isolate was susceptible, but isolates recovered during following relapses were not. All isolates were clonal, and high-MIC strains contained a mutation in the highly conserved hot spot 1 region of Fks1p.

We take advantage of an array of hybrid baboons (Papio anubis x Papio hamadryas) living in the same social group to explore the causes and consequences of different male mating strategies. Male hamadryas hold one-male units and exhibit a sustained, intense interest in adult females, regardless of the latter's reproductive state. Anubis baboons, by contrast, live in multi-male, multi-female groups where males compete for females only when the latter are estrous. These two taxa interbreed to form a hybrid zone in the Awash National Park, Ethiopia, where previous work has suggested that hybrid males have intermediate and ineffective behavior. Here, we first examine male mating strategies with respect to morphological and genetic measures of ancestry. We found significant relationships between behavioral measures and morphology; males with more hamadryas-like morphology had more hamadryas-like behavior. However, genetic ancestry was not related to behavior, and in both cases intermediates displayed a previously unreported level of behavioral variation. Furthermore, male behavior was unrelated to natal group. Second, we evaluated reproductive success by microsatellite-based paternity testing. The highest reproductive success was found for individuals exhibiting intermediate behaviors. Moreover, over nine years, some genetically and morphologically intermediate males had high reproductive success. We conclude that the behavior of hybrid males is therefore unlikely to be an absolute barrier to admixture in the region.