Showing papers by "Kettering University published in 2005"

Cilia and Hedgehog responsiveness in the mouse.

Abstract: The intraflagellar transport (IFT) proteins Ift172/Wimple and Polaris/Ift88 and the anterograde IFT motor kinesin-II are required for the production and maintenance of cilia. These proteins are also required for the activation of targets of the mouse Hedgehog (Hh) pathway by Gli transcription factors. The phenotypes of the IFT mutants, however, are not identical to mutants that lack Smoothened (Smo), an essential activator of the Hh pathway. We show here that mouse embryos that lack both Ift172 and Smo are identical to Ift172 single mutants, which indicates that Ift172 acts downstream of Smo. Ift172 mutants have a weaker neural patterning phenotype than Smo mutants, because Ift172, but not Smo, is required for proteolytic processing of Gli3 to its repressor form. Dnchc2 and Kif3a, essential subunits of the retrograde and anterograde IFT motors, are also required for both formation of Gli activator and proteolytic processing of Gli3. As a result, IFT mutants display a loss of Hh signaling phenotype in the neural tube, where Gli activators play the major role in pattern formation, and a gain of Hh signaling phenotype in the limb, where Gli3 repressor plays the major role. Because both anterograde and retrograde IFT are essential for positive and negative responses to Hh, and because cilia are present on Hh responsive cells, it is likely that cilia act as organelles that are required for all activity of the mouse Hh pathway.

Acquired Resistance to Imatinib in Gastrointestinal Stromal Tumor Occurs Through Secondary Gene Mutation

Abstract: Most gastrointestinal stromal tumors (GIST) have an activating mutation in either KIT or PDGFRA. Imatinib is a selective tyrosine kinase inhibitor and achieves a partial response or stable disease in about 80% of patients with metastatic GIST. It is now clear that some patients with GIST develop resistance to imatinib during chronic therapy. To identify the mechanism of resistance, we studied 31 patients with GIST who were treated with imatinib and then underwent surgical resection. There were 13 patients who were nonresistant to imatinib, 3 with primary resistance, and 15 with acquired resistance after initial benefit from the drug. There were no secondary mutations in KIT or PDGFRA in the nonresistant or primary resistance groups. In contrast, secondary mutations were found in 7 of 15 (46%) patients with acquired resistance, each of whom had a primary mutation in KIT exon 11. Most secondary mutations were located in KIT exon 17. KIT phosphorylation was heterogeneous and did not correlate with clinical response to imatinib or mutation status. That acquired resistance to imatinib in GIST commonly occurs via secondary gene mutation in the KIT kinase domain has implications for strategies to delay or prevent imatinib resistance and to employ newer targeted therapies.

Insights into E3 ligase activity revealed by a SUMO–RanGAP1–Ubc9–Nup358 complex

Abstract: SUMO-1 (for small ubiquitin-related modifier) belongs to the ubiquitin (Ub) and ubiquitin-like (Ubl) protein family. SUMO conjugation occurs on specific lysine residues within protein targets, regulating pathways involved in differentiation, apoptosis, the cell cycle and responses to stress by altering protein function through changes in activity or cellular localization or by protecting substrates from ubiquitination. Ub/Ubl conjugation occurs in sequential steps and requires the concerted action of E2 conjugating proteins and E3 ligases. In addition to being a SUMO E3, the nucleoporin Nup358/RanBP2 localizes SUMO-conjugated RanGAP1 to the cytoplasmic face of the nuclear pore complex by means of interactions in a complex that also includes Ubc9, the SUMO E2 conjugating protein. Here we describe the 3.0-A crystal structure of a four-protein complex of Ubc9, a Nup358/RanBP2 E3 ligase domain (IR1-M) and SUMO-1 conjugated to the carboxy-terminal domain of RanGAP1. Structural insights, combined with biochemical and kinetic data obtained with additional substrates, support a model in which Nup358/RanBP2 acts as an E3 by binding both SUMO and Ubc9 to position the SUMO-E2-thioester in an optimal orientation to enhance conjugation.

Derivation of multipotent mesenchymal precursors from human embryonic stem cells.

Abstract: Background Human embryonic stem cells provide access to the earliest stages of human development and may serve as a source of specialized cells for regenerative medicine. Thus, it becomes crucial to develop protocols for the directed differentiation of embryonic stem cells into tissue-restricted precursors. Methods and Findings Here, we present culture conditions for the derivation of unlimited numbers of pure mesenchymal precursors from human embryonic stem cells and demonstrate multilineage differentiation into fat, cartilage, bone, and skeletal muscle cells. Conclusion Our findings will help to elucidate the mechanism of mesoderm specification during embryonic stem cell differentiation and provide a platform to efficiently generate specialized human mesenchymal cell types for future clinical applications.

Friction and torque govern the relaxation of DNA supercoils by eukaryotic topoisomerase IB

Abstract: permits removal of supercoils. Using real-time single-molecule observation, we show that TopIB releases supercoils by a swivel mechanism that involves friction between the rotating DNA and the enzyme cavity: that is, the DNA does not freely rotate. Unlike a nicking enzyme, TopIB does not release all the supercoils at once, but it typically does so in multiple steps. The number of supercoils removed per step follows an exponential distribution. The enzyme is found to be torque-sensitive, as the mean number of supercoils per step increases with the torque stored in the DNA. We propose a model for topoisomerization in which the torque drives the DNA rotation over a rugged periodic energy landscape in which the topoisomerase has a small but quantifiable probability to religate the DNA once per turn. Type IB topoisomerases alter DNA topology by cleaving and rejoining one strand of the DNA duplex 1 , and are able to remove both positive and negative supercoils. In vivo, TopIB removes positive supercoils generated in advance of the replication fork 9 . Cleavage occurs via a transesterification reaction in which the scissile phosphodiester is attacked by a tyrosine of the enzyme, resulting in the formation of a DNA-(3 0

Structures of the SUMO E1 provide mechanistic insights into SUMO activation and E2 recruitment to E1

Abstract: E1 enzymes facilitate conjugation of ubiquitin and ubiquitin-like proteins through adenylation, thioester transfer within E1, and thioester transfer from E1 to E2 conjugating proteins. Structures of human heterodimeric Sae1/Sae2-Mg·ATP and Sae1/Sae2-SUMO-1-Mg·ATP complexes were determined at 2.2 and 2.75 A resolution, respectively. Despite the presence of Mg·ATP, the Sae1/Sae2-SUMO-1-Mg·ATP structure reveals a substrate complex insomuch as the SUMO C-terminus remains unmodified within the adenylation site and 35 A from the catalytic cysteine, suggesting that additional changes within the adenylation site may be required to facilitate chemistry prior to adenylation and thioester transfer. A mechanism for E2 recruitment to E1 is suggested by biochemical and genetic data, each of which supports a direct role for the E1 C-terminal ubiquitin-like domain for E2 recruitment during conjugation.

Mechanism of nonhomologous end-joining in mycobacteria: a low-fidelity repair system driven by Ku, ligase D and ligase C.

Abstract: DNA double-strand breaks (DSBs) can be repaired either via homologous recombination (HR) or nonhomologous end-joining (NHEJ). Both pathways are operative in eukaryotes, but bacteria had been thought to rely on HR alone. Here we provide direct evidence that mycobacteria have a robust NHEJ pathway that requires Ku and a specialized polyfunctional ATP-dependent DNA ligase (LigD). NHEJ of blunt-end and complementary 5'-overhang DSBs is highly mutagenic ( approximately 50% error rate). Analysis of the recombination junctions ensuing from individual NHEJ events highlighted the participation of several DNA end-remodeling activities, including template-dependent fill-in of 5' overhangs, nontemplated addition of single nucleotides at blunt ends, and nucleolytic resection. LigD itself has the template-dependent and template-independent polymerase functions in vitro that compose the molecular signatures of NHEJ in vivo. Another ATP-dependent DNA ligase (LigC) provides a backup mechanism for LigD-independent error-prone repair of blunt-end DSBs. We speculate that NHEJ allows mycobacteria to evade genotoxic host defense.

Stretch-associated injury in cervical spondylotic myelopathy: new concept and review.

Abstract: The simple pathoanatomic concept that a narrowed spinal canal causes compression of the enclosed cord, leading to local tissue ischemia, injury, and neurological impairment, fails to explain the entire spectrum of clinical findings observed in cervical spondylotic myelopathy A growing body of evidence indicates that spondylotic narrowing of the spinal canal and abnormal or excessive motion of the cervical spine results in increased strain and shear forces that cause localized axonal injury within the spinal cord During normal motion, significant axial strains occur in the cervical spinal cord At the cervicothoracic junction, where flexion is greatest, the spinal cord stretches 24% of its length This causes local spinal cord strain In the presence of pathological displacement, strain can exceed the material properties of the spinal cord and cause transient or permanent neurological injury Stretch-associated injury is now widely accepted as the principal etiological factor of myelopathy in experimental models of neural injury, tethered cord syndrome, and diffuse axonal injury Axonal injury reproducibly occurs at sites of maximal tensile loading in a well-defined sequence of intracellular events: myelin stretch injury, altered axolemmal permeability, calcium entry, cytoskeletal collapse, compaction of neurofilaments and microtubules, disruption of anterograde axonal transport, accumulation of organelles, axon retraction bulb formation, and secondary axotomy Stretch and shear forces generated within the spinal cord seem to be important factors in the pathogenesis of cervical spondylotic myelopathy

The first cleavage of the mouse zygote predicts the blastocyst axis

Abstract: One of the unanswered questions in mammalian development is how the embryonic-abembryonic axis of the blastocyst is first established. It is possible that the first cleavage division contributes to this process, because in most mouse embryos the progeny of one two-cell blastomere primarily populate the embryonic part of the blastocyst and the progeny of its sister populate the abembryonic part. However, it is not known whether the embryonic-abembryonic axis is set up by the first cleavage itself, by polarity in the oocyte that then sets the first cleavage plane with respect to the animal pole, or indeed whether it can be divorced entirely from the first cleavage and established in relation to the animal pole. Here we test the importance of the orientation of the first cleavage by imposing an elongated shape on the zygote so that the division no longer passes close to the animal pole, marked by the second polar body. Non-invasive lineage tracing shows that even when the first cleavage occurs along the short axis imposed by this experimental treatment, the progeny of the resulting two-cell blastomeres tend to populate the respective embryonic and abembryonic parts of the blastocyst. Thus, the first cleavage contributes to breaking the symmetry of the embryo, generating blastomeres with different developmental characteristics.

Analysis of mouse embryonic patterning and morphogenesis by forward genetics

Abstract: Many aspects of the genetic control of mammalian embryogenesis cannot be extrapolated from other animals. Taking a forward genetic approach, we have induced recessive mutations by treatment of mice with ethylnitrosourea and have identified 43 mutations that affect early morphogenesis and patterning, including 38 genes that have not been studied previously. The molecular lesions responsible for 14 mutations were identified, including mutations in nine genes that had not been characterized previously. Some mutations affect vertebrate-specific components of conserved signaling pathways; for example, at least five mutations affect previously uncharacterized regulators of the Sonic hedgehog (Shh) pathway. Approximately half of all of the mutations affect the initial establishment of the body plan, and several of these produce phenotypes that have not been described previously. A large fraction of the genes identified affect cell migration, cellular organization, and cell structure. The findings indicate that phenotype-based genetic screens provide a direct and unbiased method to identify essential regulators of mammalian development.

Normal Functional Capacity in Circulating Myeloid and Plasmacytoid Dendritic Cells in Patients with Chronic Hepatitis C

Abstract: Initial reports analyzing dendritic cell (DC) function in patients with hepatitis C virus (HCV) infection have been controversial. Here, we enumerate and characterize the function of circulating myeloid and plasmacytoid DCs. The results show lower percentages of myeloid DCs (0.62 vs. 0.83; P = .05) and plasmacytoid DCs (0.11 vs. 0.34; P = .004) in patients with chronic HCV infection than in healthy, non-HCV-infected individuals. Despite the lower numbers of circulating myeloid DCs present, no phenotypic or functional defects were identified. The lower percentage of plasmacytoid DCs resulted in decreased absolute interferon (IFN)-alpha production; however, when analyzed on a per-cell basis, plasmacytoid DCs from HCV-infected patients generated levels of IFN-alpha equivalent to those generated by DCs from healthy, non-HCV-infected individuals. Contrary to data from previous models (which attributed HCV pathogenesis to defects in the DC compartment), our data reveal functional DC subsets in patients with chronic HCV infection. These results are encouraging for DC-based HCV immunotherapy trials.

Live attenuated yellow fever 17D infects human DCs and allows for presentation of endogenous and recombinant T cell epitopes

Abstract: The yellow fever (YF) 17D vaccine is one of the most successful live attenuated vaccines available. A single immunization induces both long-lasting neutralizing antibody and YF-specific T cell responses. Surprisingly, the mechanism for this robust immunity has not been addressed. In light of several recent reports suggesting flavivirus interaction with dendritic cells (DCs), we investigated the mechanism of YF17D interaction with DCs and the importance of this interaction in generating T cell immunity. Our results show that YF17D can infect immature and mature human DCs. Viral entry is Ca(2+) dependent, but it is independent of DC-SIGN as well as multiple integrins expressed on the DC surface. Similar to infection of cell lines, YF infection of immature DCs is cytopathic. Although infection itself does not induce DC maturation in vitro, TNF-alpha-induced maturation protects DCs from YF-induced cytopathogenicity. Furthermore, we show that DCs infected with YF17D or YF17D carrying a recombinant epitope can process and present antigens for CD8(+) T cell stimulation. These findings offer insight into the immunologic mechanisms associated with the highly capable YF17D vaccine that may guide effective vaccine design.

Total synthesis of salinosporamide A.

Abstract: Total synthesis of potent proteasome inhibitor salinosporamide A (1) has been accomplished, which features strictly substrate-controlled operations starting with the only chiral center of (R)-pyroglutamic acid. The consecutive quaternary carbons within 1 have been efficiently constructed by manipulation of two intramolecular reactions: (1) carbonate-mediated internal acylation of imidate ester (4 --> 14) and (2) selenocyclization of aldehyde to exocyclic methylene group (5 --> 18).

On the remarkable antitumor properties of fludelone: how we got there.

Abstract: Small-molecule natural products are presumably often biosynthesized with a view to optimizing their ability to bind to strategic proteins or other biomolecular targets. Although the ultimate setting in which a drug must function may be very different, the use of such natural products as lead compounds can serve as a significant head start in the hunt for new agents of clinical value. Herein we reveal the synergistic relationship between chemical synthesis and drug optimization in the context of our research program around the epothilones: how synthesis led to the discovery of more-potent epothilone derivatives, and discovery inspired the development of new synthetic routes, thus demonstrating the value of target-directed total synthesis in the quest for new substances of material clinical benefit.

Metalloprotease‐disintegrin ADAM8: Expression analysis and targeted deletion in mice

Abstract: ADAM8 (a disintegrin and metalloprotease 8, also referred to as MS2/CD156a) is a membrane-anchored metalloprotease that was first identified in a macrophage cell line and has been implicated in neurodegenerative diseases. Here, we evaluated the expression of ADAM8 during mouse development and generated mice lacking ADAM8 (Adam8-/- mice). During early mouse development, ADAM8 is expressed by maternal cells in the decidua and by trophoblast derivatives of the embryo but not in the derivatives of the inner cell mass. At later stages, prominent expression of ADAM8 is seen in the embryo proper, in the gonadal ridge, thymus, developing cartilage and bone, brain and spinal cord, and in the mesenchyme in close proximity to the branch point between the jugular vein and developing lymphatic vessels. Examination of Adam8-/- mice, however, revealed no major defects in these or other structures during development or in adult tissues and no evident pathological phenotypes.

Mycobacterium tuberculosis appears to lack α-ketoglutarate dehydrogenase and encodes pyruvate dehydrogenase in widely separated genes

Abstract: Summary Mycobacterium tuberculosis (Mtb) persists for prolonged periods in macrophages, where it must adapt to metabolic limitations and oxidative/nitrosative stress. However, little is known about Mtb's intermediary metabolism or antioxidant defences. We recently identified a peroxynitrite reductase-peroxidase complex in Mtb that included products of the genes sucB and lpd, which are annotated to encode the dihydrolipoamide succinyltransferase (E2) and lipoamide dehydrogenase (E3) components of α-ketoglutarate dehydrogenase (KDH). However, we could detect no KDH activity in Mtb lysates, nor could we reconstitute KDH by combining the recombinant proteins SucA (annotated as the E1 component of KDH), SucB and Lpd. We therefore renamed the sucB product dihydrolipoamide acyltransferase (DlaT). Mtb lysates contained pyruvate dehydrogenase (PDH) activity, which was lost when the dlaT gene (formerly, sucB) was disrupted. Purification of PDH from Mtb yielded AceE, annotated as an E1 component of PDH, along with DlaT and Lpd. Moreover, anti-DlaT antibody coimmunoprecipitated AceE. Finally, recombinant AceE, DlaT and Lpd, although encoded by genes that are widely separated on the chromosome, reconstituted PDH in vitro with Km values typical of bacterial PDH complexes. In sum, Mtb appears to lack KDH. Instead, DlaT and Lpd join with AceE to constitute PDH.

Regulation of Mycobacterium tuberculosis cell envelope composition and virulence by intramembrane proteolysis

Abstract: Mycobacterium tuberculosis infection is a continuing global health crisis that kills 2 million people each year. Although the structurally diverse lipids of the M. tuberculosis cell envelope each have non-redundant roles in virulence or persistence, the molecular mechanisms regulating cell envelope composition in M. tuberculosis are undefined. In higher eukaryotes, membrane composition is controlled by site two protease (S2P)-mediated cleavage of sterol regulatory element binding proteins, membrane-bound transcription factors that control lipid biosynthesis. S2P is the founding member of a widely distributed family of membrane metalloproteases that cleave substrate proteins within transmembrane segments. Here we show that a previously uncharacterized M. tuberculosis S2P homologue (Rv2869c) regulates M. tuberculosis cell envelope composition, growth in vivo and persistence in vivo. These results establish that regulated intramembrane proteolysis is a conserved mechanism controlling membrane composition in prokaryotes and show that this proteolysis is a proximal regulator of cell envelope virulence determinants in M. tuberculosis.

Genetic and spectrally distinct in vivo imaging: embryonic stem cells and mice with widespread expression of a monomeric red fluorescent protein

Abstract: DsRed the red fluorescent protein (RFP) isolated from Discosoma sp. coral holds much promise as a genetically and spectrally distinct alternative to green fluorescent protein (GFP) for application in mice. Widespread use of DsRed has been hampered by several issues resulting in the inability to establish and maintain lines of red fluorescent protein expressing embryonic stem cells and mice. This has been attributed to the non-viability, or toxicity, of the protein, probably as a result of its obligate tetramerization. A mutagenesis approach directing the stepwise evolution of DsRed has produced mRFP1, the first true monomer. mRFP1 currently represents an attractive autofluorescent reporter for use in heterologous systems. We have used embryonic stem cell-mediated transgenesis to evaluate mRFP1 in embryonic stem cells and mice. We find that mRFP1 exhibits the most spatially homogenous expression when compared to the native (tetrameric) and variant dimeric forms of DsRed. High levels of mRFP1 expression do not affect cell morphology, developmental potential or viability and fertility of animals. High levels of widespread mRFP1 expression are maintained in a constitutive manner in embryonic stem cells in culture and in transgenic animals. We have used various optical imaging modalities to visualize mRFP1 expressing cells in culture, in embryos and adult mice. Moreover co-visualization of red, green and cyan fluorescent cells within a sample is easily achieved without the need for specialized methodologies, such as spectral deconvolution or linear unmixing. Fluorescent proteins with excitation and/or emission profiles in the red part of the visible spectrum represent distinct partners, or longer wavelength substitutes for GFP. Not only do DsRed-based RFPs provide a genetically and spectrally distinct addition to the available repertoire of autoflorescent proteins, but by virtue of their spectral properties they permit deeper tissue imaging. Our work in generating CAG::mRFP1 transgenic ES cells and mice demonstrates the developmental neutrality of mRFP1 in an organismal context. It paves the way for the use of DsRed-based monomeric RFPs in transgenic and gene targeted approaches which often necessitate spatially and/or temporally restricted reporter expression. Moreover animals of the CAG::mRFP1 transgenic strain serve as a source of RFP tagged tissue for the derivation of cell lines and explant, transplant and embryo chimera experiments.

Jet entrainment rate in air curtain of open refrigerated display cases

Abstract: Experimental and computational methods are used to address those parameters that have significant effects on the amount of entrained (warm) air in an open refrigerated display case. These parameters are identified, quantified, and the rate of entrainment is expressed as a function of these parameters. It is found that the turbulence intensity, the shape of the mean velocity profile at the discharge air grill, and the Reynolds number are mainly responsible for the amount of entrained air in a display case. It is also concluded that lower Reynolds numbers will reduce the amount of the entrained air in the display case, however, the trade off will be higher temperature of the food products on shelves. Digital particle image velocimetry (DPIV) was used to map the entire mean velocity flowfield and the turbulence intensity. The laser doppler velocimetry technique was also used to verify the mean velocity and turbulence intensity measurements made by DPIV. The results indicated an excellent agreement between both methods. Parametric studies for the rate of entrainment of the outside air into the display case were performed using a computational fluid dynamics tool. The results indicate that lowering the Reynolds number of the air curtain reduces the entrainment rate. However, sufficiently high momentum should still exist to enforce the integrity of the air curtain structure.

Control of Flow Past a Wing Section with Plasma-based Body Forces

Abstract: : The response of the flow past a stalled NACA 0015 airfoil at 15 deg angle of attack and Reynolds number of 45, 000 to body forces originating from radio-frequency asymmetric dielectric-barrier-discharge actuators is described via direct numerical simulations. The theoretical model couples a phenomenologically derived averaged body force with a high-order 3-D compressible Navier-Stokes solver. The body force distribution is assumed to vary linearly, diminishing away from the surface until the critical electric field limit is reached. Various magnitudes and orientations of the force field are explored, ranging from vertically upwards (away from the body) to vertically downwards (towards the body). The imposed body forces couple to the non-linear inertial terms and the pressure gradients to engender a complex sequence of events. A significant streamwise component assures the reduction or elimination of stall with the formation of a stable wall-jet. When the only component of the force vector is pointed normal to and away from the surface, no control effect is achieved. On the other hand, when the force vector is directed towards the surface, a shallower separation region is observed, accompanied by unsteady boundary layer development. At the low Mach number considered (0.1), the work done by the force has little impact on the solution, and density variations remain less than 5%. Relaxation effects are explored by abruptly switching off the force, and estimates of response times are noted. The lack of a proper spanwise breakdown mechanism for the separated shear layer in 2-D simulations results in large coherent structures, whose response in transient and unsteady asymptotic states differ significantly from those observed in 3-D. Nonetheless, if the force is sufficiently effective to eliminate separation, the flowfield becomes generally two-dimensional and steady in the vicinity of the airfoil, and the overall results from 2-D and 3-D analyses yield similar results.

Gli3 and Plzf cooperate in proximal limb patterning at early stages of limb development

Abstract: The vertebrate limb initially develops as a bud of mesenchymal cells that subsequently aggregate in a proximal to distal (P-D) sequence to give rise to cartilage condensations that prefigure all limb skeletal components. Of the three cardinal limb axes, the mechanisms that lead to establishment and patterning of skeletal elements along the P-D axis are the least understood. Here we identify a genetic interaction between Gli3 (GLI-Kruppel family member 3) and Plzf (promyelocytic leukaemia zinc finger, also known as Zbtb16 and Zfp145), which is required specifically at very early stages of limb development for all proximal cartilage condensations in the hindlimb (femur, tibia, fibula). Notably, distal condensations comprising the foot are relatively unperturbed in Gli3(-/-);Plzf(-/-) mouse embryos. We demonstrate that the cooperative activity of Gli3 and Plzf establishes the correct temporal and spatial distribution of chondrocyte progenitors in the proximal limb-bud independently of known P-D patterning markers and overall limb-bud size. Moreover, the limb defects in Gli3(-/-);Plzf(-/-) embryos correlate with the transient death of a specific subset of proximal mesenchymal cells that express bone morphogenetic protein receptor, type 1B (Bmpr1b) at the onset of limb development. These findings suggest that the development of proximal and distal skeletal elements is distinctly regulated early during limb-bud formation. The initial division of the vertebrate limb into two distinct molecular domains is consistent with fossil evidence indicating that the upper and lower extremities of the limb have different evolutionary origins.

Distinct CD4+-T-Cell Responses to Live and Heat-Inactivated Aspergillus fumigatus Conidia

Abstract: Aspergillus fumigatus is a ubiquitous mold that produces small, 2- to 3-μm spores (aka conidia) that are inhaled into lung alveoli. The hydrophobic nature of the spores and their small size allow them to remain airborne for long periods (30, 31). A. fumigatus spores are ubiquitous, and it is estimated that humans inhale several hundred conidia per day (31). In the lung airways, conidia are phagocytosed by alveolar macrophages, where they swell (the first stage of germination), thereby triggering the production of reactive oxygen intermediates (ROI) by the macrophage, a lethal event for the spore (30, 31, 34, 41, 44). Conidia that escape killing by alveolar macrophages germinate, eventually forming hyphae that invade tissues and blood vessels. Neutrophils are recruited to sites of fungal invasion, where they adhere to the hyphal surface and release ROI as well as hydrolytic enzymes that damage the fungal cell wall (22, 30, 33, 34, 44). Administration of immunosuppressive medications increases the incidence of invasive aspergillosis (IA) (31) and is responsible for the current status of Aspergillus fumigatus as one of the most prevalent airborne fungal pathogens (30, 31). The contributions of innate and adaptive immunity to protection of the immunocompetent host from invasive A. fumigatus infections are complex and incompletely defined. Clinical experience and studies in animal models implicate neutrophils and macrophages and their products, such as ROI and pentraxin 3, in innate immune defense against A. fumigatus (19, 22, 30, 31, 33, 34, 44). T cells are increasingly recognized as important mediators of protection from IA (53). Vaccination studies using dendritic cells pulsed with fungal antigens and adoptive transfer studies with T cells from A. fumigatus immune mice suggest that T cells can protect mice from invasive fungal infection (5, 6). Likewise, studies with cyclophosphamide-treated mice or with normal mice intravenously infected with conidia indicate that CD4+ T helper subsets influence the outcome of A. fumigatus infection (8-11). Inhibition of gamma interferon (IFN-γ) results in enhanced invasive disease after A. fumigatus challenge, suggesting that Th1 T cells mediate protection (8, 35). On the other hand, defense against IA is impaired by interleukin 4 (IL-4), and mice lacking this cytokine are more resistant to fungal invasion, suggesting that Th2 CD4+-T-cell responses are detrimental (7, 28, 37). A. fumigatus also causes allergic bronchopulmonary aspergillosis (ABPA), a disease that occurs in patients with asthma and exacerbates airway hyperactivity, peribronchial fibrosis, immunoglobulin E (IgE) production, and eosinophilia (15, 20, 31). A. fumigatus expresses a variety of allergens, several of which have been cloned by screening expression libraries with the sera of ABPA patients (17, 18). Most patients have been found to react to the ribotoxin Asp f I, the perixosome-like protein Asp f 3, the manganese superoxide dismutase Asp f 6, and the allergen Asp f 2 (17, 18). The presence of A. fumigatus allergen-specific antibodies in the sera of ABPA patients is an important diagnostic criterion for this disease and may play a pathogenic role (15, 20, 31). A mouse model that recapitulates the hallmarks of human ABPA has been used to dissect which components of the immune response contribute to pathogenesis (15, 20). A central role for CD4+ T cells in promoting the pathogenesis of ABPA has been demonstrated (12-14, 25, 29), with the Th2 cytokines IL-4, IL-5, and IL-13 contributing to pulmonary pathology (3, 4, 23, 27, 28, 38). The factors that determine when CD4+ T cells are activated in response to A. fumigatus exposure and whether the responding T cells will be biased to a Th1 or Th2 phenotype are unknown. In this study we assessed whether the metabolic state of the A. fumigatus spore influences CD4+-T-cell activation and differentiation by comparing responses to intratracheal challenge with live conidia or heat-inactivated conidia (HIC). We found disparate cytokine profiles in the two groups of mice, with Th1 type cytokines predominating upon exposure to live conidia while production of Th2 cytokines was more prominent following immunization with HIC. Although CD4+ T cells in draining mediastinal lymph nodes (MLN) proliferated in response to A. fumigatus antigens following immunization with live or heat-inactivated conidia, IFN-γ-producing CD4+ T cells specific for hyphae were present only in the airways of mice infected with live conidia. Humoral immune responses to A. fumigatus antigens were mounted in mice infected with live but not inactivated fungus. These results indicate that the immune system discriminates between inactivated and metabolically active spores, restricting optimal Th1 CD4+-T-cell responses and antibody generation for in vivo challenge with viable fungal spores.