We describe the complete sequence of the 16,596-nucleotide mitochondrial genome of the zebrafish (Danio rerio); contained are 13 protein genes, 22 tRNAs, 2 rRNAs, and a noncoding control region. Codon usage in protein genes is generally biased toward the available tRNA species but also reflects strand-specific nucleotide frequencies. For 19 of the 20 amino acids, the most frequently used codon ends in either A or C, with A preferred over C for fourfold degenerate codons (the lone exception was AUG: methionine). We show that rates of sequence evolution vary nearly as much within vertebrate classes as between them, yet nucleotide and amino acid composition show directional evolutionary trends, including marked differences between mammals and all other taxa. Birds showed similar compositional characteristics to the other nonmammalian taxa, indicating that the evolutionary trend in mammals is not solely due to metabolic rate and thermoregulatory factors. Complete mitochondrial genomes provide a large character base for phylogenetic analysis and may provide for robust estimates of phylogeny. Phylogenetic analysis of zebrafish and 35 other taxa based on all protein-coding genes produced trees largely, but not completely, consistent with conventional views of vertebrate evolution. It appears that even with such a large number of nucleotide characters (11,592), limited taxon sampling can lead to problems associated with extensive evolution on long phyletic branches.

Pulmonary fibrosis is characterized by alterations in fibroblast phenotypes resulting in excessive extracellular matrix accumulation and anatomic remodeling. Current therapies for this condition are largely ineffective. Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is a member of the nuclear hormone receptor superfamily, the activation of which produces a number of biological effects, including alterations in metabolic and inflammatory responses. The role of PPAR-gamma as a potential therapeutic target for fibrotic lung diseases remains undefined. In the present study, we show expression of PPAR-gamma in fibroblasts obtained from normal human lungs and lungs of patients with idiopathic interstitial pneumonias. Treatment of lung fibroblasts and myofibroblasts with PPAR-gamma agonists results in inhibition of proliferative responses and induces cell cycle arrest. In addition, PPAR-gamma agonists, including a constitutively active PPAR-gamma construct (VP16-PPAR-gamma), inhibit the ability of transforming growth factor-beta1 to induce myofibroblast differentiation and collagen secretion. PPAR-gamma agonists also inhibit fibrosis in a murine model, even when administration is delayed until after the initial inflammation has largely resolved. These observations indicate that PPAR-gamma is an important regulator of fibroblast/myofibroblast activation and suggest a role for PPAR-gamma ligands as novel therapeutic agents for fibrotic lung diseases.

Urease, a major virulence factor for Cryptococcus neoformans, promotes lethal meningitis/encephalitis in mice. The effect of urease within the lung, the primary site of most invasive fungal infections, is unknown. An established model of murine infection that utilizes either urease-producing (wt and ure1::URE1) or urease-deficient (ure1) strains (H99) of C. neoformans was used to characterize fungal clearance and the resultant immune response evoked by these strains within the lung. Results indicate that mice infected with urease-producing strains of C. neoformans demonstrate a 100-fold increase in fungal burden beginning 2 weeks post-infection (as compared with mice infected with urease-deficient organisms). Infection with urease-producing C. neoformans was associated with a highly polarized T2 immune response as evidenced by increases in the following: 1) pulmonary eosinophils, 2) serum IgE levels, 3) T2 cytokines (interleukin-4, -13, and -4 to interferon-gamma ratio), and 4) alternatively activated macrophages. Furthermore, the percentage and total numbers of immature dendritic cells within the lung-associated lymph nodes was markedly increased in mice infected with urease-producing C. neoformans. Collectively, these data define cryptococcal urease as a pulmonary virulence factor that promotes immature dendritic cell accumulation and a potent, yet non-protective, T2 immune response. These findings provide new insights into mechanisms by which microbial factors contribute to the immunopathology associated with

Successful pulmonary clearance of the encapsulated yeast Cryptococcus neoformans requires a T1 adaptive immune response. This response takes up to 3 weeks to fully develop. The role of the initial, innate immune response against the organism is uncertain. In this study, an established model of diphtheria toxin-mediated depletion of resident pulmonary dendritic cells (DC) and alveolar macrophages (AM) was used to assess the contribution of these cells to the initial host response against cryptococcal infection. The results demonstrate that depletion of DC and AM one day prior to infection results in rapid clinical deterioration and death of mice within 6 days postinfection; this effect was not observed in infected groups of control mice not depleted of DC and AM. Depletion did not alter the microbial burden or total leukocyte recruitment in the lung. Mortality (in mice depleted of DC and AM) was associated with increased neutrophil and B-cell accumulation accompanied by histopathologic evidence of suppurative neutrophilic bronchopneumonia, cyst formation, and alveolar damage. Collectively, these data define an important role for DC and AM in regulating the initial innate immune response following pulmonary infection with C. neoformans. These findings provide important insight into the cellular mechanisms which coordinate early host defense against an invasive fungal pathogen in the lung.Cryptococcus neoformans, an opportunistic fungal pathogen acquired through inhalation, causes significant morbidity and mortality primarily in patients with impairments in host defense, including those with AIDS, those with lymphoid or hematological malignancies, or those receiving immunosuppressive therapy secondary to autoimmune disease or organ transplantation (31,33,60). The development of a T1 antigenspecific immune response characterized by gamma interferon production and classical activation of macrophages is required to eradicate the organism (4,8,21,23,24,28). This adaptive immune response takes 2 to 3 weeks to develop and coincides with the CCR2-mediated recruitment of additional pulmonary dendritic cells (DC) and T cells to the lung (51,66,67). The role of initial, innate immune responses against the organism (prior to the development of adaptive immunity) is not well understood.Resident lung phagocytic cells, primarily DC and alveolar macrophages (AM), are likely the first immune cells exposed to C. neoformans upon inhalation of the organism into the lung. Both DC and AM express lectin receptors, including macrophage mannose receptor and DC-specific non-ICAM3 grabbing nonintergrin (DC-SIGN) (14, 15), which bind C. neoformans glycoantigens, including mannoproteins (42, 55). DC and AM phagocytose the organism in vitro and in vivo (29,34,63,77,78), and phagocytosis (and/or exposure to soluble glycoantigens or cryptococcal DNA) is associated with cytokine and chemokine production (5,29,40,48,49,55,61) and yeast lysis (77). It is unclear whether phagocytosis by resident DC and AM contributes to early clearance and/or the la...