Biomarker discovery: identification of a growth factor gene signature.

Gene expression signatures can be developed as comprehensive pathway readouts and used as pharmacodynamic or patient-stratification biomarkers. While a consensus on the best practices for selecting gene expression signatures from microarray data is evolving, we have developed basic guidelines to ensure consistency and quality. Here we illustrate these guidelines through the identification of a growth factor gene expression signature that is responsive to phosphatidylinositol 3-kinase (PI3K) pathway perturbations in vitro and related to phosphatase and tensin homolog (PTEN) deregulation in vivo.

PTEN and phosphorylated AKT expression and prognosis in early- and late-stage non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is the commonest cause of cancer mortality worldwide. Growth factor receptor signalling pathways constitute an important mediator for tumor growth and proliferation. PTEN and pAKT play important roles in regulating signal transduction along this pathway. Separate cohorts of stage I (n=25) and stage IV (n=34) NSCLC were examined by immunohistochemistry for PTEN and pAKT expression. There was no correlation between PTEN expression and pAKT expression and neither were associated with age, sex or smoking status. Patients with stage IV disease who overexpressed pAKT (at least 2+) or were PTEN-null had poorer overall survival and progression-free survival. This suggests that PTEN-null or pAKT-positive tumors constitute more aggressive tumors whose clinical course is not altered by therapy. There was no difference in the clinical outcome for stage I disease by PTEN or pAKT expression. A greater proportion of the stage IV patients had PTEN-null disease compared to the stage I cohort, suggesting that loss of PTEN is important in the tumor biology of advanced disease. Loss of PTEN or overexpression of pAKT predicts for an aggressive subset of lung tumors that have a poor prognosis. This will allow identification of a poor prognosis subset that can be targeted with novel treatments that either restore PTEN function or target activated AKT, mTOR and other downstream signal transduction molecules.

Kif1C, a kinesin-like motor protein, mediates mouse macrophage resistance to anthrax lethal factor.

BACKGROUND: Inbred mouse strains exhibit striking differences in the susceptibility of their macrophages to the effects of anthrax lethal toxin (LeTx). Previous data has shown that this difference in susceptibility lies downstream of toxin entry into macrophages. A locus controlling this phenotype, called Ltxs1, has been mapped to chromosome 11, but the responsible gene has not been identified. RESULTS: Here, we report the identification of the Ltxs1 gene as Kif1C, which encodes a kinesin-like motor protein of the UNC104 subfamily. Kif1C is the only gene in the Ltxs1 interval exhibiting polymorphisms between susceptible and resistant strains. Multiple alleles of Kif1C determine the susceptibility or resistance of cultured mouse macrophages to LeTx. Treatment of resistant macrophages with brefeldin-A (which alters the cellular localization of Kif1C) induces susceptibility to LeTx, while ectopic expression of a resistance allele of Kif1C in susceptible macrophages causes a 4-fold increase in the number of cells surviving LeTx treatment. We also show that cleavage of map kinase kinase 3, a target of LeTx proteolysis, occurs in resistant cells. CONCLUSIONS: We conclude that mutations in Kif1C are responsible for the differences in the susceptibility of inbred mouse macrophages to LeTx and that proper Kif1C function is required for LeTx resistance. Since the LeTx-mediated proteolysis of map kinase kinase 3 occurs even in resistant cells, Kif1C does not affect cellular entry or processing of LeTx and likely influences events occurring later in the intoxication pathway.

Genetic, physical, and transcript map of the Ltxs1 region of mouse chromosome 11.

Lethal factor (LF) is a toxin secreted by Bacillus anthracis that plays an important role in the pathogenesis of anthrax. Intoxication with LF results in a macrophage-specific cytolysis that is not well understood. Interestingly, inbred mouse strains exhibit dramatic differences in the susceptibility of their cultured macrophages to killing by LF, and a gene that influences this phenotype, called Ltxs1, has been mapped to mouse chromosome 11. Here we report a high-resolution genetic map that confines the Ltxs1 region to a 0.51-cM interval between D11Mit90 and D11Die37/D11Die38. We have also constructed a complete physical map of YAC and BAC clones covering the Ltxs1 region. In conjunction with synteny homology searching, BLAST searches of sequences obtained from the clones in the physical map have revealed 14 known genes and five ESTs that reside in the critical interval. Additionally, a region of 100 kb or more is deleted in the Ltxs1 interval of some strains. Our genetic, physical, and transcript map provides an important resource for the molecular cloning of Ltxs1.

Ltx1, a mouse locus that influences the susceptibility of macrophages to cytolysis caused by intoxication with Bacillus anthracis lethal factor, maps to chromosome 11.

The lethal factor (LF) toxin that is produced by Bacillus anthracis plays an important role in the pathogenesis of anthrax. LF has mononuclear phagocyte-specific intoxicating effects that are not well understood. We have identified genetic differences in inbred mouse strains that determine whether their cultured macrophages are susceptible to the cytolytic effect of LF intoxication. Our identification of resistant and susceptible mouse strains enabled us to analyse crosses between these strains and to map a single responsible gene (called Ltx1) to chromosome 11. Ltx1 probably influences intoxication events that occur after the delivery of LF to the cytosol, as all mouse macrophages are killed by polypeptides containing the catalytic domain of Diphtheria toxin fused to the domain of LF required for cytosolic transport. Furthermore, the susceptibility phenotype is dominant to resistance, suggesting that resistance is caused by an absence of or polymorphism in a molecule that acts jointly with, or downstream of, the activity of LF. Our mapping of Ltx1 is a crucial first step in its positional cloning, which will provide more information about the mechanism of LF intoxication.

Sequential mesenteric arteriography in pony foals during repeated inoculations of Strongylus vulgaris and treatments with ivermectin.

Semiselective mesenteric arteriography was performed at regular intervals (inoculation weeks [IW] 0, 11, 18, and 24) in 9 of 10 pony foals raised to be free of parasites. Fifty infective larvae (L3) of Strongylus vulgaris were administered weekly for 4 weeks, then every 2 weeks through the 20th week. Three ponies were given ivermectin (oral paste, 0.2 mg/kg of body weight) treatment at IW 8, 16 and 24. Four ponies were inoculated, but did not receive ivermectin, and a third group of 2 ponies acted as uninoculated controls. Control ponies did not have gross or arteriographic lesions, whereas the inoculated untreated ponies had gross and progressive arteriographic lesions typical of verminous arteritis. Arteriographic lesions in the ivermectin-treated inoculated ponies were not as severe those in the untreated inoculated group, and there was either a partial resolution or a lack of progression of arteriographic lesions in all treated ponies. One untreated inoculated pony did not have progressive arterial lesions as did the 3 others in the group, and may develop resistance to the parasite.

Polymorphism and mapping of the class II genes in the rat: RT1.B, RT1.D, and RT1.H, a new DP-like region.

The major histocompatibility complex of the rat (RT1) encodes the class II molecules involved with antigen presentation and cell to cell communication. The organization of these class II genes has been studied by Southern blot hybridization using genomic DNA from inbred and recombinant rat strains digested with various restriction endonuclease and hybridized under stringent conditions with probes for mouse class II and human class II genes. Analysis of the restriction fragment length polymorphisms has mapped the class II genes relative to each other. We have confirmed the order of the alpha- and beta-chain genes in the RT1.B region, mapped the RT1.D region relative to RT1.B and showed that it has alpha- and beta-chain loci, and identified a new HLA-DP-like locus, RT1.H, to the RT1.A side of RT1.B. The RT1.H alpha and RT1.H beta genes map to the region around the recombination point in R22, and there appears to be a hot spot of recombination in RT1.H. The H beta and D beta genes have high levels of polymorphism; B beta, B alpha, and H alpha have intermediate levels of polymorphism, and D alpha has a low level of polymorphism.

Cross-sectional area of the aditus laryngis and rima glottidis before and after transection of the left recurrent laryngeal nerve in the horse.

The ventral-to-dorsal height of the rima glottidis was measured from lateral pharyngeal radiographs after correction for magnification. The rima glottidis height was used to enlarge accurately endoscopic photographs of 5 horses taken before and after transection of the left recurrent laryngeal nerve. Areas of the rima glottidis and aditus laryngis were measured, using a computerized digitizer. Mean area of the aditus laryngis was 1,908 mm2 before neurectomy and 1,346 mm2 after neurectomy (P = 0.025). Mean area of the rima glottidis was 1,198 mm2 before neurectomy and 805 mm2 after neurectomy (P = 0.025). Mean width of the rima glottidis was 31 mm before neurectomy and 20 mm after neurectomy (P = 0.001). Significant differences were not found between the pre- and postneurectomy heights of the rima glottidis.