A KLF4-DYRK2-mediated pathway regulating self-renewal in CML stem cells.

Leukemia stem cells are a rare population with a primitive progenitor phenotype that can initiate, sustain, and recapitulate leukemia through a poorly understood mechanism of self-renewal. Here, we report that Krüppel-like factor 4 (KLF4) promotes disease progression in a murine model of chronic myeloid leukemia (CML)-like myeloproliferative neoplasia by repressing an inhibitory mechanism of preservation in leukemia stem/progenitor cells with leukemia-initiating capacity. Deletion of the Klf4 gene severely abrogated the maintenance of BCR-ABL1(p210)-induced CML by impairing survival and self-renewal in BCR-ABL1+ CD150+ lineage-negative Sca-1+ c-Kit+ leukemic cells. Mechanistically, KLF4 repressed the Dyrk2 gene in leukemic stem/progenitor cells; thus, loss of KLF4 resulted in elevated levels of dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase 2 (DYRK2), which were associated with inhibition of survival and self-renewal via depletion of c-Myc protein and p53 activation. In addition to transcriptional regulation, stabilization of DYRK2 protein by inhibiting ubiquitin E3 ligase SIAH2 with vitamin K3 promoted apoptosis and abrogated self-renewal in murine and human CML stem/progenitor cells. Altogether, our results suggest that DYRK2 is a molecular checkpoint controlling p53- and c-Myc-mediated regulation of survival and self-renewal in CML cells with leukemic-initiating capacity that can be targeted with small molecules.

Analytical validation of an enzyme-linked immunosorbent assay for the quantification of S100A12 in the serum and feces of cats.

BACKGROUND: Measuring S100A12 concentrations in serum and feces is a sensitive and specific marker of inflammation, such as seen with chronic gastrointestinal inflammation in people and dogs. Biomarkers of inflammation in cats are currently lacking. OBJECTIVES: We aimed to analytically cross-validate the canine S100A12-ELISA for the measurement of S100A12 in feline specimens. METHODS: The ELISA was analytically validated by assessing dilutional linearity, spiking/recovery, intra- and inter-assay variability. Reference intervals for serum and fecal feline S100A12 concentrations were calculated using samples from healthy cats, and the short-term biological variation of fecal S100A12 was assessed. RESULTS: Observed-to-expected ratios (O/E) for serial dilutions of serum and fecal extracts ranged from 91%-159% (mean, 120%) and 100%-128% (mean, 114%), and for the spiking/recovery method ranged from 106%-263% (mean, 154%) and 52%-171% (mean, 112%). Intra- and inter-assay CV% for serum were ≤5.6% and ≤14.0%, and for fecal extracts were ≤3.8% and ≤19.1%, repsectively. RIs for feline serum and fecal S100A12 concentrations were <43 µg/L and < 20 ng/g, respectively. A mild short-term biologic variation, but large individuality were detected when measuring fecal S100A12 concentrations in healthy cats. CONCLUSIONS: The canine S100A12-ELISA is accurate, reproducible, and sufficiently linear and precise for the measurement of S100A12 in feline serum and fecal samples. The use of this assay is a reasonable option for the measurement of S100A12 concentrations in feline specimens and provides a basis for the further evaluation of  S100A12 in cats with gastrointestinal disease. Using a population-based RI for fecal feline S100A12 appears to be of limited value.

Validation of a radioimmunoassay of serum trypsin-like immunoreactivity in ferrets.

Measurement of serum trypsin-like immunoreactivity (TLI) is used to assess exocrine pancreatic function in dogs and cats. Ferrets ( Mustela putorius furo) serve as valuable animal models for human diseases such as cystic fibrosis and other pulmonary diseases, and may be a useful model of other diseases including pancreatitis. We developed and analytically validated a competitive radioimmunoassay (RIA) for measurement of TLI in ferret serum by determination of analytical sensitivity, assay linearity, accuracy of spiking recovery, precision, and reproducibility. Analytical sensitivity of the assay was 0.55 µg/L. Observed-to-expected (O/E) ratio for dilutional parallelism was 90.2-127.9% (mean: 108.1 ± 11.9%). The O/E ratio for spiking recovery was 94.5-113.0% (mean: 103.9 ± 7.2%). The intra- and inter-assay coefficients of variation (CVs) were 2.7-5.7% and 3.5-8.2%, respectively. The reference interval (RI) for serum TLI derived from 31 healthy ferrets was 28-115 µg/L; the 90% confidence interval for the lower and upper limits of the RI were 10.0-32.1 µg/L and 103-126 µg/L, respectively. This TLI RIA is analytically sensitive, sufficiently linear, accurate, precise, and reproducible for the measurement of TLI in ferret serum samples.

Serum concentrations of canine alpha(1)-proteinase inhibitor in cobalamin-deficient Yorkshire Terrier dogs.

Fecal canine alpha1-proteinase inhibitor (cα1-PI) concentration has been reported to be increased in dogs with protein-losing enteropathy due to the loss of cα1-PI into the gastrointestinal tract. A chronic loss of cα1-PI may theoretically deplete serum cα1-PI, potentially altering the proteinase-to-proteinase inhibitor balance. Protein-losing enteropathy has been reported to occur frequently in certain dog breeds such as Yorkshire Terriers and to be associated with hypocobalaminemia. The objective was to compare serum cα1-PI concentrations in Yorkshire Terriers with and without cobalamin (COB) deficiency. Serum samples from 52 COB-deficient and 69 normocobalaminemic Yorkshire Terriers, which had been submitted to the Gastrointestinal Laboratory (2008-2011; College Station, TX), were included retrospectively. Serum cα1-PI concentrations were measured using an in-house radioimmunoassay and compared between Yorkshire Terriers with and without COB deficiency using a Mann-Whitney U test. A Fisher exact test was used to evaluate whether a decreased serum cα1-PI concentration is associated with COB deficiency in Yorkshire Terriers. Serum cα1-PI concentrations were significantly lower in COB-deficient Yorkshire Terriers (median: 1,016 mg/l, range: 315-3,945 mg/l) than in normocobalaminemic Yorkshire Terriers (median: 1,665 mg/l, range: 900-2,970 mg/l; P < 0.0001). One-fourth (n = 13) of the COB-deficient Yorkshire Terriers had a serum cα1-PI concentration below the lower limit of the reference interval (<732 mg/l), and COB deficiency was associated with decreased serum cα1-PI concentrations (P < 0.0001). In the current study, serum cα1-PI concentrations are significantly lower in COB-deficient Yorkshire Terriers when compared to normocobalaminemic Yorkshire Terriers. Further studies are needed to determine the functional and potential prognostic implications of serum cα1-PI concentrations in dogs with gastrointestinal disease.