1. Hematopoietic stimulation by a dipeptidyl peptidase inhibitor reveals a novel regulatory mechanism and therapeutic treatment for blood cell deficiencies
by Barry Jones, Sharlene Adams, Glenn T. Miller, Michael I. Jesson, Takeshi Watanabe, and Barbara P. Wallner
Blood
Volume 102(5):
September 1, 2003
©2003 by American Society of Hematology

2. PT-100 stimulates hematopoiesis in normal mice.
PT-100 stimulates hematopoiesis in normal mice. (A) Structure of PT-100. (B) Levels of erythroid and myeloid CFUs (×10–4) in BALB/c mice after oral administration of saline (□) or 5 μg PT-100 (▪) twice daily for 5 days. Erythroid and myeloid CFUs in spleens and BM were assayed as described in “Materials and methods.” CFU-Es were observed after 2 to 3 days in culture, whereas BFU-Es and CFU-GMs were observed after 7 to 9 days. Data are expressed as mean splenic CFCs ± SEM of 3 independent experiments (n = mice/treatment group) and mean BM CFCs ± SEM of 2 experiments (n = 7-8 mice/treatment group). (C) Changes in ANCs in mice administered saline (○) or 20 μg PT-100 (•) orally and twice daily. ANCs were determined by standard differential counting. Data are expressed as the mean ± SD of 5 mice/group, using discrete groups at each point in time. Changes in ANCs are representative of 2 independent experiments. (D) Changes in serum G-CSF (○,•) and IL-6 (□,▪) levels in mice administered saline (open symbols) or PT-100 (filled symbols) as described in panel C. Serum cytokine levels were determined by ELISA, and data are expressed as mean ± SD from 5 mice/group. Changes in G-CSF and IL-6 levels are representative of 3 independent experiments.
Barry Jones et al. Blood 2003;102:
©2003 by American Society of Hematology

3. PT-100 administration promotes neutrophil regeneration in mouse model of neutropenia.
PT-100 administration promotes neutrophil regeneration in mouse model of neutropenia. BALB/c mice were injected intraperitoneally with CYP (220 mg/kg) on day 0 and were administered saline or PT-100 orally and twice daily, starting on day 3. (A) Changes in ANCs in mice receiving 0.1 μg(▪), 2 μg(•), or 5 μg (▴) PT-100 or saline (○) for 5 days. (B) Changes in ANCs in mice receiving saline (○) or 2 μg PT-100 administered for 2 (▪), 3 (▴), or 5 (•) days. (C) Changes in serum G-CSF levels in mice receiving 2 μg PT-100 (•) or saline (○). (D) Changes in ANCs on days 5 (□) and 6 (▪) in mice receiving saline, 30 μg or 5 μg PT-100, or NAc-VBP as indicated on the abscissa. ANC and G-CSF levels were determined by standard differential counts and ELISA, respectively, and are expressed as mean ± SD from 4 to 5 mice per group. Data are representative of 6 independent ANC experiments and 3 independent G-CSF experiments. Significant P values are indicated in “Results.” Mean ANC and G-CSF levels in normal mice are indicated by the horizontal lines.
Barry Jones et al. Blood 2003;102:
©2003 by American Society of Hematology

4. PT-100 accelerates recovery of mice from hemolytic anemia.
PT-100 accelerates recovery of mice from hemolytic anemia. Acute hemolytic anemia was induced by intraperitoneal injection of PHZ (60 mg/kg) on days 0 and 1. Five micrograms PT-100 or saline was administered orally and twice daily to BALB/c mice for a 5-day period before PHZ treatment (ie, from day–6today–2). (A) Changes in hematocrit in mice pretreated with saline (○) or PT-100 (•). Horizontal bar indicates hematocrits of normal mice. (B) Changes in reticulocytes in mice pretreated with saline (○) or PT-100 (•). (C) Levels of splenic CFU-Es (×10–4; ▪) and BFU-Es (×10–3; □) on day 3 in mice treated with saline or PT-100 as indicated on abscissa. CFUs were assayed as described in “Materials and methods.” Data are expressed as the mean ± SD of 3 mice/group using discrete groups for each point in time, and they are representative of 2 independent experiments. Significant P values are indicated in “Results.”
Barry Jones et al. Blood 2003;102:
©2003 by American Society of Hematology

5. PT-100 stimulates the survival and growth of primitive hematopoietic progenitors in vitro and induces the production of cytokines by BM stromal cells.
PT-100 stimulates the survival and growth of primitive hematopoietic progenitors in vitro and induces the production of cytokines by BM stromal cells. (A) CFU levels after 4-week bulk culture of human CD34+ cells in the presence of varying concentrations of PT-100, as indicated on abscissa. CD34+ cells were propagated in vitro on supportive layers of X-irradiated stromal cells, and CFUs were assayed as described in “Materials and methods.” Data are expressed as mean ± SD from triplicate cultures and are representative of 6 independent experiments. (B) Clonal frequency of LTC-ICs in human CD34+ cell population cultured in the presence (•) or absence (○) of 10 nM PT-100. Limiting-dilution assay of LTC-ICs on supportive layers of stromal cells was performed as described in “Materials and methods.” Data are representative of 2 assays. (C-D) Changes in G-CSF (○,•), IL-11 (▵,▴), and IL-6 (□,▪) levels in supernatants of BM stromal cells cultured either with (filled symbols) or without (open symbols) 1 μM PT-100. Data are expressed as means from triplicate cultures and are representative of 5 replicate experiments. (E) Comparison of gene expression in BM stromal cells incubated for 6 hours in the presence (▪) or absence (□) of 1 μM PT-100. Cytokine gene expression was determined by microarray hybridization, as described in “Materials and methods,” and data were replicated in 2 independent experiments.
Barry Jones et al. Blood 2003;102:
©2003 by American Society of Hematology

6. Characterization of human BM stromal cell dipeptidyl peptidases.
Characterization of human BM stromal cell dipeptidyl peptidases. Stromal cell extracts were separated by ion-exchange chromatography into 2 fractions, E1 and E2, each containing dipeptidyl peptidase activity (A). Dipeptidyl peptidase activity eluted from the column was detected by florigenic assay with 0.1 mM Ala-Pro-AFC, and SDS-PAGE of unboiled samples identified dipeptidyl peptidases of approximately 220 kDa and 170 kDa in E1 and E2, respectively. In contrast, unfractionated stromal extract contained dipeptidyl peptidases of both molecular sizes and RPMI-7951 and CACO-2 cell lines contained only the 170-kDa dipeptidyl peptidase (B). The same samples were immunoprecipitated with mAb F19, M-A261, or a mouse IgG1 isotype control, and the immunoprecipitates were assayed for dipeptidyl peptidase activity (C). Data in panels A-C were representative of 7 independent human stromal cell cultures.
Barry Jones et al. Blood 2003;102:
©2003 by American Society of Hematology

7. PT-100 hematopoietic activity does not require CD26.
PT-100 hematopoietic activity does not require CD26. (A-B) Changes in ANCs in B6, BALB/c CD26+/+ (A) and B6, BALB/c CD26–/– (B) mice treated with CYP (250 mg/kg) on day 0 and administered 10 μg PT-100 (•) or saline (○) orally and 3 times daily from day 3 to day 6. Data are expressed as mean ± SD from 3 to 5 mice per group. Mean ANC in normal mice are indicated by horizontal bars. (C) ANCs in normal B6, BALB/c CD26+/+ (▪), and B6, BALB/c CD26–/– (□) mice after the oral administration of 25 μg PT times daily for 5 days. (D) Changes in IL-6 levels in supernatants of Fischer D+ (•) and D– (○) rat BM stromal cells cultured for 24 hours in varying concentrations of PT-100, as indicated on abscissa. Rat IL-6 levels were determined using ELISA. Data are expressed as mean ± SD from triplicate cultures and are representative of 3 separate experiments.
Barry Jones et al. Blood 2003;102:
©2003 by American Society of Hematology