Paradoxically increased FOXP3+ T cells in IBD do not preferentially express the isoform of FOXP3 lacking exon 2.

BACKGROUND: Forkhead box P3 (FOXP3)+ regulatory T cells (Tregs) are critical for controlling inflammation in the gastrointestinal tract. There is a paradoxical increase of mucosal FOXP3+ T cells in patients with inflammatory bowel disease (IBD). These FOXP3+ cells were recently shown to include interleukin (IL)-17A-producing cells in Crohn's disease, resembling Th17 cells implicated in autoimmune diseases. FOXP3 inhibits IL-17A production, but a naturally occurring splice variant of FOXP3 lacking exon 2 (Δexon2) cannot. AIMS: We hypothesized that IBD patients preferentially express the Δexon2 variant of FOXP3 so the paradoxically increased mucosal Tregs in IBD could represent cells expressing only Δexon2. METHODS: We used antibodies and primers that can distinguish between the full-length and Δexon2 splice variant of FOXP3 to evaluate expression of these isoforms in human intestinal tissue by immunohistochemistry and quantitative polymerase chain reaction (PCR), respectively. RESULTS: No difference in the expression pattern of Δexon2 relative to full-length FOXP3 was seen in ulcerative colitis or Crohn's disease versus non-IBD controls. By immunofluorescence microscopy and flow cytometry, we also did not find individual cells which expressed FOXP3 protein exclusively in the Δexon2 isoform in either IBD or control tissue. FOXP3+ mucosal CD4+ T cells from both IBD and control specimens were able to make IL-17A in vitro after phorbol myristate acetate (PMA) and ionomycin stimulation, but these cells did not preferentially express Δexon2. CONCLUSIONS: Our data do not support the hypothesis that selective expression of FOXP3 in the Δexon2 isoform accounts for the inability of copious FOXP3+ T cells to inhibit inflammation or IL-17 expression in IBD.

Limitations of a murine transgenic breast cancer model for studies of erythropoietin-induced tumor progression.

Adverse effects of erythropoietin (EPO) on tumor progression and survival were observed in recent phase 3 oncology trials. However, mechanisms remain poorly understood. We tested the effects of exogenous EPO on murine B16F10 melanoma growth in a subcutaneous tumor transplant model, and for the first time, in a model of spontaneous tumor formation within autochthonous epithelial tissues using murine mammary tumor virus promoter polyoma virus middle T antigen (MMTV-PyMT) transgenic mice. EPO receptor (EPOR) messenger RNA (mRNA) was detectable in both B16F10 tumors and mammary tumors from MMTV-PyMT mice but was 0.12 +/- 0.02% and 1.3 +/- 0.91% of the EPOR mRNA level in murine erythroid HCD-57 cells, respectively. B16F10 tumor growth rates in mice treated for 3 weeks with 30 microg/kg per week of darbepoetin alpha, 0.41 inverse days (range, 0.05-0.69 inverse days; n = 16), were similar to tumor growth rates observed in mice treated with PBS, 0.42 inverse days (range, 0.10-0.69 inverse days; n = 17). In contrast, darbepoetin alpha raised hematocrit levels to 0.593 (maximum, 0.729) compared with 0.448 (maximum, 0.532) in PBS-treated mice (P = .0004). In MMTV-PyMT mice, the weights of tumor-bearing mammary glands in mice treated for 6 weeks with 30 microg/kg per week of darbepoetin alpha, 3.37 g (range, 1.94-5.81 g; n = 27), did not significantly differ from the weights in PBS-treated mice, 3.76 g (range, 2.30-6.33 g; n = 26). In contrast, darbepoetin alpha raised hematocrit levels to 0.441 (maximum, 0.606) compared with 0.405 (maximum, 0.492) in PBS-treated mice (P = .05). Thus, effects of exogenous EPO on tumor growth were not recapitulated in these murine tumor models.

Evaluating erythropoietin-associated tumor progression using archival tissues from a phase III clinical trial.

Despite the prevalence of anemia in cancer, recombinant erythropoietin (Epo) has declined in use because of recent Phase III trials showing more rapid cancer progression and reduced survival in subjects randomized to Epo. Since Epo receptor (EpoR), Jak2, and Hsp70 are well-characterized mediators of Epo signaling in erythroid cells, we hypothesized that Epo might be especially harmful in patients whose tumors express high levels of these effectors. Because of the insensitivity of immunohistochemistry for detecting low level EpoR protein, we developed assays to measure levels of EpoR, Jak2 and Hsp70 mRNA in formalin-fixed paraffin-embedded (FFPE) tumors. We tested 23 archival breast tumors as well as 136 archival head and neck cancers from ENHANCE, a Phase III trial of 351 patients randomized to Epo versus placebo concomitant with radiotherapy following complete resection, partial resection, or no resection of tumor. EpoR, Jak2, and Hsp70 mRNA levels varied >30-fold, >12-fold, and >13-fold across the breast cancers, and >30-fold, >40-fold, and >30-fold across the head and neck cancers, respectively. Locoregional progression-free survival (LPFS) did not differ among patients whose head and neck cancers expressed above- versus below-median levels of EpoR, Jak2 or Hsp70, except in the subgroup of patients with unresected tumors (n = 28), where above-median EpoR, above-median Jak2, and below-median Hsp70 mRNA levels were all associated with significantly poorer LPFS. Our results provide a framework for exploring the relationship between Epo, cancer progression, and survival using archival tumors from other Phase III clinical trials.

Oxidation of tertiary silanes by osmium tetroxide.

In the presence of an excess of pyridine ligand L, osmium tetroxide oxidizes tertiary silanes (Et(3)SiH, (i)Pr(3)SiH, Ph(3)SiH, or PhMe(2)SiH) to the corresponding silanols. With L = 4-tert-butylpyridine ((t)Bupy), OsO(4)((t)Bupy) oxidizes Et(3)SiH and PhMe(2)SiH to yield 100 +/- 2% of silanol and the structurally characterized osmium(VI) mu-oxo dimer [OsO(2)((t)Bupy)(2)](2)(mu-O)(2) (1a). With L = pyridine (py), only 40-60% yields of R(3)SiOH are obtained, apparently because of coprecipitation of osmium(VIII) with [Os(O)(2)py(2)](2)(mu-O)(2) (1b). Excess silane in these reactions causes further reduction of the OsVI products, and similar osmium "over-reduction" is observed with PhSiH(3), Bu(3)SnH, and boranes. The pathway for OsO(4)(L) + R(3)SiH involves an intermediate, which forms rapidly at 200 K and decays more slowly to products. NMR and IR spectra indicate that the intermediate is a monomeric Os(VI)-hydroxo-siloxo complex, trans-cis-cis-Os(O)(2)L(2)(OH)(OSiR(3)). Mechanistic studies and density functional theory calculations indicate that the intermediate is formed by the [3 + 2] addition of an Si-H bond across an O=Os=O fragment. This is the first direct observation of a [3 + 2] intermediate in a sigma-bond oxidation, though such species have previously been implicated in reactions of H-H and C-H bonds with OsO(4)(L) and RuO(4).