GATA-3 dose-dependent checkpoints in early T cell commitment.

GATA-3 expression is crucial for T cell development and peaks during commitment to the T cell lineage, midway through the CD4(-)CD8(-) (double-negative [DN]) stages 1-3. We used RNA interference and conditional deletion to reduce GATA-3 protein acutely at specific points during T cell differentiation in vitro. Even moderate GATA-3 reduction killed DN1 cells, delayed progression to the DN2 stage, skewed DN2 gene regulation, and blocked appearance of the DN3 phenotype. Although a Bcl-2 transgene rescued DN1 survival and improved DN2 cell generation, it did not restore DN3 differentiation. Gene expression analyses (quantitative PCR, RNA sequencing) showed that GATA-3-deficient DN2 cells quickly upregulated genes, including Spi1 (PU.1) and Bcl11a, and downregulated genes, including Cpa3, Ets1, Zfpm1, Bcl11b, Il9r, and Il17rb with gene-specific kinetics and dose dependencies. These targets could mediate two distinct roles played by GATA-3 in lineage commitment, as revealed by removing wild-type or GATA-3-deficient early T lineage cells from environmental Notch signals. GATA-3 worked as a potent repressor of B cell potential even at low expression levels, so that only full deletion of GATA-3 enabled pro-T cells to reveal B cell potential. The ability of GATA-3 to block B cell development did not require T lineage commitment factor Bcl11b. In prethymic multipotent precursors, however, titration of GATA-3 activity using tamoxifen-inducible GATA-3 showed that GATA-3 inhibits B and myeloid developmental alternatives at different threshold doses. Furthermore, differential impacts of a GATA-3 obligate repressor construct imply that B and myeloid development are inhibited through distinct transcriptional mechanisms. Thus, the pattern of GATA-3 expression sequentially produces B lineage exclusion, T lineage progression, and myeloid-lineage exclusion for commitment.

Differences in implementation of gait analysis recommendations based on affiliation with a gait laboratory.

This study examined the extent to which gait analysis recommendations are followed by orthopedic surgeons with varying degrees of affiliation with the gait laboratory. Surgical data were retrospectively examined for 95 patients with cerebral palsy who underwent lower extremity orthopedic surgery following gait analysis. Thirty-three patients were referred by two surgeons directly affiliated with the gait laboratory (direct affiliation), 44 were referred by five surgeons from the same institution but not directly affiliated with the gait laboratory (institutional affiliation), and 18 were referred by 10 surgeons from other institutions (no affiliation). Data on specific surgeries were collected from the gait analysis referral, gait analysis report, and operative notes. Adherence to the gait analysis recommendations was calculated by dividing the number of procedures where the surgery followed the gait analysis recommendation (numerator) by the total number of procedures initially planned, recommended by gait analysis, or done (denominator). Adherence with the gait analysis recommendations was 97%, 94%, and 77% for the direct, institutional, and no affiliation groups, respectively. Procedures recommended for additions to the surgical plan were added 98%, 87%, and 77% of the time. Procedures recommended for elimination were dropped 100%, 89%, and 88% of the time. Of 81 patients who had specific surgical plans prior to gait analysis, changes were implemented in 84% (68/81) following gait analysis recommendations. Gait analysis influences the treatment decisions of surgeons regardless of affiliation with the gait laboratory, although the influence is stronger for surgeons who practice within the same institution as the gait laboratory.