Citations

Osteoporosis is an important clinical problem, affecting more than 50% of people over age 50 yr. Estrogen signaling is critical for maintaining proper bone density, and the identification of an endogenous selective estrogen receptor (ER) modulator, 27-hydroxycholesterol (27HC), suggests a mechanism by which nutritional/metabolic status can influence bone biology. With its levels directly correlated with cholesterol, a new possibility emerges wherein 27HC links estrogen and cholesterol signaling to bone homeostasis. In these studies, we found that increasing concentrations of 27HC, both by genetic and pharmacological means, led to decreased bone mineral density that was associated with decreased bone formation and increased bone resorption. Upon manipulation of endogenous estrogen levels, many of the responses to elevated 27HC were altered in such a way as to implicate ER as a likely mediator. In a model of postmenopausal bone loss, some pathologies associated with elevated 27HC were exacerbated by the absence of endogenous estrogens, suggesting that 27HC may act both in concert with and independently from classic ER signaling. These data provide evidence for interactions between estrogen signaling, cholesterol and metabolic disease, and osteoporosis. Patients with high cholesterol likely also have higher than average 27HC, perhaps putting them at a higher risk for bone loss and fracture. More studies are warranted to fully elucidate the mechanism of action of 27HC in bone and to identify ways to modulate this pathway therapeutically.

Groups of rhesus macaques that had previously been immunized with HIV-1 envelope (env) peptides and first generation adenovirus serotype 5 (FG-Ad5) vaccines expressing the same peptides were immunized intramuscularly three times with helperdependent adenovirus (HD-Ad) vaccines expressing only the HIV-1 envelope from JRFL. No gag, pol, or other SHIV genes were used for vaccination. One group of the FG-Ad5-immune animals was immunized three times with HD-Ad5 expressing env. One group was immunized by serotype-switching with HD-Ad6, HD-Ad1, and HD-Ad2 expressing env. Previous work demonstrated that serum antibody levels against env were significantly higher in the serotype-switched group than in the HD-Ad5 group. In this study, neutralizing antibody and T cell responses were compared between the groups before and after rectal challenge with CCR5-tropic SHIV-SF162P3. When serum samples were assayed for neutralizing antibodies, only weak activity was observed. T cell responses against env epitopes were higher in the serotype-switched group. When these animals were challenged rectally with SHIV-SF162P3, both the Ad5 and serotype-switch groups significantly reduced peak viral loads 2 to 10-fold 2 weeks after infection. Peak viral loads were significantly lower for the serotype-switched group as compared to the HD-Ad5-immunized group. Viral loads declined over 18 weeks after infection with some animals viremia reducing nearly 4 logs from the peak. These data demonstrate significant mucosal vaccine effects after immunization with only env antigens. These data also demonstrate HD-Ad vectors are a robust platform for vaccination.

Following infection, naïve CD8 + T cells bearing pathogen-specific T cell receptors (TCRs) differentiate into a mixed population of short-lived effector and long-lived memory T cells to mediate an adaptive immune response. How the TCR regulates memory T cell development has remained elusive. Using a mutant TCR transgenic model, we found that point mutations in the TCR β transmembrane domain (βTMD) impair the development and function of CD8 + memory T cells without affecting primary effector T cell responses. Mutant T cells are deficient in polarizing the TCR and in organizing the nuclear factor κB signal at the immunological synapse. Thus, effector and memory states of CD8 + T cells are separable fates, determined by differential TCR signaling.

AbstractAs multiple myeloma tumors universally dysregulate cyclin D genes we conducted high-throughput chemical library screens for compounds that induce suppression of cyclin D2 promoter transcription. The top-ranked compound was a natural triterpenoid, pristimerin. Strikingly, the early transcriptional response of cells treated with pristimerin closely resembles cellular responses elicited by proteosome inhibitors, with rapid induction of heat shock proteins, activating transcription factor 3 (ATF3), and CHOP. Enzymatic assays and immunoblotting confirm that pristimerin rapidly (< 90 minutes) and specifically inhibits chymotrypsin-like proteosome activity at low concentrations (< 100 nM) and causes accumulation of cellular ubiquitinated proteins. Notably, cytotoxic triterpenoids including pristimerin inhibit NF-κB activation via inhibition of IKKα or IKKβ, whereas proteosome inhibitors instead suppress NF-κB function by impairing degradation of ubiquitinated IκB. By inhibiting both IKK and the proteosome, pristimerin causes overt suppression of constitutive NF-κB activity in myeloma cells that may mediate its suppression of cyclin D. Multiple myeloma is exquisitely sensitive to proteosome or NF-κB pathway inhibition. Consistent with this, pristimerin is potently and selectively lethal to primary myeloma cells (IC50 < 100 nM), inhibits xenografted plasmacytoma tumors in mice, and is synergistically cytotoxic with bortezomib—providing the rationale for pharmaceutical development of triterpenoid dual-function proteosome/NF-κB inhibitors as therapeutics for human multiple myeloma and related malignancies.

Purpose National Cancer Institute of Canada Clinical Trials Group trial MA.17 randomly assigned 5,187 postmenopausal, hormone-receptor–positive patients with early breast cancer who completed 5 years of tamoxifen to receive either letrozole or placebo. At 30 months median follow-up, letrozole significantly improved disease-free survival (DFS) in all patients and overall survival (OS) in node-positive patients. Breast cancer incidence increases with age and more than 1,300 women age 70 years or older were enrolled onto MA.17, making it ideal to explore the benefits, toxicities, and quality of life (QOL) impact of letrozole on older women. Patients and Methods In this study, 5,169 randomly assigned patients were divided into three age groups: younger than 60 years (n = 2,152), 60 to 69 years (n = 1,694), and ≥ 70 years (n = 1,323). Log-rank test was used to compare differences in DFS, distant-disease–free survival, and OS between age and treatment groups, and Cox models were used to estimate hazard ratios and associated 95% CIs. QOL was measured using the Medical Outcomes Short Form-36 and the Menopause-Specific Quality-of-Life questionnaire. Results At 4 years, DFS demonstrated statistically significant differences favoring letrozole only in patients age younger than 60 years (hazard ratio = 0.46; P = .0004); there was no interaction between age and treatment, indicating a similar effect of letrozole among all age groups. There was no difference in toxicity or QOL at 24 months among letrozole- and placebo-treated patients age ≥ 70 years. Conclusion Healthy patients age 70 years and older completing 5 years of tamoxifen should be considered for extended adjuvant therapy with letrozole.

Purpose The National Cancer Institute of Canada Clinical Trials Group MA.17 trial examined the efficacy of letrozole (LET) started within 3 months of 5 years of adjuvant tamoxifen in postmenopausal hormone receptor–positive early-stage breast cancer. When the trial was unblinded, patients who received placebo (PLAC) were offered LET. Patients and Methods This cohort analysis describes the outcomes of women assigned PLAC at the initial random assignment after unblinding. Efficacy outcomes of women who chose LET (PLAC-LET group) were compared with those who did not (PLAC-PLAC group) by the hazard ratios and by P values calculated from Cox models that adjusted for imbalances between the groups. Toxicity analyses included only events that occurred after unblinding. Results There were 1,579 women in the PLAC-LET group (median time from tamoxifen, 2.8 years) and 804 in the PLAC-PLAC group. Patients in the PLAC-LET group were younger; had a better performance status; and were more likely to have had node-positive disease, axillary dissection, and adjuvant chemotherapy than those in the PLAC-PLAC group. At a median follow-up of 5.3 years, disease-free survival (DFS; adjusted hazard ratio [HR], 0.37; 95% CI, 0.23 to 0.61; P < .0001) and distant DFS (HR, 0.39; 95% CI, 0.20 to 0.74; P = .004) were superior in the PLAC-LET group. More self-reported new diagnoses of osteoporosis and significantly more clinical fractures occurred in the women who took LET (5.2% v 3.1%, P = .02). Conclusion Interpretation of this cohort analysis suggests that LET improves DFS and distant DFS even when there has been a substantial period of time since the discontinuation of prior adjuvant tamoxifen.

Most mechanisms of cell development, physiology, and signal transduction are controlled by protein-protein interactions. Immunoprecipitation of multiprotein complexes detected by flow cytometry (IP-FCM) is a means to quantitatively measure these interactions. The high sensitivity of this method makes it useful even when very little biomaterial is available for analysis, as in the case of rare primary cell subsets or patient samples. Detection of the T cell antigen receptor associated with the CD3 multiprotein complex from as few as 300 primary murine T cells is presented as an example. The method is compatible with quantitative flow cytometry techniques, making it possible to estimate the number of coimmunoprecipitated molecules. Both constitutive and inducible protein-protein interactions can be analyzed, as illustrated in related methodology using glutathione S -transferase–fusion protein pull-down experiments. IP-FCM represents a robust, quantitative, biochemical technique to assess native protein-protein interactions, without requiring genetic engineering or large sample sizes.