Megakaryocytes compensate for Kit insufficiency in murine arthritis.

The growth factor receptor Kit is involved in hematopoietic and nonhematopoietic development. Mice bearing Kit defects lack mast cells; however, strains bearing different Kit alleles exhibit diverse phenotypes. Herein, we investigated factors underlying differential sensitivity to IgG-mediated arthritis in 2 mast cell-deficient murine lines: KitWsh/Wsh, which develops robust arthritis, and KitW/Wv, which does not. Reciprocal bone marrow transplantation between KitW/Wv and KitWsh/Wsh mice revealed that arthritis resistance reflects a hematopoietic defect in addition to mast cell deficiency. In KitW/Wv mice, restoration of susceptibility to IgG-mediated arthritis was neutrophil independent but required IL-1 and the platelet/megakaryocyte markers NF-E2 and glycoprotein VI. In KitW/Wv mice, platelets were present in numbers similar to those in WT animals and functionally intact, and transfer of WT platelets did not restore arthritis susceptibility. These data implicated a platelet-independent role for the megakaryocyte, a Kit-dependent lineage that is selectively deficient in KitW/Wv mice. Megakaryocytes secreted IL-1 directly and as a component of circulating microparticles, which activated synovial fibroblasts in an IL-1-dependent manner. Transfer of WT but not IL-1-deficient megakaryocytes restored arthritis susceptibility to KitW/Wv mice. These findings identify functional redundancy among Kit-dependent hematopoietic lineages and establish an unanticipated capacity of megakaryocytes to mediate IL-1-driven systemic inflammatory disease.

A Multifaceted Mentoring Program for Junior Faculty in Academic Pediatrics.

PROBLEM: The departure of physician-scientists from education and research into clinical practice is a growing challenge for the future of academic medicine. Junior faculty face competing demands for clinical productivity, teaching, research, and work-life integration, which can undermine confidence in the value of an academic career. Mentorship is important to foster career development and satisfaction in junior faculty. INTERVENTION: The goals of this academic pediatrics department were to develop, implement, and evaluate a multifaceted pediatric mentoring program to promote retention and satisfaction of junior faculty. Program elements included one-on-one mentor-mentee meetings, didactic workshops, grant review assistance, and facilitated peer-group mentoring. Program effectiveness was assessed using annual surveys of mentees and structured mentee exit interviews, as well as retention data for assistant professors. CONTEXT: The mentees were instructors and assistant professors in the department of pediatrics. OUTCOME: Seventy-nine mentees participated in the program from 2007 through 2014. The response rate from seven annual surveys was 84%. Sixty-nine percent of mentees felt more prepared to advance their careers, 81% had a better understanding of the criteria for advancement, 84% were satisfied with the program, and 95% found mentors accessible. Mentees who exited the program reported they most valued the one-on-one mentoring and viewed the experience positively regardless of promotion. Retention of assistant professors improved after initiation of the program; four of 13 hired from 2002 to 2006 left the institution, whereas 18 of 18 hired from 2007 to 2014 were retained. LESSONS LEARNED: This multifaceted mentoring program appeared to bolster satisfaction and enhance retention of junior pediatric faculty. Mentees reported increased understanding of the criteria for promotion and viewed the program as a positive experience regardless of career path. Individual mentor-mentee meetings were needed at least twice yearly to establish the mentoring relationship. Identifying "next steps" at the end of individual meetings was helpful to hold both parties accountable for progress. Mentees most valued workshops fostering development of tangible skills (such as scientific writing) and those clarifying the criteria for promotion more transparent. Facilitated peer-group mentoring for mentees at the instructor rank provided valuable peer support.

A feasibility study to evaluate breast cancer patients' knowledge of their diagnosis and treatment.

OBJECTIVE: To evaluate the feasibility of an electronic survey to assess patients' knowledge of their breast cancer and treatment, and interest in receiving a medical summary. METHODS: Women undergoing breast cancer treatment completed an interviewer-administered electronic survey in person or by telephone. Medical records were abstracted to evaluate knowledge accuracy. RESULTS: Among 38 eligible patients approached for the study, 35 (92%) participated and 33 (94%) completed the survey. Participants' perceived knowledge tended to be greater than their actual knowledge. Reporting of clinicopathologic features was most accurate for stage (91%) and lymph node status (88%), and least accurate for tumor size (61%), type (61%), and grade (33%). Accurate reporting of tumor receptor over-expression varied from 76% (estrogen receptor) to 39% (progesterone receptor). Many patients correctly recalled general treatment modalities and details of surgery; fewer recalled details of radiation and chemotherapy. Importantly, nearly all (32/33) were interested in receiving a breast cancer medical summary. CONCLUSION: An electronic survey is feasible to assess breast cancer patients' knowledge. This data suggest that patients have gaps in knowledge and would like a personalized medical summary. PRACTICE IMPLICATIONS: Larger studies are needed to validate and characterize knowledge gaps, and test interventions to improve physician-patient information sharing.

Endogenous regulation of cardiovascular function by apelin-APJ.

Studies have shown significant cardiovascular effects of exogenous apelin administration, including the potent activation of cardiac contraction. However, the role of the endogenous apelin-APJ pathway is less clear. To study the loss of endogenous apelin-APJ signaling, we generated mice lacking either the ligand (apelin) or the receptor (APJ). Apelin-deficient mice were viable, fertile, and showed normal development. In contrast, APJ-deficient mice were not born in the expected Mendelian ratio, and many showed cardiovascular developmental defects. Under basal conditions, both apelin and APJ null mice that survived to adulthood manifested modest decrements in contractile function. However, with exercise stress both mutant lines demonstrated consistent and striking decreases in exercise capacity. To explain these findings, we explored the role of autocrine signaling in vitro using field stimulation of isolated left ventricular cardiomyocytes lacking either apelin or APJ. Both groups manifested less sarcomeric shortening and impaired velocity of contraction and relaxation with no difference in calcium transient. Taken together, these results demonstrate that endogenous apelin-APJ signaling plays a modest role in maintaining basal cardiac function in adult mice with a more substantive role during conditions of stress. In addition, an autocrine pathway seems to exist in myocardial cells, the ablation of which reduces cellular contraction without change in calcium transient. Finally, differences in the developmental phenotype between apelin and APJ null mice suggest the possibility of undiscovered APJ ligands or ligand-independent effects of APJ.

In vivo genetic profiling and cellular localization of apelin reveals a hypoxia-sensitive, endothelial-centered pathway activated in ischemic heart failure.

Signaling by the peptide ligand apelin and its cognate G protein-coupled receptor APJ has a potent inotropic effect on cardiac contractility and modulates systemic vascular resistance through nitric oxide-dependent signaling. In addition, there is evidence for counterregulation of the angiotensin and vasopressin pathways. Regulatory stimuli of the apelin-APJ pathway are of obvious importance but remain to be elucidated. To better understand the physiological response of apelin-APJ to disease states such as heart failure and to elucidate the mechanism by which such a response might occur, we have used the murine model of left anterior descending coronary artery ligation-induced ischemic cardiac failure. To identify the key cells responsible for modulation and production of apelin in vivo, we have created a novel apelin-lacZ reporter mouse. Data from these studies demonstrate that apelin and APJ are upregulated in the heart and skeletal muscle following myocardial injury and suggest that apelin expression remains restricted to the endothelium. In cardiac failure, endothelial apelin expression correlates with other hypoxia-responsive genes, and in healthy animals both apelin and APJ are markedly upregulated in various tissues following systemic hypoxic exposure. Experiments with cultured endothelial cells in vitro show apelin mRNA and protein levels to be increased by hypoxia, through a hypoxia-inducible factor-mediated pathway. These studies suggest that apelin-expressing endothelial cells respond to conditions associated with heart failure, possibly including local tissue hypoxia, and modulate apelin-APJ expression to regulate cardiovascular homeostasis. The apelin-APJ pathway may thus provide a mechanism for systemic endothelial monitoring of tissue perfusion and adaptive regulation of cardiovascular function.

Tartrate-resistant acid phosphatase 5b activity is a useful bone marker for monitoring bone metastases in breast cancer patients after treatment.

Metabolic markers of bone metabolism may be useful for the diagnosis and monitoring of bone metastasis in breast cancer patients. Serum tartrate-resistant acid phosphatase 5b (TRACP5b) activity is a novel bone resorption marker. The treatment response of serum TRACP5b activity, bone alkaline phosphatase (BAP) activity, and concentrations of NH(2)-terminal telopeptide of type 1 collagen (NTX) in 68 breast cancer patients with bone metastasis were determined. These patients were treated and followed up as clinically indicated. Fifty-four healthy women were recruited as control. Serum TRACP5b activity, BAP activity, and NTX level of breast cancer patients with bone metastasis were significantly higher than those of normal controls. In normal subjects, serum TRACP5b activity and NTX level are significantly correlated (P < 0.0001). Neither was correlated with BAP activity. In breast cancer patients with bone metastasis, all marker pairs correlated to each other significantly (P < 0.0001). Biomarkers were examined repeatedly in 38 patients who were evaluable for treatment response. Based on clinical criteria, 20 patients were responders and 18 were nonresponders. In the 20 responders, serum TRACP5b activity and NTX level decreased significantly (P < 0.0001 and 0.0107, respectively) after treatment. In the 18 nonresponders, only NTX level showed significant increase (P = 0.0342) after treatment; TRACP5b and BAP were unchanged. By means of multiple logistic regression with stepwise selection, we determined that TRACP5b activity has a higher probability than NTX level to indicate treatment response as a function of percent change after treatment (18 times versus 12 times). Our data support the use of either TRACP5b activity or NTX level to follow up breast cancer patients with bone metastasis after treatment instead of the prevailing BAP activity.

Development of immunoassays for serum tartrate-resistant acid phosphatase isoform 5a.

BACKGROUND: Serum tartrate-resistant acid phosphatase (TRACP) consists of 2 structurally related isoforms, TRACP 5a and 5b. TRACP 5b is from bone-resorbing osteoclasts. TRACP 5a may be a macrophage product of inflammation. We used a novel antibody to TRACP 5a to standardize immunoassays for serum TRACP 5a activity and protein. METHODS: Biotinylated anti-TRACP antibodies were used to immobilize serum TRACP isoforms. TRACP activity was measured using 4-nitrophenyl phosphate as substrate. TRACP 5a protein was measured with an independent peroxidase-conjugated anti-TRACP antibody. Immunoassays were standardized for linearity of serum dose response, sensitivity and precision. Reference ranges for TRACP 5a were established from serum of 50 healthy males and 50 healthy age-matched females. Serum TRACP 5a activity and protein were determined in 29 cases of rheumatoid arthritis. RESULTS: Serum matrix interference in both TRACP 5a assays required dilution to 10% serum to approach linearity. Intra-assay and inter-assay CV% were <10%. Mean serum TRACP 5a activity and protein were significantly higher in healthy men than women. There was a slight, but significant age related increase in both serum TRACP 5a and 5b among females, but not males, from age 20 to 70 years. TRACP 5a activity was positively correlated to TRACP 5a protein in healthy sera. Neither TRACP 5a activity nor protein was correlated strongly to TRACP-5b activity. TRACP 5a protein was significantly increased in 8/29 RA sera, whereas TRACP 5a and 5b activities were not. TRACP 5a activity and protein were not significantly correlated in RA sera. CONCLUSIONS: Although TRACP 5a and 5b are related biosynthetically, their circulating levels in healthy humans were independent, suggesting differential regulation of expression. In chronic diseases, increased TRACP 5a may represent pathological processes of inflammation unrelated to bone metabolism.

Pathway analysis of coronary atherosclerosis.

Large-scale gene expression studies provide significant insight into genes differentially regulated in disease processes such as cancer. However, these investigations offer limited understanding of multisystem, multicellular diseases such as atherosclerosis. A systems biology approach that accounts for gene interactions, incorporates nontranscriptionally regulated genes, and integrates prior knowledge offers many advantages. We performed a comprehensive gene level assessment of coronary atherosclerosis using 51 coronary artery segments isolated from the explanted hearts of 22 cardiac transplant patients. After histological grading of vascular segments according to American Heart Association guidelines, isolated RNA was hybridized onto a customized 22-K oligonucleotide microarray, and significance analysis of microarrays and gene ontology analyses were performed to identify significant gene expression profiles. Our studies revealed that loss of differentiated smooth muscle cell gene expression is the primary expression signature of disease progression in atherosclerosis. Furthermore, we provide insight into the severe form of coronary artery disease associated with diabetes, reporting an overabundance of immune and inflammatory signals in diabetics. We present a novel approach to pathway development based on connectivity, determined by language parsing of the published literature, and ranking, determined by the significance of differentially regulated genes in the network. In doing this, we identify highly connected "nexus" genes that are attractive candidates for therapeutic targeting and followup studies. Our use of pathway techniques to study atherosclerosis as an integrated network of gene interactions expands on traditional microarray analysis methods and emphasizes the significant advantages of a systems-based approach to analyzing complex disease.

Tartrate-resistant acid phosphatase 5b is a useful serum marker for extensive bone metastasis in breast cancer patients.

PURPOSE: Previous studies showed that serum tartrate-resistant acid phosphatase 5b (TRACP5b) activity was increased in 70% to 94% of breast cancer (BC) patients with bone metastasis (BM). This study aims to determine whether serum TRACP5b is useful for identifying limited or extensive BM in BC patients. EXPERIMENTAL DESIGN: Serum TRACP5b activity was measured in 168 BC patients, including 81 who were newly diagnosed with early BC, 20 with extraosseous metastasis, 24 with limited BM, and 43 with extensive BM. Serum TRACP5b activity was also measured monthly in 151 patients with early BC until they developed BM. Four hundred and twenty-seven (427) healthy women ages 18 to 90 served as control. One-way ANOVA was used to compare serum TRACP5b among groups. The sensitivity and specificity of serum TRACP5b as a marker for BM were estimated by receiver operator characteristic (ROC) curves. RESULTS: Serum TRACP5b increased with age in healthy women ( P < 0.0001). It was significantly elevated in patients with extensive BM compared with all other groups ( P < 0.0001). ROC analysis established a cutoff value of 4.026 units/L to identify patients with extensive BM with a specificity of 98% and a sensitivity of 93% (area under the curve = 0.9807; 95% CI = 0.9698-0.9915). Among the 151 patients with early BC, 6 developed limited BM and 2 developed extensive BM during the follow-up period. Serum TRACP5b remained below the cutoff value in patients with limited BM, but became significantly increased in those whose BM became extensive. CONCLUSION: Serum TRACP5b activity is a useful diagnostic marker for extensive BM in patients with BC.

Novel role for the potent endogenous inotrope apelin in human cardiac dysfunction.

BACKGROUND: Apelin is among the most potent stimulators of cardiac contractility known. However, no physiological or pathological role for apelin-angiotensin receptor-like 1 (APJ) signaling has ever been described. METHODS AND RESULTS: We performed transcriptional profiling using a spotted cDNA microarray with 12 814 unique clones on paired samples of left ventricle obtained before and after placement of a left ventricular assist device in 11 patients. The significance analysis of microarrays and a novel rank consistency score designed to exploit the paired structure of the data confirmed that natriuretic peptides were among the most significantly downregulated genes after offloading. The most significantly upregulated gene was the G-protein-coupled receptor APJ, the specific receptor for apelin. We demonstrate here using immunoassay and immunohistochemical techniques that apelin is localized primarily in the endothelium of the coronary arteries and is found at a higher concentration in cardiac tissue after mechanical offloading. These findings imply an important paracrine signaling pathway in the heart. We additionally extend the clinical significance of this work by reporting for the first time circulating human apelin levels and demonstrating increases in the plasma level of apelin in patients with left ventricular dysfunction. CONCLUSIONS: The apelin-APJ signaling pathway emerges as an important novel mediator of cardiovascular control.

Phase II trial combining paclitaxel with 24-hour infusion cisplatin for chemotherapy-naïve patients with locally advanced or metastatic breast carcinoma.

BACKGROUND: Both paclitaxel and cisplatin are active as second-line chemotherapy for patients with breast carcinoma. A synergistic cytotoxicity of these two drugs has been demonstrated in vitro. This study sought to determine the efficacy of combining these two drugs in the treatment of chemotherapy-naïve patients with breast carcinoma. METHODS: The inclusion criteria for the study were 1) women with histologically proven breast carcinoma; 2) locally advanced disease, as defined by American Joint Committee on Cancer (AJCC) Stage T4 (locally advanced breast carcinoma [LABC]) or clinically proven metastases (metastatic breast carcinoma [MBC]); and 3) no prior cytotoxic chemotherapy. The regimen consisted of paclitaxel 175 mg/m(2) intravenously by 3-hour infusion immediately followed by cisplatin 50 mg/m(2) intravenously by 24-hour infusion on Day 1 and repeated every 3 weeks. After a maximal response to chemotherapy was achieved, patients with LABC underwent resection of their primary tumor if the procedure was not contraindicated. RESULTS: From July, 1999 to January, 2001, 46 patients were enrolled into this study (28 patients with LABC and 18 patients with MBC). Their median age was 49.5 years (range, 29.8-65.5 years). A total of 205 cycles of chemotherapy were given. All patients were evaluable for toxicity, and 45 patients were evaluable for response. There were 3 complete responses (CRs) and 24 partial responses (PRs), for an overall response rate of 58.7% (95% confidence interval, 44.5-72.9%). Grade 4 hypersensitivity (asthma) to paclitaxel occurred in one patient. Grade 3-4 nausea and emesis and Grade 3-4 myelosuppression occurred in six patients and four patients, respectively. Of the 28 patients with LABC, 2 patients achieved a CR, and 14 patients achieved a PR. Twenty-seven patients underwent mastectomy patients after chemotherapy. A pathologic CR was documented in one patient. Postoperatively, 23 patients with LABC received adjuvant chemotherapy, and 18 patients with LABC received adjuvant radiotherapy. During a median follow-up of 14.6 months, 5 of 28 patients with LABC developed recurrent disease, and 2 patients died of progressive disease, whereas 3 of 18 patients with MBC died of progressive disease. CONCLUSIONS: The combination of paclitaxel by 3-hour infusion and cisplatin by 24-hour infusion appears to be an active and well-tolerated regimen for chemotherapy-naïve patients with LABC or MBC.