Doug Landsittel

Expression of Myopodin Induces Suppression of Tumor Growth and Metastasis

Myopodin was previously reported as a gene that was frequently deleted in prostate cancer. This gene shares significant homology with a cell shape-regulating gene, synaptopodin. Myopodin was shown to bind actin and to induce actin bundling when cells were stimulated. To clarify the functional role of myopodin in prostate cancer, several assays were performed to evaluate the tumor suppression activity of myopodin. Our results indicate that myopodin inhibits tumor growth and invasion both in vitro and in vivo. The activity of tumor suppression of myopodin is located at the C-terminus region. To further evaluate the role of myopodin in suppressing the invasiveness of prostate cancer, an expression analysis of myopodin protein was performed in prostate tissues. The results indicate that down-regulation of myopodin expression occurs mostly in invasive stages of prostate cancer, implying a potential invasion suppression role for myopodin in prostate cancer. In addition, hemizygous deletion and down-regulation of myopodin expression occur in three aggressive prostate cancer cell lines. All these results support the hypothesis that myopodin functions as a tumor suppressor gene to limit the growth and to inhibit the metastasis of cancer cells.

Long-term effects of alemtuzumab on regulatory and memory T-cell subsets in kidney transplantation.

Background. Induction with lymphocyte-depleting antibodies is routinely used to prevent rejection but often skews T cells toward memory. It is not fully understood which memory and regulatory T-cell subsets are most affected and how they relate to clinical outcomes. Methods. We analyzed T cells from 57 living-donor renal transplant recipients (12 reactive and 45 quiescent) 2.8±1.4 years after alemtuzumab induction. Thirty-four healthy subjects and nine patients with acute cellular rejection (ACR) were also studied. Results. We found that alemtuzumab caused protracted CD4 more than CD8 T-lymphocyte deficiency, increased proportion of CD4+ memory T cells, and decreased proportion of CD4+ regulatory T cells. Reactive patients exhibited higher proportions of CD4+ effector memory T cells (TEM) and CD8+ terminally differentiated TEM (TEMRA), with greater CD4+ TEM and CD8+ TEMRA to regulatory T cell ratios, than quiescent patients or healthy controls. Patients with ongoing ACR had profound reduction in circulating CD8+ TEMRA. Mixed lymphocyte assays showed significantly lower T-cell proliferation to donor than third-party antigens in the quiescent group, while reactive and ACR patients exhibited increased effector molecules in CD8+ T cells. Conclusions. Our findings provide evidence that T-cell skewing toward TEM may be associated with antigraft reactivity long after lymphodepletion. Further testing of TEM and TEMRA subsets as rejection predictors is warranted.

Correlation of Protease-Activated Receptor-1 With Differentiation Markers in Squamous Cell Carcinoma of the Head and Neck and Its Implication in Lymph Node Metastasis

Purpose: Protease-activated receptor-1 (PAR-1) is a G-protein-coupled receptor that contributes to multiple signal transduction pathways. Although the functions of PAR-1 in many normal cells, such as platelets and astrocytes, have been well studied, its roles in cancer progression and metastasis have not been fully elucidated, and studies to date appear contradictory. Experimental Design: To clarify the function of PAR-1 in metastasis of squamous cell carcinoma of the head and neck (SCCHN), we examined PAR-1 expression in clinical specimens by immunohistochemistry and in SCCHN cell lines by immunoblotting. Furthermore, par-1 cDNA-transfected SCCHN cell lines were also used to verify PAR-1–mediated pathway. Results: The metastatic tumors showed a lower percentage of PAR-1–positive cells (46%) and lower levels of PAR-1 expression (median weight index = 10) than node negative primary tumors (80% and median weight index = 60, respectively). In addition, expression level of PAR-1 positively correlated with levels of keratinocyte differentiation markers keratin-1, -10, and -11. Additional studies using sense and antisense par-1 cDNA–transfected SCCHN cell lines illustrated that the presence of PAR-1 was required for the expression of involucrin, a keratinocyte differentiation marker. PAR-1 expression also contributes to activation of the mitogen-activated protein kinase (MAPK) pathway. Blocking MAPK activation by a mitogen-activated protein/extracellular signal-regulated kinase inhibitor, not by a phosphatidylinositol 3′-kinase inhibitor, reduced level of involucrin, suggesting that regulation of involucrin by PAR-1 is partially through the MAPK signaling pathway. Conclusions: Our study suggests that PAR-1 signaling induces differentiation markers in SCCHN cells, and its expression is conversely correlated with cervical lymph node metastasis.

Segmentation of Individual Renal Cysts from MR Images in Patients with Autosomal Dominant Polycystic Kidney Disease

OBJECTIVE To evaluate the performance of a semi-automated method for the segmentation of individual renal cysts from magnetic resonance (MR) images in patients with autosomal dominant polycystic kidney disease (ADPKD). DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS This semi-automated method was based on a morphologic watershed technique with shape-detection level set for segmentation of renal cysts from MR images. T2-weighted MR image sets of 40 kidneys were selected from 20 patients with mild to moderate renal cyst burden (kidney volume < 1500 ml) in the Consortium for Radiologic Imaging Studies of Polycystic Kidney Disease (CRISP). The performance of the semi-automated method was assessed in terms of two reference metrics in each kidney: the total number of cysts measured by manual counting and the total volume of cysts measured with a region-based thresholding method. The proposed and reference measurements were compared using intraclass correlation coefficient (ICC) and Bland-Altman analysis. RESULTS Individual renal cysts were successfully segmented with the semi-automated method in all 20 cases. The total number of cysts in each kidney measured with the two methods correlated well (ICC, 0.99), with a very small relative bias (0.3% increase with the semi-automated method; limits of agreement, 15.2% reduction to 17.2% increase). The total volume of cysts measured using both methods also correlated well (ICC, 1.00), with a small relative bias of <10% (9.0% decrease in the semi-automated method; limits of agreement, 17.1% increase to 43.3% decrease). CONCLUSION This semi-automated method to segment individual renal cysts in ADPKD kidneys provides a quantitative indicator of severity in early and moderate stages of the disease.

Automated Segmentation of Kidneys from MR Images in Patients with Autosomal Dominant Polycystic Kidney Disease

BACKGROUND AND OBJECTIVES Our study developed a fully automated method for segmentation and volumetric measurements of kidneys from magnetic resonance images in patients with autosomal dominant polycystic kidney disease and assessed the performance of the automated method with the reference manual segmentation method. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS Study patients were selected from the Consortium for Radiologic Imaging Studies of Polycystic Kidney Disease. At the enrollment of the Consortium for Radiologic Imaging Studies of Polycystic Kidney Disease Study in 2000, patients with autosomal dominant polycystic kidney disease were between 15 and 46 years of age with relatively preserved GFRs. Our fully automated segmentation method was on the basis of a spatial prior probability map of the location of kidneys in abdominal magnetic resonance images and regional mapping with total variation regularization and propagated shape constraints that were formulated into a level set framework. T2-weighted magnetic resonance image sets of 120 kidneys were selected from 60 patients with autosomal dominant polycystic kidney disease and divided into the training and test datasets. The performance of the automated method in reference to the manual method was assessed by means of two metrics: Dice similarity coefficient and intraclass correlation coefficient of segmented kidney volume. The training and test sets were swapped for crossvalidation and reanalyzed. RESULTS Successful segmentation of kidneys was performed with the automated method in all test patients. The segmented kidney volumes ranged from 177.2 to 2634 ml (mean, 885.4±569.7 ml). The mean Dice similarity coefficient ±SD between the automated and manual methods was 0.88±0.08. The mean correlation coefficient between the two segmentation methods for the segmented volume measurements was 0.97 (P<0.001 for each crossvalidation set). The results from the crossvalidation sets were highly comparable. CONCLUSIONS We have developed a fully automated method for segmentation of kidneys from abdominal magnetic resonance images in patients with autosomal dominant polycystic kidney disease with varying kidney volumes. The performance of the automated method was in good agreement with that of manual method.

Banff Histopathological Consensus Criteria for Preimplantation Kidney Biopsies.

The Banff working group on preimplantation biopsy was established to develop consensus criteria (best practice guidelines) for the interpretation of preimplantation kidney biopsies. Digitally scanned slides were used (i) to evaluate interobserver variability of histopathologic findings, comparing frozen sections with formalin‐fixed, paraffin‐embedded tissue of wedge and needle core biopsies, and (ii) to correlate consensus histopathologic findings with graft outcome in a cohort of biopsies from international medical centers. Intraclass correlations (ICCs) and univariable and multivariable statistical analyses were performed. Good to fair reproducibility was observed in semiquantitative scores for percentage of glomerulosclerosis, arterial intimal fibrosis and interstitial fibrosis on frozen wedge biopsies. Evaluation of frozen wedge and core biopsies was comparable for number of glomeruli, but needle biopsies showed worse ICCs for glomerulosclerosis, interstitial fibrosis and tubular atrophy. A consensus evaluation form is provided to help standardize the reporting of histopathologic lesions in donor biopsies. It should be recognized that histologic parameters may not correlate with graft outcome in studies based on organs deemed to be acceptable after careful clinical assessment. Significant limitations remain in the assessment of implantation biopsies.

Menopausal Hot Flashes and Carotid Intima Media Thickness Among Midlife Women

Background and Purpose— There has been a longstanding interest in the role of menopause and its correlates in the development of cardiovascular disease (CVD) in women. Menopausal hot flashes are experienced by most midlife women; emerging data link hot flashes to CVD risk indicators. We tested whether hot flashes, measured via state-of-the-art physiologic methods, were associated with greater subclinical atherosclerosis as assessed by carotid ultrasound. We considered the role of CVD risk factors and estradiol concentrations in these associations. Methods— A total of 295 nonsmoking women free of clinical CVD underwent ambulatory physiologic hot flash assessments; a blood draw; and carotid ultrasound measurement of intima media thickness and plaque. Associations between hot flashes and subclinical atherosclerosis were tested in regression models controlling for CVD risk factors and estradiol. Results— More frequent physiologic hot flashes were associated with higher carotid intima media thickness (for each additional hot flash: &bgr; [SE]=0.004 [0.001]; P=0.0001; reported hot flash: &bgr; [SE]=0.008 [0.002]; P=0.002, multivariable) and plaque (eg, for each additional hot flash, odds ratio [95% confidence interval] plaque index ≥2=1.07 [1.003–1.14]; P=0.04, relative to no plaque, multivariable] among women reporting daily hot flashes; associations were not accounted for by CVD risk factors or by estradiol. Among women reporting hot flashes, hot flashes accounted for more variance in intima media thickness than most CVD risk factors. Conclusions— Among women reporting daily hot flashes, frequent hot flashes may provide information about a woman’s vascular status beyond standard CVD risk factors and estradiol. Frequent hot flashes may mark a vulnerable vascular phenotype among midlife women.

Physiologically assessed hot flashes and endothelial function among midlife women

Objective: Hot flashes are experienced by most midlife women. Emerging data indicate that they may be associated with endothelial dysfunction. No studies have tested whether hot flashes are associated with endothelial function using physiologic measures of hot flashes. We tested whether physiologically assessed hot flashes were associated with poorer endothelial function. We also considered whether age modified associations. Methods: Two hundred seventy-two nonsmoking women reporting either daily hot flashes or no hot flashes, aged 40 to 60 years, and free of clinical cardiovascular disease, underwent ambulatory physiologic hot flash and diary hot flash monitoring; a blood draw; and ultrasound measurement of brachial artery flow-mediated dilation to assess endothelial function. Associations between hot flashes and flow-mediated dilation were tested in linear regression models controlling for lumen diameter, demographics, cardiovascular disease risk factors, and estradiol. Results: In multivariable models incorporating cardiovascular disease risk factors, significant interactions by age (P < 0.05) indicated that among the younger tertile of women in the sample (age 40-53 years), the presence of hot flashes (beta [standard error] = −2.07 [0.79], P = 0.01), and more frequent physiologic hot flashes (for each hot flash: beta [standard error] = −0.10 [0.05], P = 0.03, multivariable) were associated with lower flow-mediated dilation. Associations were not accounted for by estradiol. Associations were not observed among the older women (age 54-60 years) or for self-reported hot flash frequency, severity, or bother. Among the younger women, hot flashes explained more variance in flow-mediated dilation than standard cardiovascular disease risk factors or estradiol. Conclusions: Among younger midlife women, frequent hot flashes were associated with poorer endothelial function and may provide information about womens vascular status beyond cardiovascular disease risk factors and estradiol.

Elevated ST2 Distinguishes Incidences of Pediatric Heart and Small Bowel Transplant Rejection.

Elevated serum soluble (s) suppressor of tumorigenicity‐2 is observed during cardiovascular and inflammatory bowel diseases. To ascertain whether modulated ST2 levels signify heart (HTx) or small bowel transplant (SBTx) rejection, we quantified sST2 in serially obtained pediatric HTx (n = 41) and SBTx recipient (n = 18) sera. At times of biopsy‐diagnosed HTx rejection (cellular and/or antibody‐mediated), serum sST2 was elevated compared to rejection‐free time points (1714 ± 329 vs. 546.5 ± 141.6 pg/mL; p = 0.0002). SBTx recipients also displayed increased serum sST2 during incidences of rejection (7536 ± 1561 vs. 2662 ± 543.8 pg/mL; p = 0.0347). Receiver operator characteristic (ROC) analysis showed that serum sST2 > 600 pg/mL could discriminate time points of HTx rejection and nonrejection (area under the curve [AUC] = 0.724 ± 0.053; p = 0.0003). ROC analysis of SBTx measures revealed a similar discriminative capacity (AUC = 0.6921 ± 0.0820; p = 0.0349). Quantitative evaluation of both HTx and SBTx biopsies revealed that rejection significantly increased allograft ST2 expression. Pathway and Network Analysis of biopsy data pinpointed ST2 in the dominant pathway modulated by rejection and predicted tumor necrosis factor‐α and IL‐1β as upstream activators. In total, our data indicate that alloimmune‐associated pro‐inflammatory cytokines increase ST2 during rejection. They also demonstrate that routine serum sST2 quantification, potentially combined with other biomarkers, should be investigated further to aid in the noninvasive diagnosis of rejection.

Changes in heart rate variability during vasomotor symptoms among midlife women

Objective:Most midlife women report vasomotor symptoms (VMS), yet their physiology remains poorly understood. This study tested whether acute decreases in cardiac vagal control would occur with VMS in a large sample of women monitored during wake and sleep. Methods:Two hundred and fifteen nonsmoking women aged 40 to 60 years with evidence of VMS were included. Women were free of a history of clinical cardiovascular disease or arrhythmia; or use of insulin, beta blockers, calcium channel blockers, or medications impacting VMS. Women underwent 24 hours of ambulatory monitoring for physiological (sternal skin conductance) and self-report (electronic diary) measurement of VMS; heart rate variability (electrocardiogram); and respiratory rate. Changes in cardiac vagal control as assessed by respiratory sinus arrhythmia during VMS, relative to periods before and after VMS, were tested in linear mixed models. Results:Significant decreases in respiratory sinus arrhythmia were observed during physiologically measured VMS relative to periods preceding (b[SE] = 0.13 (0.004), P < 0.0001) and after the vasomotor symptoms (b[SE] = 0.13 (0.004), P < 0.0001), adjusted for age, race, body mass index, and sleep/wake status. Decreases were observed for women not aware of their VMS, and differences persisted controlling for respiration rate. Interactions indicated that respiratory sinus arrhythmia decreases were most pronounced during sleep and for younger women. Conclusions:Physiologically measured VMS were accompanied by an inhibition of cardiac vagal control in a large sample of women. Changes were observed irrespective of whether the VMS were reported, were most pronounced during sleep, and were greatest among younger women. These findings contribute to the understanding of vasomotor symptom physiology.