Beware of the Film Findings!: Low Back Pain Patients and Healthy Volunteers Have A Similar X-Ray Appearance.

Low back pain (LBP) affects 20 to 30% of the United States general population, making it the fifth most common reason for all physician visits in the U.S. The resultant financial burden on the U.S. health care industry continues to rise each year with recent estimates of over $80 billion spent annually. However, despite the dramatic rise in health care utilization and costs for this complaint, self-reported outcomes have not improved. The radiographic analysis of a healthy asymptomatic population vs. a cohort of LBP patients with lumbar degenerative disc disease, specifically as it relates to posture, spinal alignments, including lumbar lordosis, as well as other findings, may provide insight to more effectively care for the LBP patient and, in return, possibly help to rein in related health care costs.

Sabutoclax, a Mcl-1 antagonist, inhibits tumorigenesis in transgenic mouse and human xenograft models of prostate cancer.

Resistance to available therapeutic agents has been a common problem thwarting progress in treatment of castrate-resistant and metastatic prostate cancer (PCa). Overexpression of the Bcl-2 family members, including Mcl-1, in PCa cells is known to inhibit intracellular mitochondrial-dependent apoptosis. Here we report the development of a novel transgenic mouse model that spontaneously develops prostatic intraepithelial neoplasia and adenocarcinoma by the inducible, conditional knockout of transforming growth factor ß receptor type II in stromal fibroblastic cells (Tgfbr2(ColTKO)). The Tgfbr2(ColTKO) prostate epithelia demonstrated down-regulation of luminal and basal differentiation markers, as well as Pten expression and up-regulation of Mcl-1. However, unlike in men, Tgfbr2(ColTKO) prostates exhibited no regression acutely after castration. The administration of Sabutoclax (BI-97C1), a pan-active Bcl-2 protein family antagonist mediated apoptosis in castrate-resistant PCa cells of Tgfbr2(ColTKO) mice and human subcutaneous, orthotopic, and intratibial xenograft PCa models. Interestingly, Sabutoclax had little apoptotic effect on benign prostate tissue in Tgfbr2(ColTKO) and wild-type mice. Sabutoclax was able to block c-Met activation, a critical axis in PCa metastatic progression. Further, Sabutoclax synergistically sensitized PC-3 cells to the cytotoxic effects of docetaxel (Taxotere). Together, these data suggest that Sabutoclax inhibits castrate-resistant PCa alone at the primary and bone metastatic site as well as support sensitivity to docetaxel treatment.

Lumbar spinal stenosis: treatment options for an aging population.

The fastest growing age group in the United States is the 45 and older population. Due to the nature of the aging lumbar spine, a significant majority of this population will experience low back pain (LBP) and symptoms associated with lumbar spinal stenosis. Accurate diagnosis and appropriate treatment is required if this particular aging group of our population is to maintain an active and productive life into their later years.

Role for stromal heterogeneity in prostate tumorigenesis.

Prostate cancer develops through a stochastic mechanism whereby precancerous lesions on occasion progress to multifocal adenocarcinoma. Analysis of human benign and cancer prostate tissues revealed heterogeneous loss of TGF-ß signaling in the cancer-associated stromal fibroblastic cell compartment. To test the hypothesis that prostate cancer progression is dependent on the heterogeneous TGF-ß responsive microenvironment, a tissue recombination experiment was designed in which the ratio of TGF-ß responsive and nonresponsive stromal cells was varied. Although 100% TGF-ß responsive stromal cells supported benign prostate growth and 100% TGF-ß nonresponsive stromal cells resulted in precancerous lesions, only the mixture of TGF-ß responsive and nonresponsive stromal cells resulted in adenocarcinoma. A computational model was used to resolve a mechanism of tumorigenic progression in which proliferation and invasion occur in two independent steps mediated by distinct stromally derived paracrine signals produced by TGF-ß nonresponsive and responsive stromal cells. Complex spatial relationships of stromal and epithelial cells were incorporated into the model on the basis of experimental data. Informed by incorporation of experimentally derived spatial parameters for complex stromal-epithelial relationships, the computational model indicated ranges for the relative production of paracrine factors by each cell type and provided bounds for the diffusive range of the molecules. Because SDF-1 satisfied model predictions for an invasion-promoting paracrine factor, a more focused computational model was subsequently used to investigate whether SDF-1 was the invasion signal. Simulations replicating SDF-1 expression data revealed the requirement for cooperative SDF-1 expression, a prediction supported biologically by heterotypic stromal interleukin-1ß signaling between fibroblastic cell populations. The cancer stromal field effect supports a functional role for the unaltered fibroblasts as a cooperative mediator of cancer progression.

Altered TGF-ß signaling in a subpopulation of human stromal cells promotes prostatic carcinogenesis.

Carcinoma-associated fibroblasts (CAF) play a critical role in malignant progression. Loss of TGF-ß receptor II (TGFßR2) in the prostate stroma is correlated with prostatic tumorigenesis. To determine the mechanisms by which stromal heterogeneity because of loss of TGFßR2 might contribute to cancer progression, we attenuated transforming growth factor beta (TGF-ß) signaling in a subpopulation of immortalized human prostate fibroblasts in a model of tumor progression. In a tissue recombination model, loss of TGFßR2 function in 50% of the stromal cell population resulted in malignant transformation of the nontumorigenic human prostate epithelial cell line BPH1. Mixing fibroblasts expressing the empty vector and dominant negative TGFßR2 increased the expression of markers of myofibroblast differentiation [coexpression of vimentin and alpha smooth muscle actin (αSMA)] through elevation of TGF-ß1 and activation of the Akt pathway. In combination, these two populations of stromal cells recapitulated the tumor inductive activity of CAFs. TGFßR2 activity in mixed stromal cell populations cultured in vitro caused secretion of factors that are known to promote tumor progression, including TGF-ß1, SDF1/CXCL12, and members of the fibroblast growth factor (FGF) and bone morphogenetic protein (BMP) families. In vivo, tissue recombination of fibroblasts overexpressing TGF-ß1 and SDF1/CXCL12 not only induced transformation of BPH1 cells, but also promoted a robust growth of highly invasive cells, similar to effects produced by CAFs. While the precise nature and/or origin of the particular stromal cell populations in vivo remain unknown, these findings strongly link heterogeneity in TGF-ß signaling to tumor promotion by tumor stromal cells.

Seeing the spine in 3D: how will it change what we do?

BACKGROUND: The Scoliosis Research Society (SRS) has appointed a committee to evaluate the clinical relevance and impact of 3D analysis on scoliotic deformities and to develop a 3D classification of adolescent idiopathic scoliosis (AIS). The goal of this article is to summarize and present the work done in recent years within this committee and show how 3D analysis of AIS has the potential to change our current methods to analyse and treat scoliosis. METHODS: A database of 600 3D reconstructions of the spine of patients with AIS has been established using calibrated PA and lateral radiographs obtained from either digital radiographs or the EOS system. The 3D reconstructions were done using dedicated software and analyzed with the "da Vinci" view, a schematic top view representation of the 3D reconstructions, which summarizes the position of the End-Apex-End vertebrae planes (planes of maximum curvature). RESULTS: Preliminary work was done using 3D reconstructions in 409 patients with AIS. Fuzzy clustering techniques were used to show that the cohort could be segmented in 5 easily differentiated curve patterns similar to those of the Lenke and King classifications. Two subsequent articles have shown that 3D reconstructions can be divided in different groups based on the location of the plane of maximum curvature of their curves. One study of 66 cases has shown a consistent loss of kyphosis within the 5 thoracic apical vertebrae. Finally, a study of 172 Lenke 1 curves analyzed by ISO Data cluster analysis has confirmed the presence of 2 statistically different subtypes according to the planes passing through the End-Apex-End vertebrae of the main thoracic curve. CONCLUSIONS: The study presented suggests that a valid and clinically useful 3D classification of AIS is within reach. 3D analysis has the potential to improve our comprehension of AIS curve types and automatic 3D classification may help decrease the known variability of current 2D classifications. LEVEL OF EVIDENCE: Level III, systematic review of retrospective comparative studies.

The central hip vertical axis: a reference axis for the Scoliosis Research Society three-dimensional classification of idiopathic scoliosis.

STUDY DESIGN: Reliability comparison of 2 radiographic axis systems by inter- and intraobserver variability. OBJECTIVE: To determine whether the central hip vertical axis (CHVA) provides a more reliable reference axis for the evaluation of scoliosis. SUMMARY OF BACKGROUND DATA: Current practices in the evaluation of the scoliotic spine use the central sacral vertical line (CSVL), a true vertical drawn upward from the middle of S1, to assess the spinal deformity. However, the CVSL is defined only in the coronal radiographic view and has no corresponding definition in the sagittal view. Therefore, it represents a 2-dimensional positioning of the scoliotic segments relative to the pelvis. In view of this limitation, the Scoliosis Research Society 3-dimensional (3D) scoliosis committee proposed the CHVA, a true vertical bisecting the line segment joining the centers of the 2 femoral heads, as a reference line for the 3D evaluation of the spinal deformity. Unlike the CSVL, the CHVA can be identified in both radiographic views (coronal and sagittal) and has been shown to represent the physiologic center of balance of the spino-pelvic unit. METHODS: A vertical axis was established on preoperative radiographs of 68 Lenke 1 main thoracic curves twice by 5 members of the Scoliosis Research Society 3D scoliosis committee assisted by dedicated software. The user digitized separately on the postero-anterior radiographs, the lateral borders of the S1 facets (for the CSVL), and 3 points on the 2 femoral heads (for the CHVA). The software then drew lines representing both axes. Then the observers determined the lumbar modifier (A, B, and C) using both axes. RESULTS.: There was no intra- and interobserver difference in the position of the CHVA (P > 0.1; SD: 0.4 mm), whereas intraobserver differences were found for the CSVL (P < 0.00007; SD: 0.9 mm). The CHVA was more reproducible and showed better intra- and interobserver agreement (kappa: 0.86/0.75; both excellent reliability), when compared with the CSVL (kappa 0.77/0.61; excellent and good reliability, respectively) for the identification of the lumbar modifier. The CSVL was on average 3.2 mm to the left, when compared with the CHVA generating a shift (A-->B-->C) in the assignment of the lumbar modifier. CONCLUSION: The CHVA is more reproducible and showed better intra- and interobserver agreement, when compared with the CSVL for the identification of the lumbar modifier. The CHVA can be easily computed in 3D and represents the physiologic center of balance of the spino-pelvic unit because it takes into account femoral head support. We recommend keeping the CSVL for 2-dimensional measurement to adapt the measures relative to the CSVL to the proposed CHVA axis and adopting CHVA as the reference axis for 3D evaluation of idiopathic scoliosis.

Three-dimensional analysis of thoracic apical sagittal alignment in adolescent idiopathic scoliosis.

STUDY DESIGN: Retrospective review of a series of adolescent idiopathic scoliosis patients. OBJECTIVE: To perform a 3-dimensional (3D) analysis of patients with thoracic scoliosis to identify differences in the thoracic sagittal alignment measured from the standard lateral projection as compared to the "true lateral" view. SUMMARY OF BACKGROUND DATA: It has been difficult to clinically obtain radiographs in the planes of maximum spinal deformity. Recently, 3D models of the spine have been developed using biplanar radiographic reconstructions that allow a more accurate assessment of spinal alignment. METHODS: Three-dimensional spinal reconstructions using biplanar radiographs were used to evaluate the apical thoracic sagittal profile. A measurement of sagittal curvature from 2 vertebral levels above and below the thoracic apex (5 vertebrae) was recorded from the standard lateral view. The 3D reconstructions were then rotated to achieve a "true lateral" view of the apical thoracic vertebra and the sagittal apical curvature was remeasured. The difference in the 2 measures of sagittal thoracic apical alignment was compared using repeated measures ANOVA, and then correlated to the coronal thoracic Cobb magnitude using a Pearson correlation analysis (P < 0.05). RESULTS: Sixty-six adolescent idiopathic scoliosis patients with right thoracic scoliosis (Cobb averaged 47 degrees +/- 10 degrees ) were evaluated. The apical thoracic sagittal curvature in the standard lateral view averaged 11 degrees +/- 10 degrees of kyphosis (range: -8 degrees to 38 degrees ). This was statistically greater (P < 0.001) than the apical sagittal curvature in the "true lateral" view that averaged 1 degrees +/- 9 degrees (range:-23 degrees to 22 degrees ). The standard lateral view was rotated an average of 13 degrees +/- 4 degrees to achieve the ideal lateral view of the thoracic apex. CONCLUSION: This 3D analysis of thoracic scoliosis demonstrated a consistent loss of kyphosis within the 5 thoracic apical vertebrae. The true apical sagittal profile was found to be overestimated by an average of 10 degrees as compared to the perceived alignment from standard lateral radiographs.

Three-dimensional classification of thoracic scoliotic curves.

STUDY DESIGN: Three-dimensional (3D) characterization of the thoracic scoliotic spine (cross-sectional study). OBJECTIVES: To investigate the presence of subgroups within Lenke type-1 curves by evaluating the thoracic segment indices extracted from 3D reconstructions of the spine, and to propose a new clinically relevant means (the daVinci representation) to report 3D spinal deformities. SUMMARY OF BACKGROUND DATA: Although scoliosis is recognized to be a 3D deformity of the spine its measurement and classification have predominantly been based on radiographs which are 2D projections in the coronal and sagittal planes. METHODS: Thoracic segment indices derived from 3D reconstructions of coronal and sagittal standing radiographs of 172 patients with right thoracic adolescent idiopathic scoliosis, reviewed by the 3D Classification Committee of the Scoliosis Research Society, were analyzed using the ISOData unsupervised clustering algorithm. Four curve indices were analyzed: Cobb angle, axial rotation of the apical vertebrae, orientation of the plane of maximum curvature of the main thoracic curve, and kyphosis (T4-T12). No assumptions were made regarding grouping tendencies in the data nor were the number of clusters predefined. RESULTS: Three primary groups were revealed wherein kyphosis and the orientation of the PMC of the main thoracic curve were the major discriminating factors with slight overlap between groups. A small group (G1) of 22 patients having smaller, nonsurgical (minor) curves was identified. Although the remaining patients had similar Cobb angles they were split into 2 groups (G2: 79 patients; G3: 71 patients) with different PMC (G2: 65 degrees -81 degrees ; G3: 76 degrees -104 degrees ) and kyphotic measures (G2: 23 degrees -43 degrees ; G3: 7 degrees -25 degrees). CONCLUSION: Two distinct subgroups within the surgical cases (major curves) of Lenke type-1 curves were found thus suggesting that thoracic curves are not always hypokyphotic. The ISOData cluster analysis technique helped to capture inherent 3D structural curve complexities that were not evident in a 2D radiographic plane. The daVinci representation is a new clinically relevant means to report 3D spinal deformities.

Gene targeting to the stroma of the prostate and bone.

Stromal-epithelial interactions mediated by paracrine signaling mechanisms dictate prostate development and progression of prostate cancer. The regulatory role of androgens in both the prostate stromal and epithelial compartments set the prostate apart from many other organs and tissues with regard to gene targeting. The identification of androgen-dependent prostate epithelial promoters has allowed successful gene targeting to the prostate epithelial compartment. Currently, there are no transgenic mouse models available to specifically alter gene expression within the prostate stromal compartment. As a primary metastatic site for prostate cancer is bone, the functional dissection of the bone stromal compartment is important for understanding stromal-epithelial interactions associated with metastatic tumor growth. Use of currently available methodologies for the expression or deletion of gene expression in recent research studies has advanced our understanding of the stroma. However, the complexity of stromal heterogeneity within the prostate remains a challenge to obtaining compartment or cell-lineage-specific in vivo models necessary for furthering our understanding of prostatic developmental, benign, tumorigenic, and metastatic growth.

CYFIP2, a direct p53 target, is leptomycin-B sensitive.

A number of target genes for the tumor suppressor, p53, have been identified, however, the mechanisms that contribute to p53-dependent apoptosis remain to be fully elucidated. In a comprehensive screen for p53 target genes, we have identified Cytoplasmic FMR Interacting Protein 2 (CYFIP2) as a p53-inducible gene. Here we show that the CYFIP2 promoter contains a p53-responsive element that confers p53 binding as well as transcriptional activation of a heterologous reporter. Inducible expression of CYFIP2 is sufficient for caspase activation and cellular apoptosis, reminiscent of p53 activation. Together, these results suggest that CYFIP2 is a direct p53 target gene that may be part of a redundant network of genes responsible for p53-dependent apoptosis. In addition, the sensitivity of CYFIP2 protein subcellular localization to Leptomycin-B, a CRM-1/Exportin inhibitor, suggests that the biological functions of CYFIP2 may extend from the cytoplasmic compartment into the nucleus of the cell.

TIS11D is a candidate pro-apoptotic p53 target gene.

A number of target genes for the tumor suppressor, p53, have been identified, however, the mechanisms that contribute to p53-dependent apoptosis remain to be fully elucidated. In a comprehensive screen for p53 target genes by differential display, we have identified TIS11D as a p53-inducible gene. Induction of TIS11D mRNA was confirmed by Northern Blot in response to p53 expression. Inducible expression of TIS11D resulted in inhibition of cell proliferation and apoptosis. These data suggest TIS11D as a candidate p53 target gene that may be part of the network of genes responsible for p53-dependent apoptosis.

Saturation screening for p53 target genes by digital fluorescent differential display.

Differential display (DD) is one of the most commonly used approaches for identifying differentially expressed genes. Despite the great impact of the method on biomedical research, there has been a lack of automation of DD technology to increase its throughput and accuracy for a systematic gene expression analysis. Most of previous DD work has taken a "shotgun" approach of identifying one gene at a time, with limited polymerase chain reaction (PCR) reactions set up manually, giving DD a low-technology and low-throughput image. With our newly created DD mathematical model, which has been validated by computer simulations, global analysis of gene expression by DD technology is no longer a shot in the dark. After identifying the "rate-limiting" factors that contribute to the "noise" level of DD method, we have optimized the DD process with a new platform that incorporates fluorescent digital readout and automated liquid handling. The resulting streamlined fluorescent DD (FDD) technology offers an unprecedented accuracy, sensitivity, and throughput in comprehensive and quantitative analysis of gene expression. We are using this newly integrated FDD technology to conduct a systematic and comprehensive screening for p53 tumor-suppressor gene targets.

NDRG1 is necessary for p53-dependent apoptosis.

Although a number of target genes for the tumor suppressor p53 have been described, the mechanism of p53-dependent apoptosis is incompletely understood. Thus, it is essential to identify and characterize additional target genes that could mediate apoptosis. In the study reported here, we isolated a p53-regulated gene named NDRG1 (N-Myc down-regulated gene 1). Its expression is induced by DNA damage in a p53-dependent fashion. The promoter region of the NDRG1 gene contains a p53 binding site that confers p53-dependent transcriptional activation via a heterologous reporter. RNA interference and inducible gene expression approaches suggest that NDRG1 is necessary but not sufficient for p53-mediated caspase activation and apoptosis. This report further supports the notion that p53 controls a network of genes that are required for its apoptotic function.

Identification of p53 target genes by fluorescent differential display.

Differential display (DD) is a method used worldwide for identifying differentially expressed genes in eukaryotic cells. The mRNA DD technology works by systematic amplification of the 3' terminal regions of mRNAs. Using anchored primers designed to bind 5' boundary of the polyA tails for reverse transcription, followed by polymerase chain reaction (PCR) amplification with additional upstream primers of arbitrary sequences, mRNA subpopulations are separated by denaturing polyacrylamide electrophoresis. This allows direct side-by-side comparison of most of the mRNAs between or among related cells. Because of its simplicity, sensitivity, and reproducibility, the mRNA DD method is finding wide-ranging and rapid applications in developmental biology, cancer research, neuroscience, pathology, endocrinology, plant physiology, and many other areas. Since the recent development of the fluorescent differential display (FDD), the first nonradioactive DD system with equivalent sensitivity to the original 33P isotopic labeling method, it is now possible with this technology to automate, which can greatly increase the throughput and accuracy of mRNA DD.

Pelvic lordosis and alignment in spondylolisthesis.

STUDY DESIGN AND OBJECTIVES: Pelvic morphology and lumbopelvic lordosis were measured on standing radiographs of 75 patients with greater than 10% L5-S1 spondylolytic spondylolisthesis. The findings were compared with those of 75 volunteers to determine significant differences between the two groups. SUMMARY OF BACKGROUND DATA: Etiology of isthmic (lytic) spondylolisthesis remains uncertain. Causation appears to be multifactorial. The relationship between pelvic morphology and spondylolisthesis deserves additional study. METHODS: Both groups had a standing lateral radiograph of the thoracolumbar spine and pelvis taken that included both hips. Three radiographic angles for pelvic morphology (pelvisacral, pelvic incidence, and pelvic lordosis) were measured by two observers. Each offered similar reliability. Measurement of the pelvic lordosis angle by the pelvic radius technique required fewer steps. It also allowed calculation of the combined angles comprising both the pelvic morphology component for lordosis (the constant pelvic lordosis angle) and the lordosis in the lumbar spine (the variable lumbar lordosis from T12-S1) that should complement the fixed pelvic lordosis (the complementary lumbopelvic lordosis). Mean values and statistical correlations were then computed for each group and compared. RESULTS: The mean slippage for patients was 30% (range, 11-85%), with 34 patients (45%) having Grade I slips, 32 (43%) having Grade II slips, and nine (12%) having Grade III and IV slips. The mean measurements between patients and volunteers were significantly different (P < 0.01) for lumbar lordosis, pelvic lordosis, and lumbopelvic lordosis. Subgroups of patients with increasingly larger slips (Grade I-III) had significantly smaller mean angles for pelvic lordosis. CONCLUSIONS: The pelvic and lumbopelvic parameters studied were different in patients compared with controls. The contribution of the pelvis to lordosis was significantly smaller in the subgroups of patients with increasingly larger grades of spondylolisthesis. Pelvic morphology may play a role in the development of spondylolisthesis. Measurement of the combined lumbar and pelvic (lumbopelvic) lordosis on standing radiographs is important.

Down-regulation of androgen receptor expression and inhibition of lacrimal gland cell proliferation by retinoic acid.

Androgens and retinoids are known to be involved in control of lacrimal gland function. Because retinoids generally antagonize androgen function it was the purpose of this study to investigate interactions of retinoic acid and androgens in rabbit lacrimal acinar cells in culture by determining effects of retinoic acid on androgen receptor (AR) mRNA expression, AR protein levels and androgen-stimulated cell proliferation. Experiments were conducted using primary rabbit lacrimal acinar cells and a transformed rabbit lacrimal acinar cell line. Exposure of primary lacrimal acinar cells in culture to 10(-10)-10(-6)M all-trans retinoic acid for 4-24hr causes an approximately 50% decrease in AR mRNA expression. Expression of AR protein in primary and transformed rabbit lacrimal acinar cells was confirmed by immunohistochemistry. Exposure of the primary cells to 10(-6)M retinoic acid for 24hr caused a 40% decrease in AR protein levels as determined by measurement of binding of(3) [H]-dihydrotestosterone (DHT) to cells in culture and Scatchard analysis. Exposure to 10(-9)-10(-6)M DHT stimulates proliferation of transformed rabbit lacrimal acinar cells. This effect is receptor mediated since it is blocked by the AR antagonist, flutamide. Proliferation of the lacrimal acinar cells is inhibited by retinoic acid, as compared to control, and retinoic acid also completely inhibits androgen stimulation of cell proliferation. This study supports the hypothesis that androgens play a supportive role in lacrimal gland function. The antagonistic influences of androgens and retinoic acid suggests that, under physiologic conditions there is a balance between the effects of androgens and retinoids in the lacrimal gland. A decrease in androgen levels in a dry eye patient may alter the balance between the effects of these important controllers of gene expression. The antagonistic effect of retinoids on androgens in the lacrimal gland must also be considered when devising pharmaceutical treatments for dye eye.