Optical molecular imaging detects changes in extracellular pH with the development of head and neck cancer

Noninvasive localized measurement of extracellular pH in cancer tissues can have a significant impact on the management of cancer. Despite its significance, there are limited approaches for rapid and noninvasive measurement of local pH in a clinical environment. In this study, we demonstrate the potential of noninvasive topical delivery of Alexa‐647 labeled pHLIP (pH responsive peptide conjugated with Alexa Fluor^® 647) to image changes in extracellular pH associated with head and neck squamous cell carcinoma using widefield and high resolution imaging. We report a series of preclinical analyses to evaluate the optical contrast achieved after topical delivery of Alexa‐647 labeled pHLIP in intact fresh human tissue specimens using widefield and high‐resolution fluorescence imaging. Using topical delivery, Alexa‐647 labeled pHLIP can be rapidly delivered throughout the epithelium of intact tissues with a depth exceeding 700 µm. Following labeling with Alexa‐647 labeled pHLIP, the mean fluorescent contrast increased four to eight fold higher in clinically abnormal tissues as compared to paired clinically normal biopsies. Furthermore, the imaging approach showed significant differences in fluorescence contrast between the cancer and the normal biopsies across diverse patients and different anatomical sites (unpaired comparison). The fluorescence contrast differences between clinically abnormal and normal tissues were in agreement with the pathologic evaluation. Topical application of fluorescently labeled pHLIP can detect and differentiate normal from cancerous tissues using both widefield and high resolution imaging. This technology will provide an effective tool to assess tumor margins during surgery and improve detection and prognosis of head and neck cancer.     

Frameshift mutation hotspot identified in Smith-Magenis syndrome: case report and review of literature

Smith-Magenis syndrome (SMS) is a complex syndrome involving intellectual disabilities, sleep disturbance, behavioural problems, and a variety of craniofacial, skeletal, and visceral anomalies. While the majority of SMS cases harbor an ~3.5 Mb common deletion on 17p11.2 that encompasses the retinoic acid induced-1 (RAI1) gene, some patients carry small intragenic deletions or point mutations in RAI1. We present data on two cases of Smith-Magenis syndrome with mutation of RAI1. Both cases are phenotypically consistent with SMS and RAI1 mutation but also have other anomalies not previously reported in SMS, including spontaneous pneumothoraces. These cases also illustrate variability in the SMS phenotype not previously shown for RAI1 mutation cases, including hearing loss, absence of self-abusive behaviours, and mild global delays. Sequencing of RAI1 revealed mutation of the same heptameric C-tract (CCCCCCC) in exon 3 in both cases (c.3103delC one case and and c.3103insC in the other), resulting in frameshift mutations. Of the seven reported frameshift mutations occurring in poly C-tracts in RAI1, four cases (~57%) occur at this heptameric C-tract. Collectively, these results indicate that this heptameric C-tract is a preferential hotspot for single nucleotide insertion/deletions (SNindels) and therefore, should be considered a primary target for analysis in patients suspected for mutations in RAI1. We expect that as more patients are sequenced for mutations in RAI1, the incidence of frameshift mutations in this hotspot will become more evident.     

De novo minimal change disease associated with reversible post-transplant nephrotic syndrome. A report of five cases and review of literature

Nephrotic syndrome (NS) is frequent in renal transplant recipients and may be related to a large variety of glomerular lesions. In some of these cases, the transplant biopsy showed no significant glomerular changes and the NS was reversible, but the primary renal disease was not minimal change disease (MCD), suggesting that MCD may develop de novo in renal transplant setting. Knowledge of this entity, however, is limited. Among 67 cases of post-transplant NS encountered in a 12-yr period, five were found to be associated with de novo MCD. A critical review of the literature revealed nine additional cases of de novo MCD. The data from these 14 cases show that patients with de novo MCD had a large variety of primary renal diseases but MCD or focal segmental glomerulosclerosis was not among them. Eight of the 14 transplanted kidneys (60%) were from living related donors, suggesting this as a risk factor. Nephrotic range proteinuria (3-76 g/d) developed immediately or shortly after transplantation (within 4 months for all reported cases, except for one at 24 months). The serum creatinine when NS was first diagnosed was normal or mildly elevated, but acute renal failure occurred in three patients. On biopsy, the glomeruli were normal or, more frequently, displayed mild, focal segmental mesangial sclerosis, hypercellularity, deposition of IgM/C3, or accumulation of mononuclear inflammatory cells in some glomerular capillaries. The tubulointerstitial compartment was normal in cases with normal renal function; displayed mild acute and/or chronic rejection that correlated with a mildly elevated serum creatinine; or showed acute changes including acute rejection, acute tubular necrosis, or acute cyclosporin A toxicity, which accounted for both acute renal failure at presentation and its subsequent reversibility. Under various treatments, including increased steroids, angiotensin converting enzyme inhibitors, calcium channel blockers and angiotensin receptor blockers, sustained remission of NS was achieved in 13 cases, within a year (0.5-12 months) in 10 and later (24, 34 and 98 months, respectively) in three. In the remaining case, the patient died of septic shock 2 months after transplantation. After remission of the NS, the grafts functioned well without or with minimal proteinuria for several years. De novo MCD has characteristic clinical and pathologic features. It represents an important but hitherto underemphasized cause of post-transplant NS, which is potentially reversible and does not adversely affect the renal transplants.     

Activation of adenosine triphosphate-sensitive potassium channels confers protection against rotenone-induced cell death: therapeutic implications for Parkinson's disease

It is anticipated that further understanding of the protective mechanism induced by ischemic preconditioning will improve prognosis for patients of ischemic injury. It is not known whether preconditioning exerts beneficial actions in neurodegenerative diseases, in which ischemic injury plays a causative role. Here we show that transient activation of ATP-sensitive potassium channels, a trigger in ischemic preconditioning signaling, confers protection in PC12 cells and SH-SY5Y cells against neurotoxic effect of rotenone and MPTP, mitochondrial complex I inhibitors that have been implicated in the pathogenesis of Parkinson's disease. The degree of protection is in proportion to the bouts of exposure to an ATP-sensitive potassium channel opener, a feature reminiscent of ischemic tolerance in vivo. Protection is sensitive to a protein synthesis inhibitor, indicating the involvement of de novo protein synthesis in the protective processes. Pretreatment of PC12 cells with preconditioning stimuli FeSO(4) or xanthine/xanthine oxidase also confers protection against rotenone-induced cell death. Our results demonstrate for the first time the protective role of ATP-sensitive potassium channels in a dopaminergic neuronal cell line against rotenone-induced neurotoxicity and conceptually support the view that ischemic preconditioning-derived therapeutic strategies may have potential and feasibility in therapy for Parkinson's disease.     

The origin of DNA replication and Fieller's problem

The paper considers the statistical problem of estimating the origin of DNA replication within the human ribosomal DNA (rDNA) locus and the issue of assessing the standard error of the estimate. Based on mapping the cumulative replication index (CRI), two different modelling schemes are suggested and investigated. The statistical problem of constructing a confidence interval for the origin of DNA replication is related to Fieller's problem of obtaining a confidence interval for the ratio of two normal means. Standard normal theory, the delta and bootstrap methods are used to estimate the standard error of the estimate of the origin of DNA replication, as well as the variation of the replication rate.     

Improving the consistency in cervical esophageal target volume definition by special training

PURPOSE: Three-dimensional conformal radiation therapy requires the precise definition of the target volume. Its potential benefits could be offset by the inconsistency in target definition by radiation oncologists. In a previous survey of radiation oncologists, a large degree of variation in target volume definition of cervical esophageal cancer was noted for the boost phase of radiotherapy. The present study evaluated whether special training could improve the consistency in target volume definitions. METHODS AND MATERIALS: A pre-training survey was performed to establish baseline values. This was followed by a special one-on-one training session on treatment planning based on the RTOG 94-05 protocol to 12 radiation oncologists. Target volumes were redrawn immediately and at 1-2 months later. Post-training vs. pre-training target volumes were compared. RESULTS: There was less variability in the longitudinal positions of the target volumes post-training compared to pre-training (p < 0.05 in 5 of 6 comparisons). One case had more variability due to the lack of a visible gross tumor on CT scans. Transverse contours of target volumes did not show any significant difference pre- or post-training. CONCLUSION: For cervical esophageal cancer, this study suggests that special training on protocol guidelines may improve consistency in target volume definition. Explicit protocol directions are required for situations where the gross tumor is not easily visible on CT scans. This may be particularly important for multicenter clinical trials, to reduce the occurrences of protocol violations.     

Reactive stroma in human prostate cancer: induction of myofibroblast phenotype and extracellular matrix remodeling.

PURPOSE: Generation of a reactive stroma environment occurs in many human cancers and is likely to promote tumorigenesis. However, reactive stroma in human prostate cancer has not been defined. We examined stromal cell phenotype and expression of extracellular matrix components in an effort to define the reactive stroma environment and to determine its ontogeny during prostate cancer progression. EXPERIMENTAL DESIGN: Normal prostate, prostatic intraepithelial neoplasia (PIN), and prostate cancer were examined by immunohistochemistry. Tissue samples included radical prostatectomy specimens, frozen biopsy specimens, and a prostate cancer tissue microarray. A human prostate stromal cell line was used to determine whether transforming growth factor beta1 (TGF-beta1) regulates reactive stroma. RESULTS: Compared with normal prostate tissue, reactive stroma in Gleason 3 prostate cancer showed increased vimentin staining and decreased calponin staining (P < 0.001). Double-label immunohistochemistry revealed that reactive stromal cells were vimentin and smooth muscle alpha-actin positive, indicating the myofibroblast phenotype. In addition, reactive stroma cells exhibited elevated collagen I synthesis and expression of tenascin and fibroblast activation protein. Increased vimentin expression and collagen I synthesis were first observed in activated periacinar fibroblasts adjacent to PIN. Similar to previous observations in prostate cancer, TGF-beta1-staining intensity was elevated in PIN. In vitro, TGF-beta1 stimulated human prostatic fibroblasts to switch to the myofibroblast phenotype and to express tenascin. CONCLUSIONS: The stromal microenvironment in human prostate cancer is altered compared with normal stroma and exhibits features of a wound repair stroma. Reactive stroma is composed of myofibroblasts and fibroblasts stimulated to express extracellular matrix components. Reactive stroma appears to be initiated during PIN and evolve with cancer progression to effectively displace the normal fibromuscular stroma. These studies and others suggest that TGF-beta1 is a candidate regulator of reactive stroma during prostate cancer progression.     

Combination immunotherapy of primary prostate cancer in a transgenic mouse model using CTLA-4 blockade

We have previously shown that antibodies to CTLA-4, an inhibitory receptor on T cells, can be effective at inducing regression of transplantable murine tumors. In this study, we demonstrate that an effective immune response against primary prostate tumors in transgenic (TRAMP) mice can be elicited using a strategy that combines CTLA-4 blockade and an irradiated tumor cell vaccine. Treatment of TRAMP mice at 14 weeks of age resulted in a significant reduction in tumor incidence (15% versus control, 75%), as assessed 2 months after treatment. Histopathological analysis revealed that treated mice had a lower tumor grade with significant accumulation of inflammatory cells in interductal spaces when treated with anti-CTLA-4 and a granulocyte-macrophage colony-stimulating factor-expressing vaccine. Vaccination of nontransgenic mice with this regimen resulted in marked prostatitis accompanied by destruction of epithelium, indicating that the immune response was, at least in part, directed against normal prostate antigens. These findings demonstrate that this combinatorial treatment can elicit a potent antiprostate response and suggest potential of this approach for treatment of prostate cancer.     

Collateralization and GAD immunoreactivity of descending pallidal efferents

The first phase of this study involves injecting a different fluorescent retrograde axonal tracer into the subthalamic nucleus, the substantia nigra, and the mesopontine tegmentum. Multiple labeled cells are found within the caudal third of the globus pallidus. The entopeduncular nucleus and adjacent basal forebrain structures such as the substantia innominata, lateral hypothalamus, bed nucleus of the stria terminalis, and central nucleus of the amygdala all exhibit some dye containing cell, although multiple labeled cells are rare. The second phase of this study involves injecting a different fluorescent retrograde tracer into either the substantia nigra, or the mesopontine tegmentum, and subsequent processing of the tissue for glutamic acid decarboxylase (GAD) immunocytochemistry. Many dye and antibody double-labeled cells could be found within the entopeduncular nucleus and the caudal third of the globus pallidus. This is in contrast to the surrounding basal forebrain regions with brainstem efferents which were rarely GAD-positive. This study indicates that the collateral pattern and immunocytochemistry of globus pallidus neurons with descending efferents are distinct from other basal forebrain neurons having similar efferents. These results also extend previous findings and suggest that the neuron of the pallidal complex are heterogeneous with respect to their patterns of projections. In particular, the present findings question previous assumptions concerning the homology of pallidal segments between primate and rodent species.