Refining the unilateral ureteral obstruction mouse model: No sham, no shame.

Fibrosis, as a common final pathway in many renal diseases, contributes significantly to the decline of organ function and to progression to end-stage renal disease. To establish therapeutic interventions that target fibrosis, animal models are essential. The most widely used model of renal fibrosis is the unilateral ureteral obstruction (UUO) model. Typically, the control for this model is a sham-operated animal. Sham surgery causes pain and distress to these control animals, and here we aim to show that there is no difference in the main read-outs of this model between sham-operated animals and non-operated C57BL/6J mice. In five experiments, quantification of Picro Sirius Red stained collagen in the renal cortex did not show any difference between 15 sham and 25 non-operated individuals. A comparison of the regulation of genes involved with fibrosis did not show any difference between sham and non-operated groups at 21 days post surgery either. We conclude that there are no significant differences between non-operated controls and sham animals with respect to collagen deposition and fibrosis pathways in the UUO mouse model.

Insights into the heparan sulphate-dependent externalisation of transglutaminase-2 (TG2) in glucose-stimulated proximal-like tubular epithelial cells.

The extracellular matrix crosslinking enzyme transglutaminase 2 (TG2) is highly implicated in tissue fibrosis that precedes end-stage kidney failure. TG2 is unconventionally secreted through extracellular vesicles in a way that depends on the heparan sulphate (HS) proteoglycan syndecan-4 (Sdc4), the deletion of which reduces experimental kidney fibrosis as a result of lower extracellular TG2 in the tubule-interstitium. Here we establish a model of TG2 externalisation in NRK-52E tubular epithelial cells subjected to glucose stress. HS-binding TG2 mutants had reduced extracellular TG2 in transfected NRK-52E, suggesting that TG2-externalisation depends on an intact TG2 heparin binding site. Inhibition of N-ethylmaleimide sensitive factor (NSF) vesicle-fusing ATPase, which was identified in the recently elucidated TG2 kidney membrane-interactome, led to significantly lower TG2-externalisation, thus validating the involvement of membrane fusion in TG2 secretion. As cyclin-G-associated kinase (GAK) had emerged as a further TG2-partner in the fibrotic kidney, we investigated whether glucose-induced TG2-externalisation was accompanied by TG2 phosphorylation in consensus sequences of cyclin-dependent kinase (CDK). Glucose stress led to intense TG2 phosphorylation in serine/threonine CDK-target. TG2 phosphorylation by tyrosine kinases was also increased by glucose. Although the precise role of glucose-induced TG2 phosphorylation is unknown, these novel data suggest that phosphorylation may be involved in TG2 membrane-trafficking.

Defining recurrence of nonmelanoma skin cancer after Mohs micrographic surgery: Report of the American College of Mohs Surgery Registry and Outcomes Committee.

BACKGROUND: Standardized definitions and methods of surveillance for local recurrence of nonmelanoma skin cancer are critical in determining cure rates attributed to treatment modalities. OBJECTIVE: We sought to offer a standard definition of local recurrence after surgical treatment of nonmelanoma skin cancer and to propose an acceptable surveillance period and tracking methods. METHODS: A literature search was performed for background definitions of local recurrence and tracking methods. The American College of Mohs Surgery (ACMS) Registry and Outcomes Committee then conducted a modified Delphi process to arrive at consensus definitions. RESULTS: We define local recurrence as a tumor with comparable histology, with contiguity to the surgical scar after treatment, and that arises within the area of the previously treated tumor. LIMITATIONS: This project reports the results of a modified Delphi method process involving members of the ACMS. The model described may not be useful for nonexcision type treatments such as topical chemotherapy, electrodessication and curettage, or radiation treatment. CONCLUSIONS: Previous definitions of recurrence and surveillance methods after surgical treatment of nonmelanoma skin cancer are variable and nonstandard. We describe consensus standards for defining and tracking recurrence that should allow for consistent scientific evaluation and development of performance data in skin cancer outcomes registries.

Nd:YAG laser capsulotomy under general anesthesia in the sitting position.

Neodymium-yttrium-aluminum-garnet (Nd:YAG) laser capsulotomy is commonly used to treat posterior capsule opacification after cataract surgery in adults. Children and adults with developmental delay, however, are not always cooperative, and the procedure must be performed under general anesthesia. We describe a technique for Nd:YAG capsulotomy under general anesthesia in the sitting position using the standard widely available Nd:YAG laser. Anesthesia was induced in the supine position and the patient was then transferred to the sitting position with chin in the slit-lamp laser delivery system. The procedure was performed without complications. We report our experience in 4 eyes of 3 patients, including 2 children. This technique is a viable option for those without access to an overhead-mounted Nd:YAG laser and an alternative to secondary posterior capsulotomy in selected patients.

Accelerated partial breast intensity-modulated radiotherapy in women who have prior breast augmentation.

PURPOSE: To examine the outcome of breast cancer patients who have prior breast augmentation treated with lumpectomy followed by accelerated partial breast external intensity-modulated radiotherapy (APBIMRT) with image-guided radiotherapy (IGRT). METHODS AND MATERIALS: Four patients with previous elective subpectoral breast augmentation were enrolled on this APBIMRT trial. These four patients were treated with 10 equal twice daily 3.85 Gy fractions over 5 consecutive days (total dose of 38.5 Gy) using APBIMRT and IGRT. Patients were assessed for pain and cosmetic outcome (physician and a patient self-assessment). RESULTS: At last follow-up, two patients reported an excellent cosmetic results (at 2 years and at 8 months, respectively), one reported good cosmetic results (at 2 years), and one reported poor cosmetic results (at 20 months). Physicians rated the cosmetic outcomes as excellent in two (CEL; at 2 years and 8 months, respectively), good in one (CEL; at 20 months) and excellent in one (KTH; at 2 years). Three patients reported no breast/chest wall pain (two at 2 years and one at 1 year) and the fourth reported mild pain (at 20 months). The mean percent volume of ipsilateral breast receiving 100%, 75%, 50%, and 25% of the prescribed dose was 7.28%, 17.55%, 24.33%, and 33.1%, respectively. The mean breast, planning target volume (PTV), and implant volumes were 399.88 cc, 43.55 cc, and 313.36 cc, respectively. The mean breast prosthesis/total volume (breast tissue plus prosthesis) ratio was 44.55%. The mean PTV/ipsilateral breast and PTV/total volume ratios were 11.1% and 6.1%, respectively. CONCLUSION: The results show that a regimen of APBIMRT with IGRT is possible in patients who have prior breast augmentation.

Finding needles in a haystack: a methodology for identifying and sampling community-based youth smoking cessation programs.

BACKGROUND: Surveys of community-based programs are difficult to conduct when there is virtually no information about the number or locations of the programs of interest. This article describes the methodology used by the Helping Young Smokers Quit (HYSQ) initiative to identify and profile community-based youth smoking cessation programs in the absence of a defined sample frame. METHODS: We developed a two-stage sampling design, with counties as the first-stage probability sampling units. The second stage used snowball sampling to saturation, to identify individuals who administered youth smoking cessation programs across three economic sectors in each county. Multivariate analyses modeled the relationship between program screening, eligibility, and response rates and economic sector and stratification criteria. Cumulative logit models analyzed the relationship between the number of contacts in a county and the number of programs screened, eligible, or profiled in a county. RESULTS: The snowball process yielded 9,983 unique and traceable contacts. Urban and high-income counties yielded significantly more screened program administrators; urban counties produced significantly more eligible programs, but there was no significant association between the county characteristics and program response rate. There is a positive relationship between the number of informants initially located and the number of programs screened, eligible, and profiled in a county. DISCUSSION: Our strategy to identify youth tobacco cessation programs could be used to create a sample frame for other nonprofit organizations that are difficult to identify due to a lack of existing directories, lists, or other traditional sample frames.

Clinical experience with image-guided radiotherapy in an accelerated partial breast intensity-modulated radiotherapy protocol.

PURPOSE: To explore the feasibility of fiducial markers for the use of image-guided radiotherapy (IGRT) in an accelerated partial breast intensity modulated radiotherapy protocol. METHODS AND MATERIALS: Nineteen patients consented to an institutional review board approved protocol of accelerated partial breast intensity-modulated radiotherapy with fiducial marker placement and treatment with IGRT. Patients (1 patient with bilateral breast cancer; 20 total breasts) underwent ultrasound guided implantation of three 1.2- x 3-mm gold markers placed around the surgical cavity. For each patient, table shifts (inferior/superior, right/left lateral, and anterior/posterior) and minimum, maximum, mean error with standard deviation were recorded for each of the 10 BID treatments. The dose contribution of daily orthogonal films was also examined. RESULTS: All IGRT patients underwent successful marker placement. In all, 200 IGRT treatment sessions were performed. The average vector displacement was 4 mm (range, 2-7 mm). The average superior/inferior shift was 2 mm (range, 0-5 mm), the average lateral shift was 2 mm (range, 1-4 mm), and the average anterior/posterior shift was 3 mm (range, 1 5 mm). CONCLUSIONS: This study shows that the use of IGRT can be successfully used in an accelerated partial breast intensity-modulated radiotherapy protocol. The authors believe that this technique has increased daily treatment accuracy and permitted reduction in the margin added to the clinical target volume to form the planning target volume.

Predictors for clinical outcomes after accelerated partial breast intensity-modulated radiotherapy.

PURPOSE: To correlate the treatment planning parameters with the clinical outcomes in patients treated with accelerated partial breast intensity-modulated radiotherapy. METHODS AND MATERIALS: A total of 105 patients with Stage I breast cancer were treated between February 2004 and March 2007 in a Phase II prospective trial and had detailed information available on the planning target volume (PTV), ipsilateral breast volume (IBV), PTV/IBV ratio, lung volume, chest wall volume, surgery to radiotherapy interval, follow-up interval, breast pain, and cosmesis. The first 7 of these patients were treated to 34 Gy, and the remaining 98 were treated to 38.5 Gy. All patients were treated twice daily for 5 consecutive days. Univariate and multivariate analyses were performed. RESULTS: The median follow-up was 13 months. No recurrences or deaths were observed. Of the 105 patients, 30 reported mild or moderate breast pain in their most recently recorded follow-up visit. The irradiated lung volume (p < 0.05) and chest wall volume receiving >35 Gy (p < 0.01) were associated with pain. The PTV, but not the PTV/IBV ratio, also correlated with pain (p < 0.01 and p = 0.42, respectively). A total of 72 patients reported excellent, 32 reported good, and 1 reported poor cosmesis. Physician-rated cosmesis reported 90 excellent and 15 good. None of the tested variables correlated with the cosmetic outcomes. CONCLUSION: Radiotherapy to the chest wall (chest wall volume receiving >35 Gy) and to lung correlated with reports of mild pain after accelerated partial breast intensity-modulated radiotherapy. Also, the PTV, but not the PTV/IBV ratio, was predictive of post-treatment reports of pain.

Plk1 activation by Ste20-like kinase (Slk) phosphorylation and polo-box phosphopeptide binding assayed with the substrate translationally controlled tumor protein (TCTP).

The mitotic protein kinase Plk1 catalyzes events associated with centrosome maturation, kinetocore function, spindle formation, and cytokinesis and is a target for anticancer drug design. It is composed of a N-terminal kinase domain and a C-terminal polo-box domain (PBD). The PBD domain serves to localize the kinase on cognate phosphorylated substrates, and this binding relieves the inhibition of the kinase by the PBD. Similar to many protein kinases, Plk1 is activated by phosphorylation on a threonine residue, Thr210, in the activation segment. In this work, we describe expression in Escherichia coli cells and purification of full-length Plk1 in quantities suitable for structural studies and use this material for quantitative characterization of the activation events with the substrate translationally controlled tumour protein (TCTP). The presence of the PBD-binding phosphopeptide enhances phosphorylation by the activating Ste20-like kinase (Slk). Native Plk1 exhibits a basal catalytic efficiency k cat/ K(M) of 9.9 x 10 (-5) s (-1) microM (-1). Association with a polo-box-binding phosphopeptide increased the catalytic efficiency by 11x largely through an increase in k(cat) with no change in K(M). Phosphorylation by Slk increases catalytic efficiency by 202x with a 2.3-fold reduction in K(M) and 88-fold increase in k(cat). Phosphorylation and the presence of the PBD-binding phosphopeptide result in an increase in catalytic efficiency of 1515x with a 2.3-fold decrease in K(M) and a 705-fold increase in k(cat) over the unmodified Plk1. Knowledge of kinase regulatory mechanisms and the structures of the Plk1 individual domains has allowed for a model to be proposed for these activatory events.

How tyrosine 15 phosphorylation inhibits the activity of cyclin-dependent kinase 2-cyclin A.

Inhibition of cyclin-dependent kinase 1 (CDK1) activity by Tyr-15 phosphorylation directly regulates entry into mitosis and is an important element in the control of the unperturbed cell cycle. Active site phosphorylation of other members of the CDK family that regulate cell cycle progression instates checkpoints that are fundamental to eukaryotic cell cycle regulation. Kinetic and crystallographic analyses of CDK2-cyclin A complexes reveal that this inhibitory mechanism operates through steric blockade of peptide substrate binding and through the creation of an environment that favors a non-productive conformation of the terminal group of ATP. By contrast, tyrosine phosphorylation of CDK2 alters neither its Km for ATP nor its significant intrinsic ATPase activity. Tyr-15-phosphorylated CDK2 retains trace protein phosphorylation activity that should be considered in quantitative and qualitative cell cycle models.

The effects of cetuximab alone and in combination with radiation and/or chemotherapy in lung cancer.

PURPOSE: The epidermal growth factor receptor (EGFR) overexpressed in approximately 80% of non-small cell lung cancers (NSCLC) is a target for novel therapeutics. Concurrent chemoradiation is the current standard of care for treatment of patients with locally advanced NSCLC. However, < 20% of patients remain disease-free at 5 years despite this aggressive treatment. Cetuximab is a humanized monoclonal antibody that recognizes the human EGFR, and in previous studies, inhibited the growth of EGFR-expressing human cancer cell lines. In this report, we investigated the cellular and molecular effects of cetuximab alone and in combination with radiation and/or chemotherapy in human NSCLC cell lines with varying levels of EGFR overexpression in vitro and in vivo. EXPERIMENTAL DESIGN: We evaluated the EGFR status of a panel of human NSCLC cancer cell lines by immunohistochemistry and flow cytometry. We then evaluated cetuximab effects on growth, cell cycle distribution, and downstream intracellular signaling molecules in this panel of NSCLC cancer cell lines. NSCLC cell lines were treated with cetuximab alone or in combination with radiation, chemotherapy, or chemoradiation to determine the cooperative effects of cetuximab both in vitro and in vivo in athymic nude mice bearing NSCLC xenografts. RESULTS: Cetuximab alone inhibited the in vitro growth of some but not all EGFR-expressing NSCLC cell lines in a dose-dependent manner. Flow cytometric analysis of cell cycle distribution after 24 hours of cetuximab treatment revealed a shift into the G(0)/G(1) phase of the cell cycle in cetuximab-sensitive EGFR-expressing cell lines and at concentrations that were growth-inhibitory. There were no cell cycle changes in the EGFR-negative cell lines. After 4 hours of exposure, cetuximab reduced epidermal growth factor (EGF)-induced phosphorylation of EGFR (pEGFR) and HER-2 (pHER2) in cetuximab-sensitive cell lines but not in cetuximab-resistant cell lines. Cetuximab reduced EGF-induced phosphorylation of extracellular signal-regulated kinase-1/2 (pERK) in all EGFR-expressing cell lines. In the absence of EGF, cetuximab alone increased the level of pEGFR and pHER2 above that seen in untreated control cells in both sensitive and resistant cell lines that were EGFR- and HER2-positive, but not in EGFR- or HER2-negative lines. Despite the cetuximab-induced increase in phosphorylation of EGFR and HER2, peak EGF-induced levels of pEGFR and pHER2 were reduced by cetuximab in the cetuximab-sensitive lines but not in the resistant lines. Cooperative (combination index values < 1.0) growth inhibitory effects were observed in vitro combination assays with cetuximab and radiation only in cetuximab-sensitive NSCLC cell lines. A lack of cooperation was seen in cetuximab-insensitive NSCLC cell lines. Similar findings were observed with in vitro combination studies of cetuximab plus cisplatin or paclitaxel. In nude mice bearing EGFR-expressing, cetuximab-sensitive, NSCLC cell line xenografts, cetuximab plus radiation induced a marked improvement in tumor growth inhibition over either agent alone. The growth inhibitory effects of cetuximab-radiation were similar to the growth inhibitory effects of concurrent chemoradiation. Triple combination therapy of cetuximab and chemoradiation yielded a nonsignificant advantage in tumor growth control over doublet combinations (cetuximab and radiation or chemoradiation) in vivo. CONCLUSIONS: Similar results in tumor growth inhibition observed in mice treated with cetuximab-radiation and cisplatin-radiation provide a rationale for the clinical investigation of cetuximab with concurrent radiation in selected patients with locally advanced NSCLC. Local tumor control and treatment toxicity should be evaluated between cetuximab-radiation and chemoradiation regimens. Proper patient selection will be critical to the success of such trials and further studies are needed to identify optimal patient selection criteria.

The magnitude of hedgehog signaling activity defines skin tumor phenotype.

Gain-of-function mutations in SMO have been implicated in constitutive activation of the hedgehog signaling pathway in human basal cell carcinomas (BCCs). We used a truncated keratin 5 (DeltaK5) promoter to assess the potential role of the human M2SMO mutant in BCC development in adult transgenic mice. DeltaK5-M2SMO mouse epidermis is hyperproliferative, ex presses BCC protein markers and gives rise to numerous epithelial downgrowths invading the underlying dermis. Lesions strikingly similar to human basaloid follicular hamartomas develop, but BCCs do not arise even in elderly mice. Hedgehog target gene transcripts were only modestly upregulated in mouse and human follicular hamartomas, in contrast to the high levels detected in BCCs. Cyclins D1 and D2 were selectively upregulated in mouse BCCs. Our data suggest that the levels of hedgehog pathway activation and G(1) cyclins are major determinants of tumor phenotype in skin, and strongly implicate deregulated hedgehog signaling in the genesis of human basaloid follicular hamartomas. Expression of an activated SMO mutant in keratinocytes appears to be insufficient for the development and/or maintenance of full-blown BCCs.

Limitations on residents' working hours at New York teaching hospitals: a status report.

Teaching hospitals in New York have been subject to regulations that limit the working hours of residency trainees since July 1989. Following a period of enhanced survey activity by the State Department of Health in the late 1990s, the state awarded a contract to a third-party organization to conduct annual audits of the state's teaching hospitals to assess compliance with the regulations. As of October 2002, preliminary results indicate that 75 of the 118 teaching hospitals in the state (63.6%) were found to be out of compliance with some component of the regulations. The most common citations for noncompliance were (1) working in excess of 24 consecutive hours (45%), and (2) working in excess of 80 hours per week, averaged over four weeks (28%). For New York teaching hospitals, the key factors identified as posing significant challenges to achieving full compliance with the regulations included (1) assuming responsibility for the work schedules of residents; (2) scheduling and monitoring difficulties; (3) the education efforts associated with the regulations; (4) the documentation requirements; (5) variations in learning abilities among the residents; and (6) mistaking verbal compliance for actual compliance. As the state begins a new round of surveys, it will be expecting better compliance efforts, and New York teaching hospitals are committed to this difficult but worthy goal.

Postoperative cognitive dysfunction in middle-aged patients.

BACKGROUND: Postoperative cognitive dysfunction (POCD) after noncardiac surgery is strongly associated with increasing age in elderly patients; middle-aged patients (aged 40-60 yr) may be expected to have a lower incidence, although subjective complaints are frequent. METHODS: The authors compared the changes in neuropsychological test results at 1 week and 3 months in patients aged 40-60 yr, using a battery of neuropsychological tests, with those of age-matched control subjects using Z-score analysis. They assessed risk factors and associations of POCD with measures of subjective cognitive function, depression, and activities of daily living. RESULTS: At 7 days, cognitive dysfunction as defined was present in 19.2% (confidence interval [CI], 15.7-23.1) of the patients and in 4.0% (CI, 1.6-8.0) of control subjects (P < 0.001). After 3 months, the incidence was 6.2% (CI, 4.1-8.9) in patients and 4.1% (CI, 1.7-8.4) in control subjects (not significant). POCD at 7 days was associated with supplementary epidural analgesia and reported avoidance of alcohol consumption. At 3 months, 29% of patients had subjective symptoms of POCD, and this finding was associated with depression. Early POCD was associated with reports of lower activity scores at 3 months. CONCLUSIONS: Postoperative cognitive dysfunction occurs frequently but resolves by 3 months after surgery. It may be associated with decreased activity during this period. Subjective report overestimates the incidence of POCD. Patients may be helped by recognition that the problem is genuine and reassured that it is likely to be transient.