bluuebunny/crossref_metadata_2025 huggingface dataset:
Rho kinase is involved in Ca2+ entry of rat penile small arteries
Authors: Villalba Nuria , Stankevicius Edgaras , Simonsen Ulf , Prieto Dolores
Published: January 2008

Tonic physiological activity of RhoA/Rho kinase contributes to the maintenance of penile flaccidity through its involvement in the Ca2+ sensitization of erectile tissue smooth muscle. The present study hypothesized that Rho kinase is also involved in the modulation of Ca2+ entry induced by α1-adrenoceptor stimulation of penile arteries. Rat penile arteries were mounted in microvascular myographs for simultaneous measurements of intracellular Ca2+ ([Ca2+]i) and force. The Rho-kinase inhibitor Y-27632 markedly reduced norepinephrine-mediated electrically induced contractions and the increases in both [Ca2+]i and tension elicited by the α1-adrenoceptor agonist phenylephrine (Phe). In contrast, the protein kinase C (PKC) inhibitor Ro-31-8220 reduced tension without altering the Phe-induced increase in [Ca2+]i. In the presence of nifedipine, Y-27632 still inhibited the non-L-type Ca2+ signal and blunted Phe contraction. Y-27632 did not impair the capacitative Ca2+ entry evoked by store depletion with cyclopiazonic acid but largely reduced the Ba2+ influx stimulated by Phe in fura-2 AM-loaded arteries. The addition of Y-27632 to arteries depolarized with high KCl markedly reduced tension without changing [Ca2+]i. In α-toxin-permeabilized penile arteries stimulated with threshold Ca2+ concentrations, Y-27632 inhibited the sensitization induced by either guanosine 5′- O-(3-thiotriphosphate) (GTPγS) or Phe in the presence of GTPγS. However, Y-27632 failed to alter contractions induced by a maximal concentration of free Ca2+. These results suggest that Rho kinase, besides its contribution to the Ca2+ sensitization of the contractile proteins, is also involved in the regulation of Ca2+ entry through a nonselective cation channel activated by α1-adenoceptor stimulation in rat penile arteries.
Functional properties of cells obtained from human cord blood CD34+ stem cells and mouse cardiac myocytes in coculture
Authors: Orlandi Alessia , Pagani Francesca , Avitabile Daniele , Bonanno Giuseppina , Scambia Giovanni , Vigna Elisa , Grassi Francesca , Eusebi Fabrizio , Fucile Sergio , Pesce Maurizio , Capogrossi Maurizio C.
Published: January 2008

Prior in vitro studies suggested that different types of hematopoietic stem cells may differentiate into cardiomyocytes. The present work examined whether human CD34+ cells from the human umbilical cord blood (hUCB), cocultured with neonatal mouse cardiomyocytes, acquire the functional properties of myocardial cells and express human cardiac genes. hUCB CD34+ cells were cocultured onto cardiomyocytes following an infection with a lentivirus-encoding enhanced green fluorescent protein (EGFP). After 7 days, mononucleated EGFP+ cells were tested for their electrophysiological features by patch clamp and for cytosolic [Ca2+] ([Ca2+]i) homeostasis by [Ca2+]i imaging of X-rhod1-loaded cells. Human Nkx2.5 and GATA-4 expression was examined in cocultured cell populations by real-time RT-PCR. EGFP+ cells were connected to surrounding cells by gap junctions, acquired electrophysiological properties similar to those of cardiomyocytes, and showed action potential-associated [Ca2+]i transients. These cells also exhibited spontaneous sarcoplasmic reticulum [Ca2+]i oscillations and the associated membrane potential depolarization. However, RT-PCR of both cell populations showed no upregulation of human-specific cardiac genes. In conclusion, under our experimental conditions, hUCB CD34+ cells cocultured with murine cardiomyocytes formed cells that exhibited excitation-contraction coupling features similar to those of cardiomyocytes. However, the expression of human-specific cardiac genes was undetectable by RT-PCR.
Diminished mesenteric vaso- and venoconstriction and elevated plasma ANP and BNP with simulated microgravity
Authors: Behnke Bradley J. , Zawieja David C. , Gashev Anatoliy A. , Ray Chester A. , Delp Michael D.
Published: January 2008

Diminished constriction of arteries and veins following exposure to microgravity or bed rest is associated with a reduced ability to augment peripheral vascular resistance (PVR) and stroke volume during orthostasis. We tested the hypothesis that small mesenteric arteries and veins, which are not exposed to large pressure shifts during simulated microgravity via head-down tail suspension (HDT), will exhibit decrements in adrenergic constriction after HDT in rats. Small mesenteric arteries and veins from control (Con; n = 41) and HDT ( n = 35) male Sprague-Dawley rats were studied in vitro. Vasoactive responsiveness to norepinephrine (NE) in arteries (10−9to 10−4M) and veins (pressure-diameter responses from 2 to 12 cmH2O after incubation in 10−6or 10−4M NE) were evaluated. Plasma concentrations of atrial (ANP) and NH2-terminal prohormone brain (NT-proBNP) natriuretic peptides were also measured. In mesenteric arteries, sensitivity and maximal responsiveness to NE were reduced with HDT. In mesenteric veins there was a diminished venoconstriction to NE at any given pressure in HDT. Plasma concentrations of both ANP and NT-proBNP were increased with HDT, and maximal arterial and venous constrictor responses to NE after incubation with 10−7M ANP or brain natriuretic peptide (BNP) were diminished. These data demonstrate that, in a vascular bed not subjected to large hydrodynamic differences with HDT, both small arteries and veins have a reduced responsiveness to adrenergic stimulation. Elevated levels of circulating ANP or NT-proBNP could adversely affect the ability of these vascular beds to constrict in vivo and conceivably could alter the intrinsic constrictor properties of these vessels with long-term exposure.
Airway contribution to alveolar nitric oxide in healthy subjects and stable asthma patients
Authors: Kerckx Yannick , Michils Alain , Van Muylem Alain
Published: January 2008

Alveolar nitric oxide (NO) concentration (FaNO), increasingly considered in asthma, is currently interpreted as a reflection of NO production in the alveoli. Recent modeling studies showed that axial molecular diffusion brings NO molecules from the airways back into the alveolar compartment during exhalation (backdiffusion) and contributes to FaNO. Our objectives in this study were 1) to simulate the impact of backdiffusion on FaNOand to estimate the alveolar concentration actually due to in situ production (FaNO,prod); and 2) to determine actual alveolar production in stable asthma patients with a broad range of NO bronchial productions. A model incorporating convection and diffusion transport and NO sources was used to simulate FaNOand exhaled NO concentration at 50 ml/s expired flow (FeNO) for a range of alveolar and bronchial NO productions. FaNOand FeNOwere measured in 10 healthy subjects (8 men; age 38 ± 14 yr) and in 21 asthma patients with stable asthma [16 men; age 33 ± 13 yr; forced expiratory volume during 1 s (FEV1) = 98.0 ± 11.9%predicted]. The Asthma Control Questionnaire (Juniper EF, Buist AS, Cox FM, Ferrie PJ, King DR. Chest 115: 1265–1270, 1999) assessed asthma control. Simulations predict that, because of backdiffusion, FaNOand FeNOare linearly related. Experimental results confirm this relationship. FaNO,prodmay be derived by FaNO,prod= (FaNO− 0.08·FeNO)/0.92 ( Eq. 1 ). Based on Eq. 1 , FaNO,prodis similar in asthma patients and in healthy subjects. In conclusion, the backdiffusion mechanism is an important determinant of NO alveolar concentration. In stable and unobstructed asthma patients, even with increased bronchial NO production, alveolar production is normal when appropriately corrected for backdiffusion.
Human respiratory muscle blood flow measured by near-infrared spectroscopy and indocyanine green
Authors: Guenette Jordan A. , Vogiatzis Ioannis , Zakynthinos Spyros , Athanasopoulos Dimitrios , Koskolou Maria , Golemati Spyretta , Vasilopoulou Maroula , Wagner Harrieth E. , Roussos Charis , Wagner Peter D. , Boushel Robert
Published: January 2008

Measurement of respiratory muscle blood flow (RMBF) in humans has important implications for understanding patterns of blood flow distribution during exercise in healthy individuals and those with chronic disease. Previous studies examining RMBF in humans have required invasive methods on anesthetized subjects. To assess RMBF in awake subjects, we applied an indicator-dilution method using near-infrared spectroscopy (NIRS) and the light-absorbing tracer indocyanine green dye (ICG). NIRS optodes were placed on the left seventh intercostal space at the apposition of the costal diaphragm and on an inactive control muscle (vastus lateralis). The primary respiratory muscles within view of the NIRS optodes include the internal and external intercostals. Intravenous bolus injection of ICG allowed for cardiac output (by the conventional dye-dilution method with arterial sampling), RMBF, and vastus lateralis blood flow to be quantified simultaneously. Esophageal and gastric pressures were also measured to calculate the work of breathing and transdiaphragmatic pressure. Measurements were obtained in five conscious humans during both resting breathing and three separate 5-min bouts of constant isocapnic hyperpnea at 27.1 ± 3.2, 56.0 ± 6.1, and 75.9 ± 5.7% of maximum minute ventilation as determined on a previous maximal exercise test. RMBF progressively increased (9.9 ± 0.6, 14.8 ± 2.7, 29.9 ± 5.8, and 50.1 ± 12.5 ml·100 ml−1·min−1, respectively) with increasing levels of ventilation while blood flow to the inactive control muscle remained constant (10.4 ± 1.4, 8.7 ± 0.7, 12.9 ± 1.7, and 12.2 ± 1.8 ml·100 ml−1·min−1, respectively). As ventilation rose, RMBF was closely and significantly correlated with 1) cardiac output ( r = 0.994, P = 0.006), 2) the work of breathing ( r = 0.995, P = 0.005), and 3) transdiaphragmatic pressure ( r = 0.998, P = 0.002). These data suggest that the NIRS-ICG technique provides a feasible and sensitive index of RMBF at different levels of ventilation in humans.
Adaptive response of pulmonary arterial smooth muscle to length change
Authors: Syyong Harley , Cheung Christine , Solomon Dennis , Seow Chun Y. , Kuo Kuo H.
Published: January 2008

Hypervasoconstriction is associated with pulmonary hypertension and dysfunction of the pulmonary arterial smooth muscle (PASM) is implicated. However, relatively little is known about the mechanical properties of PASM. Recent advances in our understanding of plastic adaptation in smooth muscle may shed light on the disease mechanism. In this study, we determined whether PASM is capable of adapting to length changes (especially shortening) and regain its contractile force. We examined the time course of length adaptation in PASM in response to step changes in length and to length oscillations mimicking the periodic stretches due to pulsatile arterial pressure. Rings from sheep pulmonary artery were mounted on myograph and stimulated using electrical field stimulation (12–16 s, 20 V, 60 Hz). The length-force relationship was determined at Lref to 0.6 Lref, where Lref was a reference length close to the in situ length of PASM. The response to length oscillations was determined at Lref, after the muscle was subjected to length oscillation of various amplitudes for 200 s at 1.5 Hz. Release (or stretch) of resting PASM from Lref to 0.6 (and vice versa) was followed by a significant force recovery (73 and 63%, respectively), characteristic of length adaptation. All recoveries of force followed a monoexponential time course. Length oscillations with amplitudes ranging from 5 to 20% Lref caused no significant change in force generation in subsequent contractions. It is concluded that, like many smooth muscles, PASM possesses substantial capability to adapt to changes in length. Under pathological conditions, this could contribute to hypervasoconstriction in pulmonary hypertension.
Absence of E-Cadherin Expression Distinguishes Noncohesive from Cohesive Pancreatic Cancer
Authors: Winter Jordan M. , Ting Angela H. , Vilardell Felip , Gallmeier Eike , Baylin Steve B. , Hruban Ralph H. , Kern Scott E. , Iacobuzio-Donahue Christine A.
Published: January 2008

Purpose: The role of E-cadherin in carcinogenesis is of great interest, but few studies have examined its relevance to pancreatic carcinoma.

Experimental Design: We evaluated E-cadherin protein expression by immunohistochemistry in pancreatobiliary cancers having a noncohesive histologic phenotype (21 undifferentiated adenocarcinomas and 7 signet ring carcinomas), comparing the results with pancreatic cancers having a cohesive phenotype (25 moderately differentiated and 14 poorly differentiated adenocarcinomas).

Results: Twenty of 21 undifferentiated cancers had complete absence of E-cadherin expression, as did two signet ring carcinomas. In contrast, cohesive cancers (n = 39) had E-cadherin labeling at the plasma membrane (P < 0.001). Subsets of cancers were also evaluated for β-catenin expression. All of the cohesive lesions (n = 28) showed a membranous β-catenin expression pattern, whereas noncohesive foci (n = 7) were characterized by either cytoplasmic labeling or complete absence of β-catenin protein expression, suggestive of a deficient zonula adherens in noncohesive cancers. E-cadherin promoter hypermethylation was observed in an undifferentiated pancreatic cancer cell line, MiaPaCa-2, whereas two pancreatic cancer cell lines derived from differentiated lesions lacked any evidence of E-cadherin promoter methylation. No pattern of E-cadherin promoter methylation could be determined in three primary cancers having mixed histologic patterns (contained both cohesive and noncohesive foci). No somatic mutations in E-cadherin were identified in noncohesive pancreatic cancers having inactivated E-cadherin.

Conclusions: Noncohesive pancreatic cancers were characterized by the loss of E-cadherin protein expression. Promoter hypermethylation is a possible mechanism of E-cadherin gene silencing in a subset of these cancers.
Association of MicroRNA Expression in Hepatocellular Carcinomas with Hepatitis Infection, Cirrhosis, and Patient Survival
Authors: Jiang Jinmai , Gusev Yuriy , Aderca Ileana , Mettler Teresa A. , Nagorney David M. , Brackett Daniel J. , Roberts Lewis R. , Schmittgen Thomas D.
Published: January 2008

Purpose: MicroRNA (miRNA) is a new class of small, noncoding RNA. The purpose of this study was to determine if miRNAs are differentially expressed in hepatocellular carcinoma (HCC).

Experimental Design: More than 200 precursor and mature miRNAs were profiled by real-time PCR in 43 and 28 pairs of HCC and adjacent benign liver, respectively, and in normal liver specimens.

Results: Several miRNAs including miR-199a, miR-21, and miR-301 were differentially expressed in the tumor compared with adjacent benign liver. A large number of mature and precursor miRNAs were up-regulated in the adjacent benign liver specimens that were both cirrhotic and hepatitis-positive compared with the uninfected, noncirrhotic specimens (P < 0.01). Interestingly, all of the miRNAs in this comparison had increased expression and none were decreased. The expression of 95 randomly selected mRNAs was not significantly altered in the cirrhotic and hepatitis-positive specimens, suggesting a preferential increase in the transcription of miRNA. Comparing the miRNA expression in the HCC tumors with patient's survival time revealed two groups of patients; those with predominantly lower miRNA expression and poor survival and those with predominantly higher miRNA expression and good survival (P < 0.05). A set of 19 miRNAs significantly correlated with disease outcome. A number of biological processes including cell division, mitosis, and G1-S transition were predicted to be targets of the 19 miRNAs in this group.

Conclusion: We show that a global increase in the transcription of miRNA genes occurs in cirrhotic and hepatitis-positive livers and that miRNA expression may prognosticate disease outcome in HCC.
Serum Proteomics and Biomarkers in Hepatocellular Carcinoma and Chronic Liver Disease
Authors: Zinkin Noah T. , Grall Franck , Bhaskar Killimangalam , Otu Hasan H. , Spentzos Dimitrios , Kalmowitz Brett , Wells Meghan , Guerrero Manuel , Asara John M. , Libermann Towia A. , Afdhal Nezam H.
Published: January 2008

Purpose: Proteomic profiling using surface enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF MS) enables the identification of biomarkers for cancer. We evaluated the sensitivity and specificity of SELDI-TOF MS for detection of established hepatocellular cancer (HCC) and compared it against α-fetoprotein (AFP), Lens culinaris agglutinin–reactive AFP (AFP-L3), and prothrombin induced by vitamin K absence-II (PIVKA-II).

Experimental Design: Forty-one patients with HCC and 51 patients with hepatitis C cirrhosis were enrolled. Serum was analyzed by SELDI-TOF MS using three Ciphergen protein array types.

Results: An 11-peak algorithm for HCC detection was identified. Using the AFP cutoff of 20 ng/mL, the sensitivity was 73% and the specificity was 71%. Using the AFP-L3 cutoff of 10% yielded a sensitivity of 63% and a specificity of 94%. Using the PIVKA-II cutoff of 125 milliabsorbance units (mAU), the sensitivity was 84% and the specificity was 69%. Overall, the sensitivity and specificity of SELDI-TOF MS for HCC were 79% and 86%, respectively. In multivariate analysis, the 11-peak SELDI profile was predictive of HCC independent of AFP, PIVKA, and AFP-L3. Among eight patients with the largest tumor size of <2 cm, SELDI-TOF MS correctly identified seven whereas AFP, AFP-L3, and PIVKA-II identified only three, one, and one, respectively. One of the 11 peaks in the SELDI-TOF MS 11-peak predictor from SELDI-TOF MS was identified as cystatin C.

Conclusions: SELDI-TOF MS accurately distinguished patients with HCC from those with hepatitis C virus cirrhosis, was more accurate than traditional biomarkers in identifying small tumors, and should be further evaluated.
Construction and Preclinical Characterization of Fc-mGITRL for the Immunotherapy of Cancer
Authors: Hu Peisheng , Arias Robyn S. , Sadun Rebecca E. , Nien Yu-Chih , Zhang Nan , Sabzevari Helen , Lutsiak M.E. Christine , Khawli Leslie A. , Epstein Alan L.
Published: January 2008

Purpose: To provide proper costimulation required for effective cancer T-cell immunity, Fc-GITRL fusion proteins were generated for use in immunotherapy protocols.

Experimental Design: Soluble fusion proteins consisting of the Fc fragment of immunoglobulin and the murine glucocorticoid-induced tumor necrosis factor–related receptor ligand (mGITRL) connected with different linkers were genetically engineered and tested for their potency in two BALB/c solid tumor models.

Results: In vivo, construct #178-14 (−5aa, −linker) showed the best activity (>90% tumor reduction) at doses ranging from 5 to 25 μg and was found to be intact by gel electrophoresis. Similar doses used with construct #175-2 (-linker) produced good but not as high tumor regression. Construct #5-1 (+linker), which was found to be relatively unstable by SDS gel electrophoresis, produced <60% tumor regression and required a higher dose (100 μg) to produce optimal results. Survival curves showed that Fc-mGITRL treatment extended the life of 80% of tumor-bearing mice to >3 months compared with controls that died by day 40. T-cell depletion studies showed that CD8+ T cells play a major role in Fc-mGITRL immunotherapy, and tumors removed from Fc-mGITRL– and DTA-1–treated mice showed a significant influx of granzyme B+ lymphocytes compared with controls. Finally, T regulatory (Treg) cell assays showed that, unlike other Fc fusion proteins, all three Fc-mGITRL constructs profoundly suppressed Treg activity.

Conclusions: These studies suggest that a stable, intact Fc-mGITRL fusion protein can provide missing costimulation for the immunotherapy of solid tumors. In addition, Fc-mGITRL may alter Treg activity to enhance its effectiveness for tumor immunotherapy.
A Novel Raji-Burkitt's Lymphoma Model for Preclinical and Mechanistic Evaluation of CD52-Targeted Immunotherapeutic Agents
Authors: Lapalombella Rosa , Zhao Xiaobin , Triantafillou Georgia , Yu Bo , Jin Yan , Lozanski Gerard , Cheney Carolyn , Heerema Nyla , Jarjoura David , Lehman Amy , Lee L. James , Marcucci Guido , Lee Robert J. , Caligiuri Michael A. , Muthusamy Natarajan , Byrd John C.
Published: January 2008

Purpose: To date, efforts to study CD52-targeted therapies, such as alemtuzumab, have been limited due to the lack of stable CD52 expressing transformed B-cell lines and animal models. We describe generation and utilization of cell lines that stably express CD52 both in vitro and in vivo.

Experimental Design: By limiting dilution, we have established several clones of Raji-Burkitt's lymphoma cell line that express surface CD52. Immunophenotype and cytogenetic characterization of these clones was done. In vivo usefulness of the CD52high cell line to evaluate the therapeutic efficacy of CD52-directed antibody was investigated using a SCID mouse xenograft model.

Results: Stable expression of CD52 was confirmed in cells cultured in vitro up to 52 weeks of continuous growth. The functional integrity of the expressed CD52 molecule was shown using alemtuzumab, which induced cytotoxic effects in vitro in the CD52high but not the CD52low clone. Compared with control antibody, alemtuzumab treatment in CD52high inoculated mice resulted in significantly increased median survival. Comparable levels of CD52-targeted direct cytotoxicity, complement-dependent cytotoxicity, and antibody-dependent cytotoxicity and anti-CD52 immunoliposome-mediated delivery of synthetic oligodeoxyribo nucleotides in CD52high clone and primary B-chronic lymphocytic leukemia cells implicated potential in vivo application of this model for evaluation of CD52-targeted antibody and immunoliposomes encapsulating therapeutic agents.

Conclusions: These results show the in vitro utility of the cloned Raji cell lines that stably express high levels CD52. The disseminated leukemia-lymphoma mouse model described herein using these stable cell lines can serve as an excellent system for in vivo therapeutic and mechanistic evaluation of existing and novel antibodies directed against CD52 molecule.
EMMPRIN Expression as a Prognostic Factor in Radiotherapy of Cervical Cancer
Authors: Ju Xing-Zhu , Yang Jin-Ming , Zhou Xiao-Yan , Li Zi-Ting , Wu Xiao-Hua
Published: January 2008

Purpose: Overexpression of extracellular matrix metalloproteinase inducer (EMMPRIN), a member of the immunoglobulin family and a glycoprotein enriched on the surface of many types of tumor cells, has been reported to be linked to invasion, metastasis, growth, and survival of malignant cells. Cervical cancer, the second most prevalent cancer in women worldwide and the fifth leading cause of cancer deaths, responds to radiotherapy variably, with 30% of cases recurring after therapy. The purpose of this study was to determine whether expression of EMMPRIN affects the response of cervical cancer to radiation therapy, and whether this membrane protein can be used as a prognostic marker for cervical cancer.

Experimental Design: The retrospective cohort study included 82 patients with invasive cervical cancer referred to the Department of Gynecologic Oncology at The Cancer Hospital of Fudan University (Shanghai) between 1991 and 2000. These patients were treated with brachytherapy at a dose of 15 Gy at point A before radical hysterectomy. Expression of EMMPRIN in cervical tumor specimens was examined by immunohistochemistry staining before and after brachytherapy and scored for both staining intensity and percentage of tumor cells stained. EMMPRIN immunoreactivity and clinicopathologic data were analyzed with respect to survival end points using univariate and multivariate approaches.

Results: The frequency of EMMPRIN overexpression was 52.4% in primary cervical cancer. After brachytherapy, EMMPRIN overexpression was significantly reduced (13.4%) compared with corresponding tumor before brachytherapy (P = 0.032). EMMPRIN expression was associated with pelvic lymph node metastasis (P = 0.026) and reduction in primary tumor volume following brachytherapy (P = 0.008). Although EMMPRIN expression before or after brachytherapy did not correlate with tumor-specific survival, but increased expression of EMMPRIN following brachytherapy tended to predict poor outcomes by univariate survival analysis (P = 0.0008). In addition, lymph vascular space invasion, deep stromal invasion, and lymph node metastasis were significantly associated with poor prognosis. In multivariate analysis, the independent prognostic factors for tumor-specific survival included the decreased expression of EMMPRIN after brachytherapy (P = 0.002; hazard ratio, 0.339; 95% confidence interval, 0.172-0.672) as well as lymph node metastasis (P = 0.044; hazard ratio, 2.053; 95% confidence interval, 1.020-4.133).

Conclusion: Expression of EMMPRIN was associated with a decrease in the reduction of cervical tumor following brachytherapy, and increased EMMPRIN expression after brachytherapy seemed to be an important predictor of poor survival in this patient cohort. Our study suggests that expression of EMMPRIN confers resistance to radiotherapy. Therefore, EMMPRIN expression in cervical cancer may be regarded both as a prognostic factor and a therapeutic target.
Effects of Plasmid-Based Stat3-Specific Short Hairpin RNA and GRIM-19 on PC-3M Tumor Cell Growth
Authors: Zhang Ling , Gao Lifang , Li Yang , Lin Guimiao , Shao Yueting , Ji Kun , Yu Hao , Hu Jiadi , Kalvakolanu Dhananjaya V. , Kopecko Dennis J. , Zhao Xuejian , Xu De-Qi
Published: January 2008

Purpose: Persistent activation of signal transducers and activators of transcription 3 (Stat3) and its overexpression contribute to the progression and metastasis of several different tumor types. For this reason, Stat3 is a reasonable target for RNA interference–mediated growth inhibition. Blockade of Stat3 using specific short hairpin RNAs (shRNA) can significantly reduce prostate tumor growth in mice. However, RNA interference does not fully ablate target gene expression in vivo, owing to the idiosyncrasies associated with shRNAs and their targets. To enhance the therapeutic efficacy of Stat3-specific shRNA, we applied a combination treatment involving gene associated with retinoid-IFN–induced mortality 19 (GRIM-19), another inhibitor of STAT3, along with shRNA.

Experimental Design: The coding sequences for GRIM-19, a cellular STAT3-specific inhibitor, and Stat3-specific shRNAs were used to create a dual expression plasmid vector and used for prostate cancer therapy in vitro and in mouse xenograft models in vivo.

Results: The coexpressed Stat3-specific shRNA and GRIM-19 synergistically and more effectively suppressed prostate tumor growth and metastases when compared with treatment with either single agent alone.

Conclusion: The simultaneous use of two specific, but mechanistically different, inhibitors of STAT3 activity exerts enhanced antitumor effects.
Rhabdoid Tumor Growth is Inhibited by Flavopiridol
Authors: Smith Melissa E. , Cimica Velasco , Chinni Srinivasa , Challagulla Kavitha , Mani Sridhar , Kalpana Ganjam V.
Published: January 2008

Purpose: Rhabdoid tumors are aggressive and incurable pediatric malignancies. INI1/hSNF5, a tumor suppressor biallelically deleted/inactivated in rhabdoid tumors, directly represses cyclin D1. Rhabdoid tumors and cells are exquisitely dependent on cyclin D1 for genesis and survival, suggesting that targeting the cyclin/cyclin-dependent kinase (cdk) axis may be an effective therapeutic strategy for these tumors. Because cdk inhibitors have not been used for preclinical or clinical testing on rhabdoid tumors, we investigated the effect of flavopiridol, a pan-cdk inhibitor with promising clinical activity, on rhabdoid tumors.

Experimental Design: The effect of flavopiridol on rhabdoid cells was tested in vitro using survival, cell cycle, and apoptosis assays. Its effect was assessed in vivo using xenografted rhabdoid tumor models. Immunoblot and immunohistochemical analysis was used to assess the effect of flavopiridol on cyclin D1 and p21 expression in vitro and in vivo, respectively.

Results: Nanomolar concentrations of flavopiridol inhibited rhabdoid cell growth (IC50 ∼200 nmol/L), induced G1 and G2 arrest, and apoptosis in vitro in a concentration-dependent manner. These effects were correlated with the down-modulation of cyclin D1, up-regulation of p21, and induction of caspase 3/7 activities. Flavopiridol (at 7.5 mg/kg) significantly inhibited the growth of xenografted rhabdoid tumors, and its effect was correlated with the induction of p21 and down-modulation of cyclin D1.

Conclusions: Flavopiridol is effective in inducing cell cycle arrest and cytotoxicity in rhabdoid tumors. Its effects are correlated with the down-regulation of cyclin D1 and the up-regulation of p21. Flavopiridol is potentially a novel chemotherapeutic agent for rhabdoid tumors.
Phase I Study of Alternate-Week Administration of Tipifarnib in Patients with Myelodysplastic Syndrome
Authors: Kurzrock Razelle , Kantarjian Hagop M. , Blascovich Michelle A. , Bucher Cindy , Verstovsek Srdan , Wright John J. , Pilat Susan R. , Cortes Jorge E. , Estey Elihu H. , Giles Francis J. , Beran Miloslav , Sebti Said M.
Published: January 2008

Purpose: To determine the safety and describe the antitumor activity of tipifarnib in patients with myelodysplastic syndrome (MDS) using an alternate-week schedule.

Experimental Design: Patients with MDS were given tipifarnib, escalating from 100 mg orally twice daily until the maximum tolerated dose for 8 weeks followed by maintenance therapy (same dose/schedule) for patients with stable disease or better.

Results: Sixty-three patients were treated. The most common toxicity was myelosuppression (60% of patients). Twenty percent of patients had no side effects. Nonhematologic toxicities included fatigue (20%), skin rash (9%), diarrhea (16%), increase in liver transaminases (14%) and bilirubin (11%), and nausea (11%). Dose-limiting toxicities of ataxia (n = 1), fatigue (n = 1), nausea (n = 1), and neutropenic fever (n = 2) occurred at tipifarnib doses above 1,200 mg/d. Sixteen of 61 (26%) evaluable patients responded (3 complete remissions and 13 hematologic improvements) with major platelet responses being most common (11 of 16 responders). There was no obvious dose-response relationship. Four of the 16 responders (25%; including a complete responder) were treated at the lowest dose level (100 mg twice daily). Only one responder had a Ras mutation. Giving tipifarnib resulted in potent inhibition of farnesyl transferase (usually more than 75%) in peripheral blood mononuclear cells regardless of dose. Partial farnesyl transferase inhibition persisted during the week off.

Conclusions: Alternate-week tipifarnib is active and well tolerated in patients with MDS at doses up to and including 600 mg orally twice daily. The biological activity of tipifarnib is not dependent on dose.
Herbal/Hormonal Dietary Supplement Possibly Associated with Prostate Cancer Progression
Authors: Shariat Shahrokh F. , Lamb Dolores J. , Iyengar Radha G. , Roehrborn Claus G. , Slawin Kevin M.
Published: January 2008

Background: Patients seek herbal/hormonal dietary supplements (HHDS) to prevent and/or solve health and aging issues. After two men developed an unusual course of clinically aggressive prostate cancer within months of starting daily consumption of the same HHDS product, we investigated the effect of this product on prostate cancer progression.

Methods: We evaluated serum levels of total testosterone, luteinizing hormone, and follicle-stimulating hormone and screened prostate biopsy and metastatic specimens for androgen receptor protein expression and mutations. We did hormone analyses and capillary electrophoresis. We tested the effect of the HHDS product on androgen receptor-negative (DU-145 and PC-3) and androgen receptor-positive (LNCaP) human prostate cancer cell lines.

Results: Both patients had low hormone levels. The androgen receptor was expressed in all primary and metastatic prostate cancer tissues and no mutations were identified. Hormone analysis revealed that the HHDS contained testosterone and estradiol. The HHDS product was a more potent dose-dependent stimulator of cancer cell growth than testosterone both in androgen receptor-negative and receptor-positive cell lines. Blocking experiments with increasing concentrations of bicalutamide did not prevent the HHDS product–stimulated growth. We filed an adverse event report with the Food and Drug Administration who issued a warning letter. The manufacturer responded by removing this HHDS product from the market.

Conclusions: The HHDS product contained one or more endocrinologically active tumor-promoting components that had cellular androgen receptor status–independent activity. The HHDS product exhibited potent prostate cancer growth stimulatory activity that was more powerful than that of testosterone, independent of the androgen-receptor status of prostate cancer cells, and resistant to antiandrogen blockade.
Development of a Real-time RT-PCR Assay for Detecting EGFRvIII in Glioblastoma Samples
Authors: Yoshimoto Koji , Dang Julie , Zhu Shaojun , Nathanson David , Huang Tiffany , Dumont Rebecca , Seligson David B. , Yong William H. , Xiong Zhenggang , Rao Nagesh , Winther Henrik , Chakravarti Arnab , Bigner Darell D. , Mellinghoff Ingo K. , Horvath Steve , Cavenee Webster K. , Cloughesy Timothy F. , Mischel Paul S.
Published: January 2008

Purpose: Epidermal growth factor receptor variant III (EGFRvIII) is an oncogenic, constitutively active mutant form of the EGFR that is commonly expressed in glioblastoma and is also detected in a number of epithelial cancers. EGFRvIII presents a unique antigenic target for anti-EGFRvIII vaccines and it has been shown to modulate response to EGFR kinase inhibitor therapy. Thus, detection in clinical samples may be warranted. Existing patents preclude the use of anti-EGFRvIII antibodies for clinical detection. Further, frozen tissue is not routinely available, particularly for patients treated in the community. Thus, detection of EGFRvIII in formalin-fixed paraffin-embedded (FFPE) clinical samples is a major challenge.

Experimental Design: We developed a real-time reverse transcription-PCR (RT-PCR) assay for detecting EGFRvIII in FFPE samples and analyzed 59 FFPE glioblastoma clinical samples with paired frozen tissue from the same surgical resection. We assessed EGFRvIII protein expression by immunohistochemistry using two distinct specific anti-EGFRvIII antibodies and examined EGFR gene amplification by fluorescence in situ hybridization.

Results: The FFPE RT-PCR assay detected EGFRvIII in 16 of 59 (27%) samples, exclusively in cases with EGFR amplification, consistent with the expected frequency of this alteration. The FFPE RT-PCR assay was more sensitive and specific for detecting EGFRvIII than either of the two antibodies alone, or in combination, with a sensitivity of 93% (95% confidence interval, 0.78-1.00) and a specificity of 98% (95% confidence interval, 0.93-1.00).

Conclusion: This assay will facilitate accurate assessment of EGFRvIII in clinical samples and may aid in the development of strategies for stratifying patients for EGFRvIII-directed therapies.
Serial Development of Pulmonary Hypertension in Patients with Idiopathic Pulmonary Fibrosis
Authors: Nathan Steven D. , Shlobin Oksana A. , Ahmad Shahzad , Koch Jim , Barnett Scott D. , Ad Niv , Burton Nelson , Leslie Kevin
Published: January 2008

Background: Idiopathic pulmonary fibrosis (IPF) is a disease with very high mortality. Objective: We sought to characterize serial changes in pulmonary artery pressures (PAP) in patients with advanced IPF who survive to transplant. Methods: Retrospective analysis of IPF patients comparing mean PAP at the time of initial evaluation for transplan- tation (mPAPbaseline) with mPAP at the time of transplant (mPAPfollow-up). The measurements were correlated with New York Heart Association (NYHA) functional class and oxygen requirements. Results: The final cohort consisted of 44 patients with serial right heart catheterization data. The mean mPAPbaseline and mPAPfollow-up were 22.5 and 32.7 mm Hg, respectively. 38.6% (17/44) of the patients had pulmonary hypertension (PH) at baseline. The majority of the non-PH patients developed PH during the serial time interval with a subsequent incidence of 77.8%. At the time of transplant, 86.4% of the patients had PH. There was a significant association between transplant NYHA class, severity of PH and oxygen requirements. Transplant NYHA class IV patients had a higher rate of mPAP change. The severity of PH at the time of transplant did not affect transplant outcomes. Conclusion: PH is common and progressive in patients with advanced IPF who are transplant candidates. Serial change and severity of PAP elevations have a significant association with oxygen requirements and functional status, but not transplant outcomes. Whether or not progressive PH has a significant impact on outcomes without transplantation requires further study.
Regulation of Cardiomyocyte Proliferation and Myocardial Growth During Development by FOXO Transcription Factors
Authors: Evans-Anderson Heather J. , Alfieri Christina M. , Yutzey Katherine E.
Published: January 2008

Cardiomyocytes actively proliferate during embryogenesis and withdraw from the cell cycle during neonatal stages. FOXO (Forkhead O) transcription factors are a direct target of phosphatidylinositol-3 kinase/AKT signaling in skeletal and smooth muscle and regulate expression of the Cip/Kip family of cyclin kinase inhibitors in other cell types; however, the interaction of phosphatidylinositol-3 kinase/AKT signaling, FOXO transcription factors, and cyclin kinase inhibitor expression has not been reported for the developing heart. Here, we show that FOXO1 and FOXO3 are expressed in the developing myocardium concomitant with increased cyclin kinase inhibitor expression from embryonic to neonatal stages. Cell culture studies show that embryonic cardiomyocytes are responsive to insulin-like growth factor 1 stimulation, which results in the induction of the phosphatidylinositol-3 kinase/AKT pathway, cytoplasmic localization of FOXO proteins, and increased myocyte proliferation. Likewise, adenoviral-mediated expression of AKT promotes cardiomyocyte proliferation and cytoplasmic localization of FOXO. In contrast, increased expression of FOXO1 negatively affects myocyte proliferation. In vivo myocyte-specific transgenic expression of FOXO1 during heart development causes embryonic lethality at embryonic day 10.5 because of severe myocardial defects that coincide with premature activation of
p21

cip1

,
p27

kip1

, and
p57

kip2

and decreased myocyte proliferation. Transgenic expression of dominant negative FOXO1 in cardiomyocytes does not obviously affect heart development at embryonic day 10.5, but results in abnormal morphology of the myocardium by embryonic day 18.5 along with decreased cyclin kinase inhibitor expression and increased myocyte proliferation. These data support FOXO transcription factors as negative regulators of cardiomyocyte proliferation and promoters of neonatal cell cycle withdrawal during heart development.
Survival and reproduction of Meloidogyne incognita on tomato as affected by humic acid
Authors: Al-Shalaby Mona , Kesba Hosny
Published: January 2008

The commercial products humic acid (Actosol®), humic acid (Actosol®) + NPK and humic acid (Actosol®) + micro-elements (Fe, Mn, Cu) were tested against the root-knot nematode, Meloidogyne incognita, in vitro and in vivo on tomato and compared with the nematicide, Vydate. In vitro, Vydate at 2 ml l–1 water was the best treatment for inhibiting hatch (30.8% inhibition) and was significantly more effective than all other treatments, except Vydate at 4 ml l–1. Humic acid + micro-elements (2 ml l–1 water) was the least effective treatment for inhibiting hatch. Humic acid supplemented with micro-elements was significantly better than other products in reducing the number of surviving juveniles and achieving the highest percentage of nematode inhibition (49.2%), followed by the two concentrations of Vydate, which were not significantly different from one another. In vivo, all treatments significantly reduced the numbers of galls, embedded stages in roots, final population and population build-up (Pf/Pi) in all types of soil as compared to the untreated control. Materials appeared to be more effective at preventing nematode penetration of roots in sandy loam soil than in the other two soil types (sandy and clay). The double application of humic acid + NPK gave the best plant growth (fresh and dry weights) in sandy loam soil. All treatments reduced plant content of NPK in sandy soil except for humic acid + micro-elements and the two doses of Vydate.
Stability of Heterodera glycines (Tylenchida: Heteroderidae) juvenile hatching from eggs obtained from different sources of soybean, Glycine max
Authors: Masler Edward , Sardanelli Sandra , Donald Patricia
Published: January 2008

The hatching behaviours of Heterodera glycines second-stage juveniles (J2) from eggs obtained from laboratory and glasshouse cultures, and from the field, were examined in vitro using large scale (Baermann funnel) and small-scale (depression slide) assay designs. Hatching of J2 from cultured eggs was robust, whereas J2 from field eggs hatched very poorly in vitro (<1% total cumulative percentage hatch). Qualitative aspects of hatch were stable as hatching of J2 from eggs from all sources was linear from 2 through to 8-10 days. By contrast, quantitative aspects were more variable. Total cumulative percentage hatch typically ranged from 45 to 70% but approached 90% depending upon the source of the cultured eggs. Egg density (eggs/cm2) affected hatch of J2 in the large scale in vitro system, with total cumulative percentage hatch significantly greater at 3100 eggs/cm2 than at 420 eggs/cm2. The poor hatch of J2 from field eggs was lost after two generations in culture and replaced by the typical hatch characteristics of J2 from eggs from an established culture. This included both qualitative and quantitative elements. Stability of H. glycines hatching behaviour and its reflection of dormancy and diapause of J2 are discussed.
Histological study of syncytia induced in cereals by the Mediterranean cereal cyst nematode Heterodera latipons
Authors: Oka Yuji , Mor Mishael , Spiegel Yitzhak
Published: January 2008

Feeding sites of Heterodera latipons in wheat, barley and Lolium rigidum were studied and compared with those of Heterodera avenae in wheat. Juveniles of H. latipons penetrated mainly differentiated roots and chose a root cortical cell as their initial syncytium cell (ISC) in wheat and barley, whilst H. avenae chose a vascular parenchymal cell as an ISC. The cell associated with females in wheat root underwent hypertrophy, and incorporated endodermis, pericycle and parenchyma cells of the vascular cylinder by cell wall dissolution. Syncytia of H. latipons contained a high number of organelles, including endoplasmic reticulum, mitochondria, plastids and amoeboid nuclei, whilst those of H. avenae were highly vacuolated. The syncytium partly occupied both the cortex and the vascular cylinder in contrast to those of H. avenae, which occupied most of the space within the vascular cylinder near the infection site. The cortical cell associated with H. latipons males did not undergo hypertrophy, but had dense cytoplasm, and incorporation of other cells by cell wall dissolution was rare. A similar syncytium formation process was observed with barley, but a cortical cell, several cell layers away from the endodermis, was chosen for the ISC. In resistant L. rigidum, H. latipons juveniles chose an endodermal cell or its neighbouring cortical cell as an ISC. Partial dissolution of cell wall was observed in neighbouring parenchymal cells, although the nematode did not develop into adults.
Scavenging or infection? Possible host choosing by entomopathogenic nematodes
Authors: San-Blas Ernesto , Pembroke Barbara , Gowen Simon
Published: January 2008

Entomopathogenic nematodes are able to survive by scavenging. We tested Steinernema feltiae, S. affine and Heterorhabditis megidis alone or in different combinations to evaluate the responses of these nematodes when dead or live Galleria mellonella larvae were offered. Steinernema feltiae and S. affine scavenged upon dead G. mellonella larvae and about 30% more dead larvae were penetrated than live ones. By contrast, H. megidis penetrated more live larvae than dead ones. When the nematode species were combined, the results varied among the combinations, but the dead larvae were always used as a host. The behaviour of natural field populations of S. feltiae and S. affine was also compared. Steinernema feltiae showed no difference between scavenging and performing 'normal infections', whereas S. affine scavenged to a reduced amount (around 60% less); this difference could be related to the particular foraging strategy of these nematodes.
Salvation by Bricks: Theological Reflections on the Planning Process
Authors: Gorringe Timothy
Published: January 2008

This article proposes that planning is driven by values. Since Christian doctrine implies values, theology has a prima facie interest in town planning. Seven marks of the church are proposed as criteria by which to measure planning proposals.
The City's Grace? Recycling the Urban Ecology
Authors: Scott Peter Manley
Published: January 2008

This article argues for the importance of a theology of 'recycling' as a form of public theology for an urban context. The argument begins by noting some of the difficulties in assessing the urban environment: the quality of some urban ecologies is improving although this goes hand-in-hand with the displacing of nature in wealthier cities. In response a theology of the urban ecology rather than a theology of the urban environment is proposed. This ecological interpretation better explains problems in efforts at urban regeneration and the resistance of urban neighbourhoods to change. The concept of the 'translocal'—a Eucharistic notion—is then introduced as a way of grasping the ecological situatedness of urban living and elaborating on the notion of a recycled city. The article concludes with a recommendation of six principles derived from this theology of 'recycling' that would aid the development of cities as recycled and promote the repeatability of cities.
On the History of the North Caucasian Diaspora in Turkey
Authors: Chochiev Georgi
Published: January 2008

The preservation of the ethno-linguistic and cultural identity of the majority of the North Caucasian ethnic groups for almost one and a half centuries following their migration to Turkey and other Near Eastern countries is a unique ethno-social phenomenon. The paper discusses several aspects of the history, socio-political life, and the ethnic development of the North Caucasian migrants in the Ottoman Empire (resp. the Republic of Turkey). The author takes into account the following factors in discussing the problem: the state regimes and the legal status of the ethnic minorities in Turkey; the ethnic characteristics and the level of social, economic, and cultural development of the local population; the intensity of the contacts with the historical homeland; and the character of interrelations between the country of habitation (Turkey) and Russia/USSR.