http://www.translational-medicine.com The latest research articles published by Journal of Translational Medicine 2012-05-16T00:00:00Z Background: In vitro fabricated tissue engineered vascular constructs could provide an alternative to conventional substitutes. A crucial factor for tissue engineering of vascular constructs is an appropriate cell source. Vascular cells from the human umbilical cord can be directly isolated and cryopreserved until needed. Currently no cell bank for human vascular cells is available. Therefore, the establishment of a future human vascular cell bank conforming to good manufacturing practice (GMP) conditions is desirable for therapeutic applications such as tissue engineered cardiovascular constructs.Materials and MethodsA fundamental step was the adaption of conventional research and development starting materials to GMP compliant starting materials. Human umbilical cord artery derived cells (HUCAC) and human umbilical vein endothelial cells (HUVEC) were isolated, cultivated, cryopreserved (short- and long-term) directly after primary culture and recultivated subsequently. Cell viability, expression of cellular markers and proliferation potential of fresh and cryopreserved cells were studied using trypan blue staining, flow cytometry analysis, immunofluorescence staining and proliferation assays. Statistical analyses were performed using Student's t-test. Results: Sufficient numbers of isolated cells with acceptable viabilities and homogenous expression of cellular markers confirmed that the isolation procedure was successful using GMP compliant starting materials. The influence of cryopreservation was marginal, because cryopreserved cells mostly maintain phenotypic and functional characteristics similar to those of fresh cells. Phenotypic studies revealed that fresh cultivated and cryopreserved HUCAC were positive for alpha smooth muscle actin, CD90, CD105, CD73, CD29, CD44, CD166 and negative for smoothelin. HUVEC expressed CD31, CD146, CD105 and CD144 but not alpha smooth muscle actin. Functional analysis demonstrated acceptable viability and sufficient proliferation properties of cryopreserved HUCAC and HUVEC. Conclusion: Adaptation of cell isolation, cultivation and cryopreservation to GMP compliant starting materials was successful. Cryopreservation did not influence cell properties with lasting impact, confirming that the application of vascular cells from the human umbilical cord is feasible for cell banking. A specific cellular marker expression profile was established for HUCAC and HUVEC using flow cytometry analysis, applicable as a GMP compliant quality control. Use of these cells for the future fabrication of advanced therapy medicinal products GMP conditions are required by the regulatory authority. http://www.translational-medicine.com/content/10/1/98 Bianca Polchow Kati Kebbel Gerno Schmiedeknecht Anne Reichardt Wolfgang Henrich Roland Hetzer Cora Lueders Journal of Translational Medicine 2012, null:98 2012-05-16T00:00:00Z doi:10.1186/1479-5876-10-98 /content/figures/1479-5876-10-98-toc.gif Journal of Translational Medicine 1479-5876 ${item.volume} 98 2012-05-16T00:00:00Z PDF Background: Genomic DNA amplification is a genetic factor involved in cancer, and some oncogenes, such as ERBB2, are highly amplified in gastric cancer. We searched for the possible amplification of other genes in gastric cancer.Methods and ResultsA genome-wide single nucleotide polymorphism microarray analysis was performed using three cell lines of differentiated gastric cancers, and 22 genes (including ERBB2) in five highly amplified chromosome regions (with a copy number of more than 6) were identified. Particular attention was paid to the CRKL gene, the product of which is an adaptor protein containing Src homology 2 and 3 (SH2/SH3) domains. An extremely high CRKL copy number was confirmed in the MKN74 gastric cancer cell line using fluorescence in situ hybridization (FISH), and a high level of CRKL expression was also observed in the cells. The RNA-interference-mediated knockdown of CRKL in MKN74 disclosed the ability of CRKL to upregulate gastric cell proliferation. An immunohistochemical analysis revealed that CRKL protein was overexpressed in 24.4% (88/360) of the primary gastric cancers that were analyzed. The CRKL copy number was also examined in 360 primary gastric cancers using a FISH analysis, and CRKL amplification was found to be associated with CRKL overexpression. Finally, we showed that MKN74 cells with CRKL amplification were responsive to the dual Src/BCR-ABL kinase inhibitor BMS354825, likely via the inhibition of CRKL phosphorylation, and that the proliferation of MKN74 cells was suppressed by treatment with a CRKL-targeting peptide. Conclusion: These results suggested that CRKL protein is overexpressed in a subset of gastric cancers and is associated with CRKL amplification in gastric cancer. Furthermore, our results suggested that CRKL protein has the ability to regulate gastric cell proliferation and has the potential to serve as a molecular therapy target for gastric cancer. http://www.translational-medicine.com/content/10/1/97 Hiroko Natsume Kazuya Shinmura Hong Tao Hisaki Igarashi Masaya Suzuki Kiyoko Nagura Masanori Goto Hidetaka Yamada Matsuyoshi Maeda Hrioyuki Konno Satoki Nakamura Haruhiko Sugimura Journal of Translational Medicine 2012, null:97 2012-05-16T00:00:00Z doi:10.1186/1479-5876-10-97 /content/figures/1479-5876-10-97-toc.gif Journal of Translational Medicine 1479-5876 ${item.volume} 97 2012-05-16T00:00:00Z PDF Background: Heat shock protein 70, a stress protein, has been implicated in tumor progression. However, its role in nasopharyngeal carcinoma (NPC) progression has not yet been clearly investigated. Methods: Immunohistochemistry (IHC) was employed to examine the expression patterns of Hsp70, human leukocyte antigen -A (HLA-A) in NPC tissue samples. Results: The expression of Hsp70 exhibited different spatial patterns among nuclear, membrane and cytoplasm in 507 NPC tumor tissues. Kaplan-Meier survival analysis demonstrated that different Hsp70 expression patterns are correlated with different patient outcomes. High membranal and cytoplasmic levels of Hsp70 predicted good survival of patients. In contrast, high nuclear abundance of Hsp70 correlated with poor survival. Moreover, the membranal and cytoplasmic levels of Hsp70 were positively correlated with levels of the MHC I molecule HLA-A. Conclusions: Different Hsp70 expression patterns had distinct predictive values. The different spatial abundance of Hsp70 may imply its important role in NPC development and provide insight for the development of novel therapeutic strategies involving immunotherapy for NPC. http://www.translational-medicine.com/content/10/1/96 Man-Bo Cai Xiao-Pai Wang Jia-Xing Zhang Hui-Qiong Han Chao-Chun Liu Jin-Xin Bei Ruo-Jun Peng Yi Liang Qi-Sheng Feng Hai-Yun Wang Li-Zhen Chen Sha Fu Tie-bang Kang Jian-Yong Shao Yi-Xin Zeng Journal of Translational Medicine 2012, null:96 2012-05-16T00:00:00Z doi:10.1186/1479-5876-10-96 /content/figures/1479-5876-10-96-toc.gif Journal of Translational Medicine 1479-5876 ${item.volume} 96 2012-05-16T00:00:00Z PDF Background: T cell activation is associated with a rapid increase in intracellular fructose-2,6-bisphosphate (F2,6BP), an allosteric activator of the glycolytic enzyme, 6-phosphofructo-1-kinase. The steady state concentration of F2,6BP in T cells is dependent on the expression of the bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFB1-4) and the fructose-2,6-bisphosphatase, TIGAR. Of the PFKFB family of enzymes, PFKFB3 has the highest kinase:bisphosphatase ratio and has been demonstrated to be required for T cell proliferation. A small molecule antagonist of PFKFB3, 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO), recently has been shown to reduce F2,6BP synthesis, glucose uptake and proliferation in transformed cells. We hypothesized that the induction of PFKFB3 expression may be required for the stimulation of glycolysis in T cells and that exposure to the PFKFB3 antagonist, 3PO, would suppress T cell activation. Methods: We examined PFKFB1-4 and TIGAR expression and F2,6BP concentration in purified CD3+ T cells stimulated with microbead-conjugated agonist antibodies specific for CD3 and the co-stimulatory receptor, CD28. We then determined the effect of 3PO on anti-CD3/anti-CD28-induced T cell activation, F2,6BP synthesis, 2-[1-14C]-deoxy-D-glucose uptake, TNF-alpha secretion and proliferation. Finally, we examined the effect of 3PO administration on the development of delayed type hypersensitivity to methylated BSA and on imiquimod-induced psoriasis in mice. Results: We found that purified human CD3+ T cells express PFKFB2, PFKFB3, PFKFB4 and TIGAR, and that anti-CD3/anti-CD28 conjugated microbeads stimulated a >20-fold increase in F2,6BP with a coincident increase in protein expression of the PFKFB3 family member and a decrease in TIGAR protein expression. We then found that exposure to the PFKFB3 small molecule antagonist, 3PO (1-10 uM), markedly attenuated the stimulation of F2,6BP synthesis, 2-[1-14C]-deoxy-D-glucose uptake, lactate secretion, TNF-alpha secretion and T cell aggregation and proliferation. We examined the in vivo effect of 3PO on the development of delayed type hypersensitivity to methylated BSA and on imiquimod-induced psoriasis in mice and found that 3PO suppressed the development of both T cell-dependent models of immunity in vivo. Conclusions: Our data demonstrate that inhibition of the PFKFB3 kinase activity attenuates the activation of T cells in vitro and suppresses T-cell dependent immunity in vivo and indicate that small molecule antagonists of PFKFB3 may prove effective as T cell immunosuppressive agents. http://www.translational-medicine.com/content/10/1/95 Sucheta Telang Brian Clem Alden Klarer Amy Clem John Trent Richard Bucala Jason Chesney Journal of Translational Medicine 2012, null:95 2012-05-16T00:00:00Z doi:10.1186/1479-5876-10-95 /content/figures/1479-5876-10-95-toc.gif Journal of Translational Medicine 1479-5876 ${item.volume} 95 2012-05-16T00:00:00Z PDF Background: The immune system reconstitution in HIV-1- infected patients undergoing combined antiretroviral therapy is routinely evaluated by T-cell phenotyping, even though the infection also impairs the B-cell mediated immunity. To find new laboratory markers of therapy effectiveness, both B- and T- immune recovery were evaluated by means of a follow-up study of long-term treated HIV-1- infected patients, with a special focus on the measure of new B- and T-lymphocyte production. Methods: A longitudinal analysis was performed in samples obtained from HIV-1-infected patients before therapy beginning and after 6, 12, and 72 months with a duplex real-time PCR allowing the detection of K-deleting recombination excision circles (KRECs) and T-cell receptor excision circles (TRECs), as measures of bone-marrow and thymic output, respectively. A cross sectional analysis was performed to detect B- and T-cell subsets by flow cytometry in samples obtained at the end of the follow-up, which were compared to those of untreated HIV-1-infected patients and uninfected controls. Results: The kinetics and the timings of B- and T-cell release from the bone marrow and thymus during antiretroviral therapy were substantially different, with a decreased B-cell release and an increased thymic output after the prolonged therapy. The multivariable regression analysis showed that a longer pre-therapy infection duration predicts a minor TREC increase and a major KREC reduction. Conclusions: The quantification of KRECs and TRECs represent an improved method to monitor the effects of therapies capable of influencing the immune cell pool composition in HIV-1-infected patients. http://www.translational-medicine.com/content/10/1/94 Eugenia Quiros-Roldan Federico Serana Marco Chiarini Cinzia Zanotti Alessandra Sottini Daria Gotti Carlo Torti Luigi Caimi Luisa Imberti Journal of Translational Medicine 2012, null:94 2012-05-16T00:00:00Z doi:10.1186/1479-5876-10-94 /content/figures/1479-5876-10-94-toc.gif Journal of Translational Medicine 1479-5876 ${item.volume} 94 2012-05-16T00:00:00Z PDF Background: Nasopharyngeal carcinoma (NPC) is well-known for its highly metastatic characteristics, but little is known of its molecular mechanisms. New biomarkers that predict clinical outcome, in particular the ability of the primary tumor to develop metastatic tumors are urgently needed. The aim of this study is to investigate the role of FLJ10540 in human NPC development. Methods: A bioinformatics approach was used to explore the potentially important regulatory genes involved in the growth/metastasis control of NPC. FLJ10540 was chosen for this study. Two co-expression strategies from NPC microarray were employed to identify the relationship between FLJ10540 and osteopontin. Quantitative-RT-PCR, immunoblotting, and immunohistochemistry analysis were used to investigate the mRNA and protein expression profiles of FLJ10540 and osteopontin in the normal and NPC tissues to confirm microarray results. TW01 and Hone1 NPC cells with overexpression FLJ10540 or siRNA to repress endogenous FLJ10540 were generated by stable transfection to further elucidate the molecular mechanisms of FLJ10540-elicited cell growth and metastasis under osteopontin stimulation. Results: We found that osteopontin expression exhibited a positive correlation with FLJ10540 in NPC microarray. We also demonstrated comprehensively that FLJ10540 and osteopontin were not only overexpressed in NPC specimens, but also significantly correlated with advanced tumor and lymph node-metastasis stages, and had a poor 5-year survival rate, respectively. Stimulation of NPC parental cells with osteopontin results in an increase in FLJ10540 mRNA and protein expressions. Functionally, FLJ10540 transfectant alone, or stimulated with osteopontin, exhibited fast growth and increased metastasis as compared to vehicle control with or without osteopontin stimulation. Conversely, knockdown of FLJ10540 by siRNA results in the suppression of NPC cell growth and motility. Treatment with anti-CD44 antibodies in NPC parental cells not only resulted in a decrease of FLJ10540 protein, but also affected the abilities of FLJ10540-elicited cell growth and motility in osteopontin stimulated-NPC cells. Conclusions: These findings suggest that FLJ10540 may be critical regulator of disease progression in NPC, and the underlying mechanism may involve in the osteopontin/CD44 pathway. http://www.translational-medicine.com/content/10/1/93 Chang-Han Chen Li-Yen Shiu Li-Jen Su Chi-Ying Huang Shun-Chen Huang Chao-Cheng Huang Yu-Fang Yin Wei-Sheng Wang Hsin-Ting Tsai Fu-Min Fang Wan-Chu Chuang Hong-Chang Kang Chung-Feng Hwang Journal of Translational Medicine 2012, null:93 2012-05-16T00:00:00Z doi:10.1186/1479-5876-10-93 /content/figures/1479-5876-10-93-toc.gif Journal of Translational Medicine 1479-5876 ${item.volume} 93 2012-05-16T00:00:00Z PDF This Editorial announces a new section in the Journal of Translational Medicine: Patient-Targeted Molecular Therapies. This section is dedicated to the dissemination of targeted molecular therapies in context of patient centered outcomes research and evidence-based clinical decisions. The focus on patient-targeted molecular therapies - spanning small molecules and biomolecules alike - stems from the unprecedented growth in this arena. This is consonant with the overall objective of the Journal of Translational Medicine, which seeks out to expand firmly to other vast areas of medicine in the domain of translational science, viewed here as the transaction between translational research and translational effectiveness. As we inaugurate this new section in Journal of Translational Medicine, with its mission described in detail in this Editorial, we invite interested scientists to submit their work for publication. http://www.translational-medicine.com/content/10/1/92 Adrian Bot Francesco Chiappelli Journal of Translational Medicine 2012, null:92 2012-05-15T00:00:00Z doi:10.1186/1479-5876-10-92 /content/figures/1479-5876-10-92-toc.gif Journal of Translational Medicine 1479-5876 ${item.volume} 92 2012-05-15T00:00:00Z PDF Background: The process of malignant transformation, progression and metastasis of melanoma is not completely understood. Recently, the microarray technology has been used to survey transcriptional differences that might provide insight into the metastatic process, but the validation of changing gene expression during metastatic transition period is poorly investigated. A large body of literature has been produced on the role of the HOX genes network in tumour evolution, suggesting the involvement of HOX genes in several types of human cancers. Deregulated paralogous group 13 HOX genes expression has been detected in melanoma, cervical cancer and odonthogenic tumors. Among these, Hox C13 is also involved in the expression control of the human keratin genes hHa5 and hHa2, and recently it was identified as a member of human DNA replication complexes. Methods: In this study, to investigate HOX C13 expression in melanoma progression, we have compared its expression pattern between naevi, primary melanoma and metastasis. In addition HOXC13 prophile pattern of expression has been evaluated in melanoma cell lines. Results: Our results show the strong and progressive HOX C13 overexpression in metastatic melanoma tissues and cytological samples compared to nevi and primary melanoma tissues and cells. Conclusions: The data presentated in the paper suggest a possible role of HOX C13 in metastatic melanoma switch. http://www.translational-medicine.com/content/10/1/91 Monica Cantile Giosuè Scognamiglio AnnaMaria Anniciello Marisa Farina Giusy Gentilcore Clemente Santonastaso Franco Fulciniti Clemente Cillo Renato Franco Paolo Ascierto Gerardo Botti Journal of Translational Medicine 2012, null:91 2012-05-14T00:00:00Z doi:10.1186/1479-5876-10-91 /content/figures/1479-5876-10-91-toc.gif Journal of Translational Medicine 1479-5876 ${item.volume} 91 2012-05-14T00:00:00Z PDF Background: At recurrence the use of nitrosoureas is widely-used as a therapeutic option for glioblastoma (GBM) patients. The efficacy of fotemustine (FTM) has been demonstrated in phase II clinical trials; however, these papers report a wide range of progression-free-survival (PFS-6m) rates, ranging from 21% to 52%. We investigated whether FTM could have a different response pattern in respect to time to adjuvant temozolomide failure, or whether specific independent risk factors could be responsible for the wide range of response rates observed. Methods: Recurrent GBM patients have been treated with fotemustine 75-100mg/sqm at day 1, 8, 15 and after 4/5 weeks of rest with 100 mg/sqm every 21 days. Patients were stratified in 4 groups according to time to temozolomide failure: before starting (B0), during the first 6 months (B1), after more than 6 months of therapy (B2), and after a treatment-free interval (B3). Primary endpoint was PFS-6m. A multivariable analysis was performed to identify whether gender, time after radiotherapy, second surgery and number of TMZ cycles could be independent predictors of the clinical benefit to FTM treatment. Results: 163 recurrent GBM patients were included in the analysis. PFS-6m rates for the B0, B1, B2 and B3 groups were 25%, 28%, 31.1% and 43.8%, respectively. The probability of disease control was higher in patients with a longer time after radiotherapy (p=0.0161) and in those who had undergone a second surgery (p=0.0306). Conclusions: FTM is confirmed as a valuable therapeutic option for patients with recurrent GBM and was active in all study patient groups. Time after the completion of radiotherapy and second surgery are independent treatment-related risk factors that were predictive of clinical benefit. http://www.translational-medicine.com/content/10/1/90 Alessandro Paccapelo Ivan Lolli Maria Fabrini Giovanni Silvano Beatrice Detti Franco Perrone Giuseppina Savio Erminio Bonizzoni Tania Perrone Silvia Scoccianti Journal of Translational Medicine 2012, null:90 2012-05-14T00:00:00Z doi:10.1186/1479-5876-10-90 /content/figures/1479-5876-10-90-toc.gif Journal of Translational Medicine 1479-5876 ${item.volume} 90 2012-05-14T00:00:00Z PDF Background: Translational Medicine focuses on "bench to bedside", converting experimental results into clinical use. The "bedside to bench" transition remains challenging, requiring clinicians to define true clinical need for laboratory study. In this study, we show how observational data (an eleven-year data survey program on adolescent smoking behaviours), can identify knowledge gaps and research questions leading directly to clinical implementation and improved health care. We studied gender-specific trends (2000-2010) in Italian students to evaluate the specific impact of various anti-smoking programs, including evaluation of perceptions of access to cigarettes and health risk. Methods: The study used, ESPAD-Italia (European School Survey Project on Alcohol and other Drugs), is a nationally representative sample of high-school students. The permutation test for joinpoint regression was used to calculate the annual percent change in smoking. Changes in smoking habits by age, perceived availability and risk over a 11-year period were tested using a gender-specific logistic model and a multinomial model. Results: Gender-stratified analysis showed 1) decrease of lifetime prevalence, then stabilization (both genders); 2) decrease in last month and occasional use (both genders); 3) reduction of moderate use (females); 4) no significant change in moderate use (males) and in heavy use (both genders). Perceived availability positively associates with prevalence, while perceived risk negatively associates, but interact with different effects depending on smoking patterns. In addition, government implementation of public policies concerning access to tobacco products in this age group during this period presented a unique background to examine their specific impact on behaviours. Conclusion: Large observational databases are a rich resource in support of translational research. From these observations, key clinically relevant issues can be identified and form the basis for further clinical studies. The ability to identify patterns of behaviour and gaps in available data translates into new experiments, but also impacts development of public policy and reveals patterns of clinical reality.The observed global decrease in use is countered by stabilization in number of heavy smokers. Increased cigarette cost has not reduced use. While perceived risk of smoking may prevent initial experimentation, how government policies impact the perception of risk is not easily quantifiable. http://www.translational-medicine.com/content/10/1/89 Valeria Siciliano Annalisa Pitino Mercedes Gori Olivia Curzio Loredana Fortunato Michael Liebman Sabrina Molinaro Journal of Translational Medicine 2012, null:89 2012-05-14T00:00:00Z doi:10.1186/1479-5876-10-89 /content/figures/1479-5876-10-89-toc.gif Journal of Translational Medicine 1479-5876 ${item.volume} 89 2012-05-14T00:00:00Z PDF