Calcium Oxalate Stone Fragment and Crystal Phagocytosis by Human Macrophages.

PURPOSE: In murine and human hyperoxaluric conditions macrophages can be seen surrounding renal calcium oxalate crystal deposits. We hypothesized that macrophages have a role in degrading and destroying these deposits. We investigated the inflammatory response and phagocytic mechanisms when macrophages were exposed to human kidney stones and inorganic crystals. MATERIALS AND METHODS: Human monocytes were differentiated into resting, fully differentiated macrophages by treatment with recombinant human macrophage colony-stimulating factor (M-CSF) or GM-CSF (granulocyte M-CSF) for 6 days. After confirming phenotype by flow cytometry the macrophages were exposed for 20 hours to fragments of sterile human calcium oxalate stones or calcium oxalate crystals. Crystal uptake was determined, and supernatant cytokine and chemokine profiles were analyzed using antibody arrays. Quantitative reverse transcriptase-polymerase chain reaction was done to validate mRNA profile expression. RESULTS: Under direct vision fluorescence microscopy activated human macrophages were noted to surround stone fragments and synthesized crystals, and destroy them in a step-by-step process that involved clathrin mediated endocytosis and phagocytosis. An inflammatory cascade was released by macrophages, including the chemokines chemokine ligand (CCL)2, CCL3, interleukin (IL)-1 receptor antagonist (IL-1ra), complement component C5/C5a and IL-8. Response patterns to stone and crystal material depended on macrophage phenotype and activation status. CONCLUSIONS: In our in vitro study macrophages differentiated with M-CSF showed greater ability to phagocytize crystal deposits than those treated with GM-CSF. Following clathrin mediated endocytosis macrophages released a number of cytokines that are crucial for the inflammatory immune response. This suggests that tissue macrophages have an important role in preventing kidney stone disease by removing and digesting interstitial renal crystal deposits.

Clinical practice guidelines on prostate cancer: a critical appraisal.

PURPOSE: Clinical practice guidelines are increasingly being used by leading organizations to promote high quality evidence-based patient care. However, the methodological quality of clinical practice guidelines developed by different organizations varies considerably. We assessed published clinical practice guidelines on the treatment of localized prostate cancer to evaluate the rigor, applicability and transparency of their recommendations. MATERIALS AND METHODS: We searched for English based clinical practice guidelines on treatment of localized prostate cancer from leading organizations in the 15-year period from 1999 to 2014. Clinical practice guidelines limited to early detection, screening, staging and/or diagnosis of prostate cancer were excluded from analysis. Four independent reviewers used the validated AGREE II instrument to assess the quality of clinical practice guidelines in 6 domains, including 1) scope and purpose, 2) stakeholder involvement, 3) rigor of development, 4) clarity of presentation, 5) applicability and 6) editorial independence. RESULTS: A total of 13 clinical practice guidelines met inclusion criteria. Overall the highest median scores were in the AGREE II domains of clarity of presentation, editorial independence, and scope and purpose. The lowest median score was for applicability (28.1%). Although the median score of editorial independence was high (85.4%), variability was also substantial (IQR 12.5-100). NICE and AUA clinical practice guidelines consistently scored well in most domains. CONCLUSIONS: Clinical practice guidelines from different organizations on treatment of localized prostate cancer are of variable quality and fall short of current standards in certain areas, especially in applicability and stakeholder involvement. Improvements in these key domains can enhance the impact and implementation of clinical practice guidelines.

Cell-based selection provides novel molecular probes for cancer stem cells.

Cancer stem cells (CSC) represent a malignant subpopulation of cells in hierarchically organized tumors. They constitute a subpopulation of malignant cells within a tumor mass and possess the ability to self-renew giving rise to heterogeneous tumor cell populations with a complex set of differentiated tumor cells. CSC may be the cause of metastasis and therapeutic refractory disease. Because few markers exist to identify and isolate pure CSC, we used cell-based Systematic Evolution of Ligands by EXponential enrichment (cell-SELEX) to create DNA aptamers that can identify novel molecular targets on the surfaces of live CSC. Out of 22 putative DNA sequences, 3 bound to ~90% and 5 bound to ~15% of DU145 prostate cancer cells. The 15% of cells that were positive for the second panel of aptamers expressed high levels of E-cadherin and CD44, had high aldehyde dehydrogenase 1 activity, grew as spheroids under nonadherent culture conditions, and initiated tumors in immune-compromised mice. The discovery of the molecular targets of these aptamers could reveal novel CSC biomarkers.

Trifecta nerve complex: potential anatomical basis for microsurgical denervation of the spermatic cord for chronic orchialgia.

PURPOSE: We identified structural abnormalities in the spermatic cord nerves that may explain how microsurgical denervation of the spermatic cord provides pain relief in patients with chronic orchialgia. MATERIALS AND METHODS: We retrospectively reviewed a prospective database to compare spermatic cord biopsy specimens from 56 men treated with a total of 57 procedures for microsurgical denervation of the spermatic cord for chronic orchialgia vs a control group of men without pain treated with cord surgery, including varicocelectomy in 4 and radical orchiectomy in 6. Tissue biopsies were obtained from mapped regions of the spermatic cord in all cases. Biopsies stained with hematoxylin and eosin were examined by an independent pathologist. Three human cadaveric spermatic cords were dissected to confirm localization of the nerve distribution identified on pathological mapping. RESULTS: We identified a median of 25 small diameter (less than 1 mm) nerve fibers in the spermatic cord. Of the 57 procedures for orchialgia 48 (84%) showed wallerian degeneration in 1 or more of these nerves but only 2 of 10 controls (20%) had such degeneration (p = 0.0008). In decreasing order of nerve density the 3 primary sites (trifecta nerve complex) of these changes were the cremasteric muscle fibers (19 nerves per patient), perivasal tissues and vasal sheath (9 nerves per patient), and posterior cord lipomatous/perivessel tissues (3 nerves per patient). Cord nerve distribution mapped by the biopsies was confirmed by cadaveric dissection. CONCLUSIONS: In men with chronic orchialgia there appears to be wallerian degeneration in reproducible patterns in the spermatic cord nerve fibers. Transection of these nerves may explain the effect of the denervation procedure.

Circulating and tumor-infiltrating myeloid cell subsets in patients with bladder cancer.

Both cancer-related inflammation and tumor-induced immune suppression are associated with expansion of myeloid cell subsets including myeloid-derived suppressor cells. However, little known regarding characteristics of myeloid cells in patients with bladder cancer. In this study, we analyzed myeloid cells from peripheral blood (PBMC) and tumor tissue that were collected from patients with superficial noninvasive and invasive urothelial carcinomas. Our results demonstrate that PBMC from bladder cancer patients contain two major CD11b myeloid cell subsets: granulocyte-type CD15(high) CD33(low) cells and monocyte-type CD15(low) CD33(high) cells. The number of circulating granulocytic but not monocytic myeloid cells in cancer patients was markedly increased when compared to healthy individuals. Both myeloid cell subsets from cancer patients were highly activated and produced substantial amounts of proinflammatory chemokines/cytokines including CCL2, CCL3, CCL4, G-CSF, IL-8 and IL-6. Granulocytic myeloid cells were able to inhibit in vitro T cell proliferation through induction of CD4(+) Foxp3(+) T regulatory cells. Analysis of bladder cancer tissues revealed that tumors were infiltrated with monocyte-macrophage CD11b(+) HLA-DR(+) and granulocytic CD11b(+) CD15(+) HLA-DR(-) myeloid cells. Collectively, this study identifies myeloid cell subsets in patients with bladder cancer. We demonstrate that these highly activated inflammatory myeloid cells represent a source of multiple chemokines/cytokines and may contribute to inflammation and immune dysfunction in bladder cancer.

Disciplinary Intersection of Medicine and Business: A Novel Population Health Management Premedical Pathway for Medicine and Other Health Care Professions.

Premedical students typically pursue undergraduate specialization in basic biological and other sciences, learning to understand living systems at a microscopic, genetic, or molecular level. However, curricula in the basic sciences do not traditionally include courses that enable students to learn about living systems at the macro level-understanding individuals within their environment, as well as understanding and managing the health of populations, especially those with underlying conditions or the underserved. This disconnect can be bridged by creating novel curricular programs intersecting medicine and business. Population health management is a multidisciplinary field that aims to improve the patient experience, reduce health care costs, and improve treatment outcomes. Though related to public health, the 2 fields differ in significant ways. Population health management emphasizes health outcomes and their measurements and seeks to improve health outcomes based on a full understanding of risk, behaviors, lifestyle, environment, and all social determinants of health. A shift in premedical education that connects the medical sciences with business requires curricular reform with the emerging field of population health management at their intersection, allowing students to be better prepared for future medical practices and to understand expectations, benchmarks, business, and economics in a new value-based health care system.

Variations in Opioid Use Following Robotic Radical Prostatectomy in South Florida.

Introduction: Opioid dependency has been a persistent issue in the United States over the past two decades. Increased efforts have been made to reduce opioid prescribing. Our objective was to quantify at-home opioid requirements following radical prostatectomy. Methods: Written questionnaires were administered to patients 1 week following robot-assisted laparoscopic radical prostatectomy (RALP). Patients provided data on opioid use, pain levels, and demographic characteristics. Results: Sixty-five patients were included. Median age (interquartile range [IQR]) was 69 (62-72) years. The majority were white (85%) and hispanic (67%). Prescriptions ranged from 6 to 15 pills of 5-mg oxycodone equivalents. Twenty-two percent (145/663) of the prescribed pills in the study were consumed. Fifty-four percent (35/65) of patients did not take opioids. Of the 30 patients who took opioids, median use (IQR) was 4.5 (3-6) pills. Forty-six percent (30/65) reported catheter-related pain. Patients who took opioids reported higher levels of pain. On generalized linear regression, younger age, lower levels of education, and living with a family member were factors associated with increased risk for opioid use (all p < 0.05). Conclusions: Despite the Florida Department of Health's restriction on narcotic prescriptions to 3-day supplies, opioids are still overprescribed in our region. The majority of patients do not require opioids after RALP, and patients who do require an opioid analgesic can be adequately managed with less than 6 pills of 5-mg oxycodone equivalents.

Redesign of US Medical Schools: A Shift from Health Service to Population Health Management.

The integration of medical schools and clinical partners is effectively established through the formation of academic medical centers (AMCs). The tripartite mission of AMCs emphasizes the importance of providing critical clinical services, medical innovation through research, and the education of future health care leaders. Although AMCs represent only 5% of all hospitals, they contribute substantially to serving disadvantaged populations of patients, including an estimated 37% of all charity care and 26% of all Medicaid hospitalizations. Currently, most AMCs use a business model centered upon revenue generated from hospital services and/or practice plans. In the last decade, mounting financial demands have placed significant pressure on AMC finances because of the rising costs associated with complex clinical care and operating diverse graduate medical education programs. A shift toward population health-centric health care management strategies will profoundly influence the predominant forms of health care delivery in the United States in the foreseeable future. Health systems are increasingly pursuing new strategies to manage financial risk, such as forming Accountable Care Organizations and provider-sponsored plans to provide value-based care. Refocusing research and operational capacity toward population health management fosters collaboration and enables reintegration with hospital and clinical partners across care networks, and can potentially create new revenue streams for AMCs. Despite the benefits of population health integration, current literature lacks a blueprint to guide AMCs in the transformation toward sustainable population health management models. The purpose of this paper is to propose a modern conceptual framework that can be operationalized by AMCs in order to achieve a sustainable future.

Combination therapy for renal cell cancer: what are possible options?

Antiangiogenic therapy has shown promise in the treatment of patients with renal cell carcinoma (RCC). Two classes of antiangiogenic drugs, the anti-vascular endothelial growth factor antibody bevacizumab and the tyrosine kinase inhibitors sorafenib, sunitinib and pazopanib, have shown efficacy in patients with RCC and are approved by the US Food and Drug Administration for treatment of this cancer. In practice, the clinical benefit of antiangiogenic drugs in RCC has been heterogeneous, and in patients who do respond, benefits are modest and/or short-lived. To improve efficacy, combination targeted therapy has been attempted, but with either very limited additional efficacy or nontolerable toxicities. Recent advances in the molecular understanding of tumor angiogenesis and mechanism of resistance, along with the rapid development of targeted drug discovery, have made it possible to further explore novel combination therapy for RCC.

Altered expression of 15-hydroxyprostaglandin dehydrogenase in tumor-infiltrated CD11b myeloid cells: a mechanism for immune evasion in cancer.

Many cancers are known to produce high amounts of PGE(2), which is involved in both tumor progression and tumor-induced immune dysfunction. The key enzyme responsible for the biological inactivation of PGE(2) in tissue is NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH). It is well established that cancer cells frequently show down-regulated expression of 15-PGDH, which plays a major role in catabolism of the PGE(2). Here we demonstrate that tumor-infiltrated CD11b cells are also deficient for the 15-PGDH gene. Targeted adenovirus-mediated delivery of 15-PGDH gene resulted in substantial inhibition of tumor growth in mice with implanted CT-26 colon carcinomas. PGDH-mediated antitumor effect was associated with attenuated tumor-induced immune suppression and substantially reduced secretion of immunosuppressive mediators and cytokines such as PGE(2), IL-10, IL-13, and IL-6 by intratumoral CD11b cells. We show also that introduction of 15-PGDH gene in tumor tissue is sufficient to redirect the differentiation of intratumoral CD11b cells from immunosuppressive M2-oriented F4/80(+) tumor-associated macrophages (TAM) into M1-oriented CD11c(+) MHC class II-positive myeloid APCs. Notably, the administration of the 15-PGDH gene alone demonstrated a significant therapeutic effect promoting tumor eradication and long-term survival in 70% of mice with preestablished tumors. Surviving mice acquired antitumor T cell-mediated immune response. This study for the first time demonstrates an important role of the 15-PGDH in regulation of local antitumor immune response and highlights the potential to be implemented to enhance the efficacy of cancer therapy and immunotherapy.

Generation of antigen-presenting cells from tumor-infiltrated CD11b myeloid cells with DNA demethylating agent 5-aza-2'-deoxycytidine.

Tumor-recruited CD11b myeloid cells, including myeloid-derived suppressor cells, play a significant role in tumor progression, as these cells are involved in tumor-induced immune suppression and tumor neovasculogenesis. On the other hand, the tumor-infiltrated CD11b myeloid cells could potentially be a source of immunostimulatory antigen-presenting cells (APCs), since most of these cells represent common precursors of both dendritic cells and macrophages. Here, we investigated the possibility of generating mature APCs from tumor-infiltrated CD11b myeloid cells. We demonstrate that in vitro exposure of freshly excised mouse tumors to DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (decitabine, AZA) results in selective elimination of tumor cells, but, surprisingly it also enriches CD45(+) tumor-infiltrated cells. The majority of "post-AZA" surviving CD45(+) tumor-infiltrated cells were represented by CD11b myeloid cells. A culture of isolated tumor-infiltrated CD11b cells in the presence of AZA and GM-CSF promoted their differentiation into mature F4/80/CD11c/MHC class II-positive APCs. These tumor-derived myeloid APCs produced substantially reduced amounts of immunosuppressive (IL-13, IL-10, PGE(2)), pro-angiogenic (VEGF, MMP-9) and pro-inflammatory (IL-1beta, IL-6, MIP-2) mediators than their precursors, freshly isolated tumor-infiltrated CD11b cells. Vaccinating naïve mice with ex vivo generated tumor-derived APCs resulted in the protection of 70% mice from tumor outgrowth. Importantly, no loading of tumor-derived APC with exogenous antigen was needed to stimulate T cell response and induce the anti-tumor effect. Collectively, our results for the first time demonstrate that tumor-infiltrated CD11b myeloid cells can be enriched and differentiated in the presence of DNA demethylating agent 5-aza-2'-deoxycytidine into mature tumor-derived APCs, which could be used for cancer immunotherapy.

Reporting quality and information consistency of randomized, controlled trials presented as abstracts at the American Urological Association annual meetings.

PURPOSE: We assessed the quality of randomized, controlled trial reporting in abstracts from the annual meetings of the American Urological Association and determined whether the information provided is consistent with subsequent full text publications. MATERIALS AND METHODS: All randomized, controlled trials presented in abstract form at the 2002 and 2003 American Urological Association annual meetings were identified for review. A systematic PubMed search based on authorship and key words from the study title was done to identify all subsequent full text publications. A standardized evaluation form was developed based on the published literature, pilot tested in a separate sample and applied by 2 independent reviewers. RESULTS: A total of 126 randomized, controlled trials were identified for review, including 56 in 2002 and 70 in 2003. Approximately a third of the trials (43 or 34.1%) identified the study design as a randomized, controlled trial in the abstract title. The method of randomization, allocation concealment and blinding was reported in 0% (0), 0% (0) and 40.5% (51) of studies, respectively. Mean/median followup was provided in 27.0% of studies (34). Of 126 randomized, controlled trials presented in abstract form 62.7% (79) were subsequently published as full text articles. Study sample size and the number of randomized subjects differed in 24.1% and 28.9% of abstracts, respectively. From the small proportion of randomized, controlled trials (23 or 29.1%) that identified a single primary end point results differed in 9 of 23 (39.1%). CONCLUSIONS: Most abstracts fail to provide the necessary information to assess methodological quality. Organizers of urological meetings should consider implementing a more structured abstract format that requires authors to provide the necessary study details, thereby allowing urologists to critically appraise study validity.

Expression of pluripotent stem cell reprogramming factors by prostate tumor initiating cells.

PURPOSE: We identified a discrete population of stem cell-like tumor cells expressing 5 essential transcription factors required to reprogram pluripotency in prostate tumor cell lines and primary prostate cancer tissue. MATERIALS AND METHODS: DU145 and PC3 human prostate cancer cell lines (ATCC), tumor tissue from patients with prostate cancer and normal prostate tissue were evaluated for the reprogramming factors OCT3/4 (Cell Signaling Technology), SOX2, Klf4 (Santa Cruz Biotechnology, Santa Cruz, California), Nanog (BioLegend) and c-Myc (Cell Signaling) by semiquantitative reverse transcriptase-polymerase chain reaction, histological and immunohistochemical analysis. Stem cell-like tumor cells were enriched by flow cytometric cell sorting using E-cadherin (R&D Systems) as a surface marker, and soft agar, spheroid and tumorigenicity assays to confirm cancer stem cell-like characteristics. RESULTS: mRNA expression of transcription factors OCT3/4 and SOX2 highly correlated in primary prostate tumor tissue samples. The number of OCT3/4 or SOX2 expressing cells was significantly increased in prostate cancer tissue compared to that in normal prostate or benign prostate hyperplasia tissue (p <0.05). When isolated from the DU145 and PC3 prostate cancer cell lines by flow cytometry, stem cell-like tumor cells expressing high OCT3/4 and SOX2 levels showed high tumorigenicity in immunodeficient mice. In vivo growth of the parental DU145 and PC3 prostate cancer cell lines was inhibited by short hairpin RNA knockdown of OCT3/4 or SOX2. CONCLUSIONS: Data suggest that prostate tumor cells expressing pluripotent stem cell transcription factors are highly tumorigenic. Identifying such cells and their importance in prostate cancer growth could provide opportunities for novel targeting strategies for prostate cancer therapy.

Transitioning to Value-Based Diabetes Care: A Call for Action Derived from Primary Care Providers in South Florida.

Background: In Florida, 2.4 million people have diabetes and 5.8 million are pre-diabetic. Not only has the prevalence of diabetes doubled over the past 20 years from 5.2 in 1992 to 11.2 in 2014, but the Centers for Disease Control and Prevention expects 1 out of every 3 adults will have diabetes by the year 2050. In addition, in every year since 1996, Florida well exceeds the national levels in terms of prevalence of diabetes, and the gap is getting wider. A study was conducted to gather information from key physician stakeholders as to how to address unmet needs of patients at risk for, or whom already have, diabetes in a tri-county region of South Florida where the prevalence of diabetes is very high. Objective: The goal was to catalyze innovation and generate solutions for high quality and affordable diabetes care by convening community physicians in South Florida and querying them about solutions for delivering value-based care. Methods: A physician-led task force of community physicians was convened to uncover unmet needs in the diabetes care continuum, identify areas of improvement for coordinating care across the continuum and effectively accessing specialty care. Focus groups were convened with 30 participants to capture qualitative data relative to unmet needs, utilizing the Rapid Ideation Technique. A survey instrument was designed and administered to the twenty-one community clinicians on the task force to augment the qualitative data with quantitative data. The first part of the survey captured characteristics of the participating clinicians, their practices, their diabetes services and management approaches. The second part of the survey captured individual ratings of the importance and merit of needs and/or potential solutions generated. Results: The focus groups generated a wealth of information regarding challenges, issues, areas of opportunities, and potential solutions that could be organized within eight main themes: care coordination and integration; patient engagement, education and behavioral change; physician and practice support; EMR and data issues; telehealth solutions; health informatics and data analytics; and access to care. The surveys culminated in the formation of a Call-For-Action Agenda for immediate work. Conclusions: The ultimate goal of the taskforce was to catalyze innovation and generate solutions for high quality and affordable care. This article reports the findings and provides a roadmap for the future.

Management Principles to Drive the Creation of a 21st Century Medical School.

Introduction: There are currently no data, blueprints, best practices, or financial models available to guide the creation of a new medical school. Yet, the United States is experiencing unprecedented growth of new allopathic medical schools. Findings: This article brings logic to the process. It converts the complexity of what is often regarded as an administrative exercise into the first published framework of management principles. Those principles were then translated into a process map and a financial optimization model. All three elements can be successfully implemented for establishing an accredited, value-driven medical education program that minimizes time from inception to implementation, and ensures sustainability over time. Outcomes: This case report provides a blueprint for planning and implementation of a new medical school. Outcomes include both process and optimization models, as well as valuable insights that have utility when considering a new medical school to mitigate the projected nationwide shortage of physicians.

Duplicate presentations on prostate cancer at American Urological Association and European Association of Urology annual meetings.

PURPOSE: We determined the rate of duplicate research presentations at recent American Urological Association and European Urological Association annual meetings. MATERIALS AND METHODS: We cross-referenced all clinical research presentations related to prostate cancer presented at the 2006 American Urological Association and European Urological Association annual meetings with those presented at the corresponding annual meetings in 2005, 2006 and 2007 using a defined search strategy based on author names, abstract titles, study design and objectives. All data abstraction was performed in duplicate by 2 independent reviewers to ensure accuracy. RESULTS: We identified 282 and 312 abstracts on prostate cancer clinical research at the 2006 European Urological Association and American Urological Association annual meetings, respectively. The overall duplication rate of American Urological Association abstracts was 19.2% (60 of 312). Of duplicated abstracts 80.0% (48 of 60) were presented at the European Urological Association annual meeting the same year. Duplication of European Urological Association abstracts was identified in 20.9% (59 of 282). Authors who presented the same research (71 duplicate abstracts) at the 2 meetings altered the presentations in various ways, including a different study title in 40.8%, a different first and senior author in 14.1% and 18.3%, and increased or decreased sample size in 8.5% and 14.1%, respectively. CONCLUSIONS: Approximately a fifth of clinical research abstracts on prostate cancer presented at the American Urological Association annual meeting were also presented at the European Urological Association meeting and vice versa. Inconsistencies between duplicate abstracts raise concerns about the integrity of the underlying studies. Stricter submission guidelines and improved dissemination of research findings from the 2 meetings may help limit this practice.

Evaluating the evidence: the methodological and reporting quality of comparative observational studies of surgical interventions in urological publications.

OBJECTIVE: To develop and apply a standardized evaluation form for assessing the methodological and reporting quality of observational studies of surgical interventions in urology. METHODS: An evaluation standard was developed using the Consolidated Standards for Reporting Trials statement and previously reported surgical reporting quality instruments. Consensus scoring among three reviewers was developed using two distinct sets of studies. All comparative observational trials involving therapeutic surgical procedures published in four major urological journals in 1995 and 2005 were randomly assigned to each reviewer. Categories of reporting adequacy included background, intervention, statistical analysis, results and discussion. RESULTS: Twenty-seven articles in 1995 and 62 in 2005 met the inclusion criteria; 90% of studies were retrospective. From 1995 to 2005, the overall reporting quality score increased by 3.9 points (95% confidence interval, CI, 2.7-5.9; P = 0.001), from a mean (SD) of 19.1 (3.9) to 23.0 (4.2) on a scale of 0-42. There were significant improvements in the reporting categories of study background (+0.7 points, 95% CI 0.1-1.3, P = 0.043, 0-8-point scale), intervention (+1.6 points, 0.8-2.3, P = 0.001, 0-9-point scale), and statistical analysis (+0.8 points, 0.2-1.4, P = 0.006, 0-9-point scale). There were smaller and statistically insignificant improvements for results (+0.5 points, -0.3 to 1.2, P = 0.217, 0-10-point scale) and discussion reporting (+0.4 points, -0.1 to 0.8, P = 0.106, 0-6-point scale). CONCLUSIONS: There have been minor improvements in the reporting of observational studies of surgical intervention between 1995 and 2005. However, reporting quality remains suboptimal. Clinical investigators, reviewers and journal editors should continue to strive for transparent reporting of the observational studies representing the bulk of the clinical evidence for urological procedures.

Differentiation of human embryonic stem cells into immunostimulatory dendritic cells under feeder-free culture conditions.

PURPOSE: The objective of this study was to develop a scalable and broadly applicable active immunotherapy approach against cancer, circumventing the limitations typically encountered with autologous vaccination strategies. We hypothesized that human embryonic stem cells (hESC) can serve as a virtually unlimited source for generating dendritic cells (DC) with potent antigen-presenting function. Here, we investigated the developmental processes and requirements for generating large numbers of mature, antigen-presenting DC from pluripotent hESC. EXPERIMENTAL DESIGN: A feeder cell-free culture system was developed to differentiate hESC into mature DC sequentially through hematopoietic and myeloid precursor stages. RESULTS: Using this method, we were able to yield large numbers of mature immunostimulatory DC from hESC to enable clinical investigation. Upon activation, the hESC-derived DC secreted interleukin-12p70, migrated in response to MIP-3beta, and exhibited allostimulatory capacity. Most importantly, antigen-loaded, hESC-derived DC were capable of stimulating potent antigen-specific CD8(+) T-cell responses in an HLA class I-matched semiallogeneic assay system. Moreover, HLA class II-mismatched hESC-derived DC induced a potent Th1-type cytokine response without expanding FOXP3(+) regulatory T cells in vitro. CONCLUSIONS: These data suggest the development of a novel active immunotherapy platform to stimulate potent T-cell immunity in patients with intractable diseases, such as cancer or viral infection.

Reversal of myeloid cell-mediated immunosuppression in patients with metastatic renal cell carcinoma.

PURPOSE: Tumor-induced immunosuppression remains a significant obstacle that limits the efficacy of biological therapy for renal cell carcinoma. Here we evaluate the role of CD33 myeloid-derived suppressor cells (MDSC) in the regulation of T-cell responses in renal cell carcinoma patients. We also examine effect of all-trans-retinoic acid (ATRA) on MDSC-mediated immune suppression. EXPERIMENTAL DESIGN: CD33-positive myeloid cells were isolated from the peripheral blood of renal cell carcinoma patients with magnetic beads and tested in vitro for their ability to inhibit T-cell responses. T-cell function was evaluated using ELISPOT and CTL assays. RESULTS: MDSC isolated from renal cell carcinoma patients, but not from healthy donors, were capable of suppressing antigen-specific T-cell responses in vitro through the secretion of reactive oxygen species and nitric oxide upon interaction with CTL. MDSC-mediated immune suppression and IFN-gamma down-regulation was reversible in vitro by exposing cells to the reactive oxygen species inhibitors. Moreover, ATRA was capable of abrogating MDSC-mediated immunosuppression and improving T-cell function by direct differentiation into antigen-presenting cell precursors. CONCLUSIONS: These results may have significant implications regarding the future design of active immunotherapy protocols that may include differentiation agents as part of a multimodal approach to renal cell carcinoma immunotherapy.

Enhancement of vaccine-mediated antitumor immunity in cancer patients after depletion of regulatory T cells.

In this study, we investigated whether elimination of CD4+/CD25+ Tregs using the recombinant IL-2 diphtheria toxin conjugate DAB(389)IL-2 (also known as denileukin diftitox and ONTAK) is capable of enhancing the immunostimulatory efficacy of tumor RNA-transfected DC vaccines. We show that DAB(389)IL-2 is capable of selectively eliminating CD25-expressing Tregs from the PBMCs of cancer patients without inducing toxicity on other cellular subsets with intermediate or low expression of CD25. DAB(389)IL-2-mediated Treg depletion resulted in enhanced stimulation of proliferative and cytotoxic T cell responses in vitro but only when DAB(389)IL-2 was omitted during T cell priming. DAB(389)IL-2 significantly reduced the number of Tregs present in the peripheral blood of metastatic renal cell carcinoma (RCC) patients and abrogated Treg-mediated immunosuppressive activity in vivo. Moreover, DAB(389)IL-2-mediated elimination of Tregs followed by vaccination with RNA-transfected DCs significantly improved the stimulation of tumor-specific T cell responses in RCC patients when compared with vaccination alone. Our findings may have implications in the design of immune-based strategies that may incorporate the Treg depletion strategy to achieve potent antitumor immunity with therapeutic impact.