Stephan Rogalla, M.D. PhD

All Publications

• Automated spatial omics landscape analysis approach reveals novel tissue architectures in ulcerative colitis. Scientific reports Holman, D. R., Rubin, S. J., Ferenc, M., Holman, E. A., Koron, A. N., Daniel, R., Boland, B. S., Nolan, G. P., Chang, J. T., Rogalla, S. 2024; 14 (1): 18934

  Abstract

  The utility of spatial omics in leveraging cellular interactions in normal and diseased states for precision medicine is hampered by a lack of strategies for matching disease states with spatial heterogeneity-guided cellular annotations. Here we use a spatial context-dependent approach that matches spatial pattern detection to cell annotation. Using this approach in existing datasets from ulcerative colitis patient colonic biopsies, we identified architectural complexities and associated difficult-to-detect rare cell types in ulcerative colitis germinal-center B cell follicles. Our approach deepens our understanding of health and disease pathogenesis, illustrates a strategy for automating nested architecture detection for highly multiplexed spatial biology data, and informs precision diagnosis and therapeutic strategies.

  View details for DOI 10.1038/s41598-024-68397-5

  View details for PubMedID 39147769

  View details for PubMedCentralID PMC11327370

• Exploring the effects of taurolidine on tumor weight and microvessel density in a murine model of osteosarcoma. Oncology research Neijenhuis, L. K., Naumann, L. L., Ferkel, S. A., Rubin, S. J., Rogalla, S. 2024; 32 (7): 1163-1172

  Abstract

  Osteosarcoma is the most common malignant primary bone tumor. The prognosis for patients with disseminated disease remains very poor despite recent advancements in chemotherapy. Moreover, current treatment regimens bear a significant risk of serious side effects. Thus, there is an unmet clinical need for effective therapies with improved safety profiles. Taurolidine is an antibacterial agent that has been shown to induce cell death in different types of cancer cell lines.In this study, we examined both the antineoplastic and antiangiogenic effects of taurolidine in animal models of osteosarcoma. K7M2 murine osteosarcoma cells were injected, both intramuscular and intraperitoneal, into 60 BALB/c mice on day zero. Animals were then randomized to receive treatment with taurolidine 2% (800 mg/kg), taurolidine 1% (400 mg/kg), or NaCl 0.9% control for seven days by intravenous or intraperitoneal administration.After 35 days, mice were euthanized, and the tumors were harvested for analysis. Eighteen mice were excluded from the analysis due to complications. Body weight was significantly lower in the 2% taurolidine intraperitoneal treatment group from day 9 to 21, consistent with elevated mortality in this group. Intraperitoneal tumor weight was significantly lower in the 1% (p = 0.003) and 2% (p = 0.006) intraperitoneal taurolidine treatment groups compared to the control. No antineoplastic effects were observed on intramuscular tumors or for intravenous administration of taurolidine. There were no significant differences in microvessel density or mitotic rate between treatment groups. Reduced body weight and elevated mortality in the 2% taurolidine intraperitoneal group suggest that the lower 1% dose is preferable.In conclusion, there is no evidence of antiangiogenic activity, and the antitumor effects of taurolidine on osteosarcoma observed in this study are limited. Moreover, its toxic profile grants further evaluation. Given these observations, further research is necessary to refine the use of taurolidine in osteosarcoma treatment.

  View details for DOI 10.32604/or.2024.050907

  View details for PubMedID 38948019

  View details for PubMedCentralID PMC11209741

• Automated Spatial Omics Landscape Analysis Approach Reveals Novel Tissue Architectures in Ulcerative Colitis. Research square Rogalla, S., Holman, D., Rubin, S., Ferenc, M., Holman, E., Koron, A., Daniel, R., Boland, B., Nolan, G., Chang, J. 2024

  Abstract

  The utility of spatial omics in leveraging cellular interactions in normal and diseased states for precision medicine is hampered by a lack of strategies for matching disease states with spatial heterogeneity-guided cellular annotations. Here we use a spatial context-dependent approach that matches spatial pattern detection to cell annotation. Using this approach in existing datasets from ulcerative colitis patient colonic biopsies, we identified architectural complexities and associated difficult-to-detect rare cell types in ulcerative colitis germinal-center B cell follicles. Our approach deepens our understanding of health and disease pathogenesis, illustrates a strategy for automating nested architecture detection for highly multiplexed spatial biology data, and informs precision diagnosis and therapeutic strategies.

  View details for DOI 10.21203/rs.3.rs-3965505/v1

  View details for PubMedID 38559236

  View details for PubMedCentralID PMC10980100

• Looking to Future Applications of Large Language Models. The American journal of gastroenterology Liu, X., Rubin, S. J., Rogalla, S. 2023; 118 (12): 2305

  View details for DOI 10.14309/ajg.0000000000002401

  View details for PubMedID 38033226

• GPR15 in colon cancer development and anti-tumor immune responses. Frontiers in oncology Namkoong, H., Lee, B., Swaminathan, G., Koh, S. J., Rogalla, S., Paraskevopoulou, M. D., Tang, J., Mikhail, D., Becker, L. S., Habtezion, A. 2023; 13: 1254307

  Abstract

  The chemoattractant receptor, G protein-coupled receptor 15 (GPR15), promotes colon homing of T cells in health and colitis. GPR15 function in colon cancer is largely unexplored, motivating our current studies.In human study, immune cells were isolated from tumor tissues and healthy surgical tumor margins (STM), and their proportions as well as expression of GPR15 was analyzed by flow cytometry. In mouse studies, colon cancer was induced in GPR15-deficient (KO) and GPR15-suficient (Het) mice using azoxymethane (AOM) and dextran sulfate sodium (DSS) solution in drinking water. Serial endoscopy was performed in mice to monitor and visualize the distal region of colon. Mice were euthanized 10 weeks after the initial DSS administration, and the colon length and the number of polyps were recorded. Next, we identified the effects of GPR15L on established tumors in the MC38-colorectal cancer (CRC) mouse model. Immune cells were isolated from the mice colons or tumors and assessed by flow cytometry.Our analysis of human CRC tissue revealed a significant reduction in GPR15+ immune cell frequencies in tumors compared to 'tumor-free' surgical margins. Similarly, our data analysis using The Cancer Genome Atlas (TCGA) indicated that lower GPR15 expression is associated with poor survival in human colon cancer. In the AOM/DSS colitis-associated colon cancer model, we observed increased colonic polyps and lower survival in Gpr15 +-KO compared to Gpr15-Het mice. Analysis of immune cell infiltrates in the colonic polyps showed significantly decreased CD8+ T cells and increased IL-17+ CD4+ and IL-17+ CD8+ T cells in Gpr15-KO than in Het mice. Consistent with a protective role of GPR15, administration of GPR15L to established tumors in the MC38-CRC model increased CD45+ cell infiltration, enhanced TNFa expression on CD4+ and CD8+ T cells at the tumor site and dramatically reduced tumor burden.Our findings highlight an important, unidentified role of the GPR15-GPR15L axis in promoting a tumor-suppressive immune microenvironment and unveils a novel, colon-specific therapeutic target for CRC.

  View details for DOI 10.3389/fonc.2023.1254307

  View details for PubMedID 38074634

  View details for PubMedCentralID PMC10708945

• A tissue atlas of ulcerative colitis revealing evidence of sex-dependent differences in disease-driving inflammatory cell types and resistance to TNF inhibitor therapy SCIENCE ADVANCES Mayer, A. T., Holman, D. R., Sood, A., Tandon, U., Bhate, S. S., Bodapati, S., Barlow, G. L., Chang, J., Black, S., Crenshaw, E. C., Koron, A. N., Streett, S. E., Gambhir, S. S., Sandborn, W. J., Boland, B. S., Hastie, T., Tibshirani, R., Chang, J. T., Nolan, G. P., Schuerch, C. M., Rogalla, S. 2023; 9 (3)

  View details for Web of Science ID 000964550100033

• A tissue atlas of ulcerative colitis revealing evidence of sex-dependent differences in disease-driving inflammatory cell types and resistance to TNF inhibitor therapy. Science advances Mayer, A. T., Holman, D. R., Sood, A., Tandon, U., Bhate, S. S., Bodapati, S., Barlow, G. L., Chang, J., Black, S., Crenshaw, E. C., Koron, A. N., Streett, S. E., Gambhir, S. S., Sandborn, W. J., Boland, B. S., Hastie, T., Tibshirani, R., Chang, J. T., Nolan, G. P., Schürch, C. M., Rogalla, S. 2023; 9 (3): eadd1166

  Abstract

  Although literature suggests that resistance to TNF inhibitor (TNFi) therapy in patients with ulcerative colitis (UC) is partially linked to immune cell populations in the inflamed region, there is still substantial uncertainty underlying the relevant spatial context. Here, we used the highly multiplexed immunofluorescence imaging technology CODEX to create a publicly browsable tissue atlas of inflammation in 42 tissue regions from 29 patients with UC and 5 healthy individuals. We analyzed 52 biomarkers on 1,710,973 spatially resolved single cells to determine cell types, cell-cell contacts, and cellular neighborhoods. We observed that cellular functional states are associated with cellular neighborhoods. We further observed that a subset of inflammatory cell types and cellular neighborhoods are present in patients with UC with TNFi treatment, potentially indicating resistant niches. Last, we explored applying convolutional neural networks (CNNs) to our dataset with respect to patient clinical variables. We note concerns and offer guidelines for reporting CNN-based predictions in similar datasets.

  View details for DOI 10.1126/sciadv.add1166

  View details for PubMedID 36662860

• Expanding hyperspectral imaging applications to the clinical scene: non-invasive, label-free approaches for early diagnostics and precision medicine Frontiers in Imaging Holman, E. A., Holman, D. R., Rogalla, S. 2023; 2

  View details for DOI 10.3389/fimag.2023.1175860

• The Tumor Immune Microenvironment in Pancreatic Ductal Adenocarcinoma: Neither Hot nor Cold. Cancers Rubin, S. J., Sojwal, R. S., Gubatan, J., Rogalla, S. 2022; 14 (17)

  Abstract

  Pancreatic ductal adenocarcinoma (PDAC) is the most common pancreatic tumor and is associated with poor prognosis and treatment response. The tumor microenvironment (TME) is recognized as an important factor in metastatic progression across cancers. Despite extensive study of the TME in PDAC, the cellular and molecular signaling networks remain poorly understood, largely due to the tremendous heterogeneity across tumors. While earlier work characterized PDAC as an immunologically privileged tumor poorly recognized by the immune system, recent studies revealed the important and nuanced roles of immune cells in the pathogenesis of PDAC. Distinct lymphoid, myeloid, and stromal cell types in the TME exert opposing influences on PDAC tumor trajectory, suggesting a more complex organization than the classical "hot" versus "cold" tumor distinction. We review the pro- and antitumor immune processes found in PDAC and briefly discuss their leverage for the development of novel therapeutic approaches in the field.

  View details for DOI 10.3390/cancers14174236

  View details for PubMedID 36077772

• In vivo visualization and molecular targeting of the cardiac conduction system. The Journal of clinical investigation Goodyer, W. R., Beyersdorf, B. M., Duan, L., van den Berg, N. S., Mantri, S., Galdos, F. X., Puluca, N., Buikema, J. W., Lee, S., Salmi, D., Robinson, E. R., Rogalla, S., Cogan, D. P., Khosla, C., Rosenthal, E. L., Wu, S. M. 2022

  Abstract

  Accidental injury to the cardiac conduction system (CCS), a network of specialized cells embedded within the heart and indistinguishable from the surrounding heart muscle tissue, is a major complication in cardiac surgeries. Here, we addressed this unmet need by engineering targeted antibody-dye conjugates directed against CCS, allowing for the visualization of the CCS in vivo following a single intravenous injection in mice. These optical imaging tools showed high sensitivity, specificity, and resolution, with no adverse effects to CCS function. Further, with the goal of creating a viable prototype for human use, we generated a fully human monoclonal Fab, that similarly targets the CCS with high specificity. We demonstrate that, when conjugated to an alternative cargo, this Fab can also be used to modulate CCS biology in vivo providing a proof-of-principle for targeted cardiac therapeutics. Finally, in performing differential gene expression analyses of the entire murine CCS at single-cell resolution, we uncovered and validated a suite of additional cell surface markers that can be used to molecularly target the distinct subcomponents of the CCS, each prone to distinct life-threatening arrhythmias. These findings lay the foundation for translational approaches targeting the CCS for visualization and therapy in cardiothoracic surgery, cardiac imaging and arrhythmia management.

  View details for DOI 10.1172/JCI156955

  View details for PubMedID 35951416

• Gut Microbiome in Inflammatory Bowel Disease: Role in Pathogenesis, Dietary Modulation, and Colitis-Associated Colon Cancer. Microorganisms Gubatan, J., Boye, T. L., Temby, M., Sojwal, R. S., Holman, D. R., Sinha, S. R., Rogalla, S. R., Nielsen, O. H. 2022; 10 (7)

  Abstract

  The gut microbiome has increasingly been recognized as a critical and central factor in inflammatory bowel disease (IBD). Here, we review specific microorganisms that have been suggested to play a role in the pathogenesis of IBD and the current state of fecal microbial transplants as a therapeutic strategy in IBD. We discuss specific nutritional and dietary interventions in IBD and their effects on gut microbiota composition. Finally, we examine the role and mechanisms of the gut microbiome in mediating colitis-associated colon cancer.

  View details for DOI 10.3390/microorganisms10071371

  View details for PubMedID 35889090

• Highlighting the Undetectable - Fluorescence Molecular Imaging in Gastrointestinal Endoscopy. Molecular imaging and biology Stibbe, J. A., Hoogland, P., Achterberg, F. B., Holman, D. R., Sojwal, R. S., Burggraaf, J., Vahrmeijer, A. L., Nagengast, W. B., Rogalla, S. 2022

  Abstract

  Flexible high-definition white-light endoscopy is the current gold standard in screening for cancer and its precursor lesions in the gastrointestinal tract. However, miss rates are high, especially in populations at high risk for developing gastrointestinal cancer (e.g., inflammatory bowel disease, Lynch syndrome, or Barrett's esophagus) where lesions tend to be flat and subtle. Fluorescence molecular endoscopy (FME) enables intraluminal visualization of (pre)malignant lesions based on specific biomolecular features rather than morphology by using fluorescently labeled molecular probes that bind to specific molecular targets. This strategy has the potential to serve as a valuable tool for the clinician to improve endoscopic lesion detection and real-time clinical decision-making. This narrative review presents an overview of recent advances in FME, focusing on probe development, techniques, and clinical evidence. Future perspectives will also be addressed, such as the use of FME in patient stratification for targeted therapies and potential alliances with artificial intelligence. KEY MESSAGES: • Fluorescence molecular endoscopy is a relatively new technology that enables safe and real-time endoscopic lesion visualization based on specific molecular features rather than on morphology, thereby adding a layer of information to endoscopy, like in PET-CT imaging. • Recently the transition from preclinical to clinical studies has been made, with promising results regarding enhancing detection of flat and subtle lesions in the colon and esophagus. However, clinical evidence needs to be strengthened by larger patient studies with stratified study designs. • In the future fluorescence molecular endoscopy could serve as a valuable tool in clinical workflows to improve detection in high-risk populations like patients with Barrett's esophagus, Lynch syndrome, and inflammatory bowel syndrome, where flat and subtle lesions tend to be malignant up to five times more often. • Fluorescence molecular endoscopy has the potential to assess therapy responsiveness in vivo for targeted therapies, thereby playing a role in personalizing medicine. • To further reduce high miss rates due to human and technical factors, joint application of artificial intelligence and fluorescence molecular endoscopy are likely to generate added value.

  View details for DOI 10.1007/s11307-022-01741-1

  View details for PubMedID 35764908

• Antimicrobial peptides and the gut microbiome in inflammatory bowel disease. World journal of gastroenterology Gubatan, J., Holman, D. R., Puntasecca, C. J., Polevoi, D., Rubin, S. J., Rogalla, S. 2021; 27 (43): 7402-7422

  Abstract

  Antimicrobial peptides (AMP) are highly diverse and dynamic molecules that are expressed by specific intestinal epithelial cells, Paneth cells, as well as immune cells in the gastrointestinal (GI) tract. They play critical roles in maintaining tolerance to gut microbiota and protecting against enteric infections. Given that disruptions in tolerance to commensal microbiota and loss of barrier function play major roles in the pathogenesis of inflammatory bowel disease (IBD) and converge on the function of AMP, the significance of AMP as potential biomarkers and novel therapeutic targets in IBD have been increasingly recognized in recent years. In this frontier article, we discuss the function and mechanisms of AMP in the GI tract, examine the interaction of AMP with the gut microbiome, explore the role of AMP in the pathogenesis of IBD, and review translational applications of AMP in patients with IBD.

  View details for DOI 10.3748/wjg.v27.i43.7402

  View details for PubMedID 34887639

  View details for PubMedCentralID PMC8613745

• Antimicrobial peptides and the gut microbiome in inflammatory bowel disease WORLD JOURNAL OF GASTROENTEROLOGY Gubatan, J., Holman, D. R., Puntasecca, C. J., Polevoi, D., Rubin, S. S., Rogalla, S. 2021; 27 (43): 7402-7422

  View details for DOI 10.3748/wjg.v27.i43.7402

  View details for Web of Science ID 000723262500001

• SPECT/CT Imaging, Biodistribution and Radiation Dosimetry of a Lu-177-DOTA-Integrin alpha v beta 6 Cystine Knot Peptide in a Pancreatic Cancer Xenograft Model FRONTIERS IN ONCOLOGY Sachindra, S., Hellberg, T., Exner, S., Prasad, S., Beindorff, N., Rogalla, S., Kimura, R., Gambhir, S., Wiedenmann, B., Groetzinger, C. 2021; 11: 684713

  Abstract

  Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignant neoplasms, as many cases go undetected until they reach an advanced stage. Integrin αvβ6 is a cell surface receptor overexpressed in PDAC. Consequently, it may serve as a target for the development of probes for imaging diagnosis and radioligand therapy. Engineered cystine knottin peptides specific for integrin αvβ6 have recently been developed showing high affinity and stability. This study aimed to evaluate an integrin αvβ6-specific knottin molecular probe containing the therapeutic radionuclide 177Lu for targeting of PDAC.The expression of integrin αvβ6 in PDAC cell lines BxPC-3 and Capan-2 was analyzed using RT-qPCR and immunofluorescence. In vitro competition and saturation radioligand binding assays were performed to calculate the binding affinity of the DOTA-coupled tracer loaded with and without lutetium to BxPC-3 and Capan-2 cell lines as well as the maximum number of binding sites in these cell lines. To evaluate tracer accumulation in the tumor and organs, SPECT/CT, biodistribution and dosimetry projections were carried out using a Capan-2 xenograft tumor mouse model.RT-qPCR and immunofluorescence results showed high expression of integrin αvβ6 in BxPC-3 and Capan-2 cells. A competition binding assay revealed high affinity of the tracer with IC50 values of 1.69 nM and 9.46 nM for BxPC-3 and Capan-2, respectively. SPECT/CT and biodistribution analysis of the conjugate 177Lu-DOTA-integrin αvβ6 knottin demonstrated accumulation in Capan-2 xenograft tumors (3.13 ± 0.63%IA/g at day 1 post injection) with kidney uptake at 19.2 ± 2.5 %IA/g, declining much more rapidly than in tumors.177Lu-DOTA-integrin αvβ6 knottin was found to be a high-affinity tracer for PDAC tumors with considerable tumor accumulation and moderate, rapidly declining kidney uptake. These promising results warrant a preclinical treatment study to establish therapeutic efficacy.

  View details for DOI 10.3389/fonc.2021.684713

  View details for Web of Science ID 000661097300001

  View details for PubMedID 34136410

  View details for PubMedCentralID PMC8200818

• A protease-activated, near-infrared fluorescent probe for early endoscopic detection of premalignant gastrointestinal lesions. Proceedings of the National Academy of Sciences of the United States of America Yim, J. J., Harmsen, S., Flisikowski, K., Flisikowska, T., Namkoong, H., Garland, M., van den Berg, N. S., Vilches-Moure, J. G., Schnieke, A., Saur, D., Glasl, S., Gorpas, D., Habtezion, A., Ntziachristos, V., Contag, C. H., Gambhir, S. S., Bogyo, M., Rogalla, S. 2021; 118 (1)

  Abstract

  Fluorescence imaging is currently being actively developed for surgical guidance; however, it remains underutilized for diagnostic and endoscopic surveillance of incipient colorectal cancer in high-risk patients. Here we demonstrate the utility and potential for clinical translation of a fluorescently labeled cathepsin-activated chemical probe to highlight gastrointestinal lesions. This probe stays optically dark until it is activated by proteases produced by tumor-associated macrophages and accumulates within the lesions, enabling their detection using an endoscope outfitted with a fluorescence detector. We evaluated the probe in multiple murine models and a human-scale porcine model of gastrointestinal carcinogenesis. The probe provides fluorescence-guided surveillance of gastrointestinal lesions and augments histopathological analysis by highlighting areas of dysplasia as small as 400 m, which were visibly discernible with significant tumor-to-background ratios, even in tissues with a background of severe inflammation and ulceration. Given these results, we anticipate that this probe will enable sensitive fluorescence-guided biopsies, even in the presence of highly inflamed colorectal tissue, which will improve early diagnosis to prevent gastrointestinal cancers.

  View details for DOI 10.1073/pnas.2008072118

  View details for PubMedID 33443161

• AND-gate contrast agents for enhanced fluorescence-guided surgery. Nature biomedical engineering Widen, J. C., Tholen, M. n., Yim, J. J., Antaris, A. n., Casey, K. M., Rogalla, S. n., Klaassen, A. n., Sorger, J. n., Bogyo, M. n. 2020

  Abstract

  Surgical resection of tumours requires precisely locating and defining the margins between lesions and normal tissue. However, this is made difficult by irregular margin borders. Although molecularly targeted optical contrast agents can be used to define tumour margins during surgery in real time, the selectivity of the contrast agents is often limited by the target being expressed in both healthy and tumour tissues. Here, we show that AND-gate optical imaging probes that require the processing of two substrates by multiple tumour-specific enzymes produce a fluorescent signal with significantly improved specificity and sensitivity to tumour tissue. We evaluated the performance of the probes in mouse models of mammary tumours and of metastatic lung cancer, as well as during fluorescence-guided robotic surgery. Imaging probes that rely on multivariate activation to selectively target complex patterns of enzymatic activity should be useful in disease detection, treatment and monitoring.

  View details for DOI 10.1038/s41551-020-00616-6

  View details for PubMedID 32989286

• Biodegradable fluorescent nanoparticles for endoscopic detection of colorectal carcinogenesis. Advanced functional materials Rogalla, S., Flisikowski, K., Gorpas, D., Mayer, A. T., Flisikowska, T., Mandella, M. J., Ma, X., Casey, K. M., Felt, S. A., Saur, D., Ntziachristos, V., Schnieke, A., Contag, C. H., Gambhir, S. S., Harmsen, S. 2019; 29 (51)

  Abstract

  Early and comprehensive endoscopic detection of colonic dysplasia - the most clinically significant precursor lesion to colorectal adenocarcinoma - provides an opportunity for timely, minimally-invasive intervention to prevent malignant transformation. Here, the development and evaluation of biodegradable near-infrared fluorescent silica nanoparticles (FSN) is described that have the potential to improve adenoma detection during fluorescence-assisted white-light colonoscopic surveillance in rodent and human-scale models of colorectal carcinogenesis. FSNs are biodegradable (t1/2 of 2.7 weeks), well-tolerated, and enable detection and delineation of adenomas as small as 0.5 mm2 with high tumor-to-background ratios. Furthermore, in the human-scale, APC 1311/+ porcine model, the clinical feasibility and benefit of using FSN-guided detection of colorectal adenomas using video-rate fluorescence-assisted white-light endoscopy is demonstrated. Since nanoparticles of similar size (e.g., 100-150-nm) or composition (i.e., silica, silica/gold hybrid) have already been successfully translated to the clinic, and, clinical fluorescent/white light endoscopy systems are becoming more readily available, there is a viable path towards clinical translation of the proposed strategy for early colorectal cancer detection and prevention in high-risk patients.

  View details for DOI 10.1002/adfm.201904992

  View details for PubMedID 33041743

  View details for PubMedCentralID PMC7546531

• Biodegradable Fluorescent Nanoparticles for Endoscopic Detection of Colorectal Carcinogenesis ADVANCED FUNCTIONAL MATERIALS Rogalla, S., Flisikowski, K., Gorpas, D., Mayer, A. T., Flisikowska, T., Mandella, M. J., Ma, X., Casey, K. M., Felt, S. A., Saur, D., Ntziachristos, V., Schnieke, A., Contag, C. H., Gambhir, S. S., Harmsen, S. 2019; 29 (51)

  View details for DOI 10.1002/adfm.201904992

  View details for Web of Science ID 000516572400007

• Detection of Premalignant Gastrointestinal Lesions Using Surface-Enhanced Resonance Raman Scattering-Nanoparticle Endoscopy. ACS nano Harmsen, S., Rogalla, S., Huang, R., Spaliviero, M., Neuschmelting, V., Hayakawa, Y., Lee, Y., Tailor, Y., Toledo-Crow, R., Kang, J. W., Samii, J. M., Karabeber, H., Davis, R. M., White, J. R., van de Rijn, M., Gambhir, S. S., Contag, C. H., Wang, T. C., Kircher, M. F. 2019; 13 (2): 1354–64

  Abstract

  Cancers of the gastrointestinal (GI) tract are among the most frequent and most lethal cancers worldwide. An important reason for this high mortality is that early disease is typically asymptomatic, and patients often present with advanced, incurable disease. Even in high-risk patients who routinely undergo endoscopic screening, lesions can be missed due to their small size or subtle appearance. Thus, current imaging approaches lack the sensitivity and specificity to accurately detect incipient GI tract cancers. Here we report our finding that a single dose of a high-sensitivity surface-enhanced resonance Raman scattering nanoparticle (SERRS-NP) enables reliable detection of precancerous GI lesions in animal models that closely mimic disease development in humans. Some of these animal models have not been used previously to evaluate imaging probes for early cancer detection. The studies were performed using a commercial Raman imaging system, a newly developed mouse Raman endoscope, and finally a clinically applicable Raman endoscope for larger animal studies. We show that this SERRS-NP-based approach enables robust detection of small, premalignant lesions in animal models that faithfully recapitulate human esophageal, gastric, and colorectal tumorigenesis. This method holds promise for much earlier detection of GI cancers than currently possible and could lead therefore to marked reduction of morbidity and mortality of these tumor types.

  View details for PubMedID 30624916

• Detection of Premalignant Gastrointestinal Lesions Using Surface-Enhanced Resonance Raman Scattering-Nanoparticle Endoscopy ACS NANO Harmsen, S., Rogalla, S., Huang, R., Spaliviero, M., Neuschmelting, V., Hayakawa, Y., Lee, Y., Tailor, Y., Toledo-Crow, R., Kang, J., Samii, J. M., Karabeber, H., Davis, R. M., White, J. R., van de Rijn, M., Gambhir, S. S., Contag, C. H., Wang, T. C., Kircher, M. F. 2019; 13 (2): 1354–64

  View details for DOI 10.1021/acsnano.8b06808

  View details for Web of Science ID 000460199400038

• Emerging Intraoperative Imaging Modalities to Improve Surgical Precision MOLECULAR IMAGING AND BIOLOGY Alam, I. S., Steinberg, I., Vermesh, O., van den Berg, N. S., Rosenthal, E. L., van Dam, G. M., Ntziachristos, V., Gambhir, S. S., Hernot, S., Rogalla, S. 2018; 20 (5): 705–15

  View details for DOI 10.1007/s11307-018-1227-6

  View details for Web of Science ID 000444754000003

• Emerging Intraoperative Imaging Modalities to Improve Surgical Precision. Molecular imaging and biology : MIB : the official publication of the Academy of Molecular Imaging Alam, I. S., Steinberg, I., Vermesh, O., van den Berg, N. S., Rosenthal, E. L., van Dam, G. M., Ntziachristos, V., Gambhir, S. S., Hernot, S., Rogalla, S. 2018

  Abstract

  Intraoperative imaging (IOI) is performed to guide delineation and localization of regions of surgical interest. While oncological surgical planning predominantly utilizes x-ray computed tomography (CT), magnetic resonance imaging (MRI), and ultrasound (US), intraoperative guidance mainly remains on surgeon interpretation and pathology for confirmation. Over the past decades however, intraoperative guidance has evolved significantly with the emergence of several novel imaging technologies, including fluorescence-, Raman, photoacoustic-, and radio-guided approaches. These modalities have demonstrated the potential to further optimize precision in surgical resection and improve clinical outcomes for patients. Not only can these technologies enhance our understanding of the disease, they can also yield large imaging datasets intraoperatively that can be analyzed by deep learning approaches for more rapid and accurate pathological diagnosis. Unfortunately, many of these novel technologies are still under preclinical or early clinical evaluation. Organizations like the Intra-Operative Imaging Study Group of the European Society for Molecular Imaging (ESMI) support interdisciplinary interactions with the aim to improve technical capabilities in the field, an approach that can succeed only if scientists, engineers, and physicians work closely together with industry and regulatory bodies to resolve roadblocks to clinical translation. In this review, we provide an overview of a variety of novel IOI technologies, discuss their challenges, and present future perspectives on the enormous potential of IOI for oncological surgical navigation.

  View details for PubMedID 29916118

• Intraoperative Molecular Imaging in Lung Cancer: The State of the Art and the Future. Molecular therapy : the journal of the American Society of Gene Therapy Rogalla, S. n., Joosten, S. C., Alam, I. S., Gambhir, S. S., Vermesh, O. n. 2018; 26 (2): 338–41

  View details for PubMedID 29398484

• The tyrosine kinase inhibitor imatinib mesylate suppresses uric acid crystal-induced acute gouty arthritis in mice PLOS ONE Reber, L. L., Starkl, P., Balbino, B., Sibilano, R., Gaudenzio, N., Rogalla, S., Sensarn, S., Kang, D., Raghu, H., Sokolove, J., Robinson, W. H., Contag, C. H., Tsai, M., Galli, S. J. 2017; 12 (10): e0185704

  Abstract

  Gouty arthritis is caused by the deposition of monosodium urate (MSU) crystals in joints. Despite many treatment options for gout, there is a substantial need for alternative treatments for patients unresponsive to current therapies. Tyrosine kinase inhibitors have demonstrated therapeutic benefit in experimental models of antibody-dependent arthritis and in rheumatoid arthritis in humans, but to date, the potential effects of such inhibitors on gouty arthritis has not been evaluated. Here we demonstrate that treatment with the tyrosine kinase inhibitor imatinib mesylate (imatinib) can suppress inflammation induced by injection of MSU crystals into subcutaneous air pouches or into the ankle joint of wild type mice. Moreover, imatinib treatment also largely abolished the lower levels of inflammation which developed in IL-1R1-/- or KitW-sh/W-sh mice, indicating that this drug can inhibit IL-1-independent pathways, as well as mast cell-independent pathways, contributing to pathology in this model. Imatinib treatment not only prevented ankle swelling and synovial inflammation when administered before MSU crystals but also diminished these features when administrated after the injection of MSU crystals, a therapeutic protocol more closely mimicking the clinical situation in which treatment occurs after the development of an acute gout flare. Finally, we also assessed the efficiency of local intra-articular injections of imatinib-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles in this model of acute gout. Treatment with low doses of this long-acting imatinib:PLGA formulation was able to reduce ankle swelling in a therapeutic protocol. Altogether, these results raise the possibility that tyrosine kinase inhibitors might have utility in the treatment of acute gout in humans.

  View details for PubMedID 28982129

• Neutrophil myeloperoxidase diminishes the toxic effects and mortality induced by lipopolysaccharide JOURNAL OF EXPERIMENTAL MEDICINE Reber, L. L., Gillis, C. M., Starkl, P., Joensson, F., Sibilano, R., Marichal, T., Gaudenzio, N., Berard, M., Rogalla, S., Contag, C. H., Bruhns, P., Galli, S. J. 2017; 214 (5): 1249-1258

  Abstract

  Neutrophils have crucial antimicrobial functions but are also thought to contribute to tissue injury upon exposure to bacterial products, such as lipopolysaccharide (LPS). To study the role of neutrophils in LPS-induced endotoxemia, we developed a new mouse model, PMN(DTR) mice, in which injection of diphtheria toxin induces selective neutrophil ablation. Using this model, we found, surprisingly, that neutrophils serve to protect the host from LPS-induced lethal inflammation. This protective role was observed in conventional and germ-free animal facilities, indicating that it does not depend on a particular microbiological environment. Blockade or genetic deletion of myeloperoxidase (MPO), a key neutrophil enzyme, significantly increased mortality after LPS challenge, and adoptive transfer experiments confirmed that neutrophil-derived MPO contributes importantly to protection from endotoxemia. Our findings imply that, in addition to their well-established antimicrobial properties, neutrophils can contribute to optimal host protection by limiting the extent of endotoxin-induced inflammation in an MPO-dependent manner.

  View details for DOI 10.1084/jem.20161238

  View details for Web of Science ID 000400379300006

  View details for PubMedID 28385925

• Dose-dependent role of novel agents emodin and BTB14431 in colonic cancer treatment in rats. Acta chirurgica Belgica Braumann, C. n., Koplin, G. n., Geier, C. n., Höhn, P. n., Pohlenz, J. n., Dubiel, W. n., Rogalla, S. n. 2017: 1–9

  Abstract

  BTB14431 is an in silico homolog to emodin. Both were found to possess anti-tumor effects in vitro. The aim of this work was to analyze the tumor suppressing effects of both molecules in an intraperitoneal (ip) and intravenous (iv) treated rat model (WAG-Rij).A tumor cell suspension (CC531) was applied at the cecum after laparotomy and at the back. The rats where treated twice a day over 1 week with BTB14431, emodin and isotone sodium chloride solution (control). Treatment was applied iv or ip in a variety of dosages. Peripheral blood samples were taken before tumor application and on day 7. Twenty-one days after the last day of therapy animals were euthanized and tumor growth was evaluated.Data showed an insignificant decrease of tumor growth after iv and ip treatment with low doses of BTB14431 and emodin. Differential blood analysis showed apoptosis. Increased doses of emodin clearly raised mortality rate.Apoptosis was verified but no tumor-suppressing effects could be observed for iv and ip treatment with both agents in contrast to in vitro studies in our model. Establishing a successful ip treatment model for emotion and BTB14331 requires further studies.

  View details for DOI 10.1080/00015458.2017.1341145

  View details for PubMedID 28669313

• A Clinical Wide-Field Fluorescence Endoscopic Device for Molecular Imaging Demonstrating Cathepsin Protease Activity in Colon Cancer. Molecular imaging and biology Sensarn, S., Zavaleta, C. L., Segal, E., Rogalla, S., Lee, W., Gambhir, S. S., Bogyo, M., Contag, C. H. 2016; 18 (6): 820-829

  Abstract

  Early and effective detection of cancers of the gastrointestinal tract will require novel molecular probes and advances in instrumentation that can reveal functional changes in dysplastic and malignant tissues. Here, we describe adaptation of a wide-field clinical fiberscope to perform wide-field fluorescence imaging while preserving its white-light capability for the purpose of providing wide-field fluorescence imaging capability to point-of-care microscopes.We developed and used a fluorescent fiberscope to detect signals from a quenched probe, BMV109, that becomes fluorescent when cleaved by, and covalently bound to, active cathepsin proteases. Cathepsins are expressed in inflammation- and tumor-associated macrophages as well as directly from tumor cells and are a promising target for cancer imaging. The fiberscope has a 1-mm outer diameter enabling validation via endoscopic exams in mice, and therefore we evaluated topically applied BMV109 for the ability to detect colon polyps in an azoxymethane-induced colon tumor model in mice.This wide-field endoscopic imaging device revealed consistent and clear fluorescence signals from BMV109 that specifically localized to the polypoid regions as opposed to the normal adjacent colon tissue (p < 0.004) in the murine colon carcinoma model.The sensitivity of detection of BMV109 with the fluorescence fiberscope suggested utility of these tools for early detection at hard-to-reach sites. The fiberscope was designed to be used in conjunction with miniature, endoscope-compatible fluorescence microscopes for dual wide-field and microscopic cancer detection.

  View details for PubMedID 27154508

• A Clinical Wide-Field Fluorescence Endoscopic Device for Molecular Imaging Demonstrating Cathepsin Protease Activity in Colon Cancer MOLECULAR IMAGING AND BIOLOGY Sensarn, S., Zavaleta, C. L., Segal, E., Rogalla, S., Lee, W., Gambhir, S. S., Bogyo, M., Contag, C. H. 2016; 18 (6): 820–29

  View details for DOI 10.1007/s11307-016-0956-7

  View details for Web of Science ID 000387367100004

• SU-E-J-274: Responses of Medulloblastoma Cells to Radiation Dosimetric Parameters in Intensity-Modulated Radiation Therapy. Medical physics Park, J., Park, J., Rogalla, S., Woo, D., Lee, D., Park, H., Contag, C., Suh, T. 2015; 42 (6): 3330-?

  Abstract

  To evaluate radiation responses of the medulloblastoma cell line Daoy in intensity-modulated radiation therapy (IMRT), quantitative variations to variable radiation dosimetic parameters were tracked by bioluminescent images (BLIs).The luciferase and green fluorescent protein positive Daoy cells were cultured on dishes. The medulloblastoma cells irradiated to different dose rate, interval of fractionated doses, field margin and misalignment, and dose uniformity in IMRT were monitored using bioluminescent images. The cultured cells were placed into a dedicated acrylic phantom to deliver intensity-modulated fluences and calculate accurate predicted dose distribution. The radiation with dose rate from 0.5 Gy/min to 15 Gy/min was irradiated by adjusting monitor unit per minute and source-to-surface distances. The intervals of fractionated dose delivery were changed considering the repair time of double strand breaks (DSB) revealed by straining of gamma-H2AX.The effect of non-uniform doses on the cells were visualized by registering dose distributions and BLIs. The viability according to dosimetric parameters was correlated with bioluminescent intensities for cross-check of radiation responses.The DSB and cell responses due to the first fractionated dose delivery significantly affected final tumor control rather than other parameters. The missing tumor volumes due to the smaller field margin than the tumor periphery or field misalignment caused relapse of cell responses on BLIs. The dose rate and gradient had effect on initial responses but could not bring out the distinguishable killing effect on cancer cells.Visualized and quantified bioluminescent images were useful to correlate the dose distributions with spatial radiation effects on cells. This would derive the effective combination of dose delivery parameters and fractionation. Radiation responses in particular IMRT configuration could be reflected to image based-dose re-optimization.

  View details for DOI 10.1118/1.4924360

  View details for PubMedID 26127667

• Early Cancer Detection at the Epithelial Surface CANCER JOURNAL Rogalla, S., Contag, C. H. 2015; 21 (3): 179-187

  Abstract

  Malignant neoplastic lesions derived from epithelial tissue, carcinomas, account for 80% to 100% of all human cancers including some of the most deadly diseases such as cervical and non-small cell lung cancer. Many of these carcinomas present at readily accessible epithelial surfaces offering unique detection opportunities. Effective clinical management of carcinomas is enabled by early detection, at a time when full surgical resection is possible and before invasion of adjacent tissue or significant intravasation into blood vessels leading to metastasis. Good prognosis with long-term disease-free survival is more likely after early detection when progression is limited. At present, detection of carcinomas at epithelial surfaces largely relies on routine inspection with the naked eye (e.g., skin and oropharynx) or simple white light tools (e.g., cervix and colon). Emerging optical tools based on differential refraction, absorption, reflection, scattering, or fluorescence of carcinomas relative to normal tissues enable label-free visualization of neoplasia. However, the differences in intrinsic optical properties of normal and malignant tissues can be subtle, and relying on these may lead to high miss rates. Enhanced optical contrast offered by molecularly targeted agents can be used to improve early detection; and given that optical imaging and sensing tools can be readily combined, integrated systems that image over a range of scales, or detect multiple parameters, can be developed to aid in early detection. Diagnosis is, at present, made by histologic examination of tissue biopsies after identification of suspicious lesions. Miniature and handheld microscopic imaging tools have recently been developed, and integration of these tools with wide-field optical surveillance devices offers both rapid detection and confirmatory histologic examination at the point-of-care, that can provide guidance for biopsy and/or resection. A wide variety of targeted probe strategies have been described with demonstrated benefit in preclinical models and in a limited number of human studies. Here, we present examples of integrated multimodality optical imaging and sensing tools that use combinations of intrinsic and extrinsic optical contrast for early detection or margin delineation for carcinomas at epithelial surfaces. We will discuss several new technologies that have use in detecting the most common carcinomas that derive from the epithelium of the skin, gastrointestinal and urogenital tracts, and bronchoalveoli.

  View details for DOI 10.1097/PPO.0000000000000122

  View details for Web of Science ID 000355658000008

  View details for PubMedID 26049697

• A Real-Time Clinical Endoscopic System for Intraluminal, Multiplexed Imaging of Surface-Enhanced Raman Scattering Nanoparticles PLOS ONE Garai, E., Sensarn, S., Zavaleta, C. L., Loewke, N. O., Rogalla, S., Mandella, M. J., Felt, S. A., Friedland, S., Liu, J. T., Gambhir, S. S., Contag, C. H. 2015; 10 (4)

  Abstract

  The detection of biomarker-targeting surface-enhanced Raman scattering (SERS) nanoparticles (NPs) in the human gastrointestinal tract has the potential to improve early cancer detection; however, a clinically relevant device with rapid Raman-imaging capability has not been described. Here we report the design and in vivo demonstration of a miniature, non-contact, opto-electro-mechanical Raman device as an accessory to clinical endoscopes that can provide multiplexed molecular data via a panel of SERS NPs. This device enables rapid circumferential scanning of topologically complex luminal surfaces of hollow organs (e.g., colon and esophagus) and produces quantitative images of the relative concentrations of SERS NPs that are present. Human and swine studies have demonstrated the speed and simplicity of this technique. This approach also offers unparalleled multiplexing capabilities by simultaneously detecting the unique spectral fingerprints of multiple SERS NPs. Therefore, this new screening strategy has the potential to improve diagnosis and to guide therapy by enabling sensitive quantitative molecular detection of small and otherwise hard-to-detect lesions in the context of white-light endoscopy.

  View details for DOI 10.1371/journal.pone.0123185

  View details for Web of Science ID 000353711600032

  View details for PubMedID 25923788

  View details for PubMedCentralID PMC4414592

• Atherosclerotic Plaque Targeting Mechanism of Long-Circulating Nanoparticles Established by Multimodal Imaging ACS NANO Lobatto, M. E., Calcagno, C., Millon, A., Senders, M. L., Fay, F., Robson, P. M., Ramachandran, S., Binderup, T., Paridaans, M. P., Sensarn, S., Rogalla, S., Gordon, R. E., Cardoso, L., Storm, G., Metselaar, J. M., Contag, C. H., Stroes, E. S., Fayad, Z. A., Mulder, W. J. 2015; 9 (2): 1837-1847

  Abstract

  Atherosclerosis is a major cause of global morbidity and mortality that could benefit from novel targeted therapeutics. Recent studies have shown efficient and local drug delivery with nanoparticles, although the nanoparticle targeting mechanism for atherosclerosis has not yet been fully elucidated. Here we used in vivo and ex vivo multimodal imaging to examine permeability of the vessel wall and atherosclerotic plaque accumulation of fluorescently labeled liposomal nanoparticles in a rabbit model. We found a strong correlation between permeability as established by in vivo dynamic contrast enhanced magnetic resonance imaging and nanoparticle plaque accumulation with subsequent nanoparticle distribution throughout the vessel wall. These key observations will enable the development of nanotherapeutic strategies for atherosclerosis.

  View details for DOI 10.1021/nn506750r

  View details for Web of Science ID 000349940500080

  View details for PubMedID 25619964

• A real-time clinical endoscopic system for intraluminal, multiplexed imaging of surface-enhanced Raman scattering nanoparticles. PloS one Garai, E., Sensarn, S., Zavaleta, C. L., Loewke, N. O., Rogalla, S., Mandella, M. J., Felt, S. A., Friedland, S., Liu, J. T., Gambhir, S. S., Contag, C. H. 2015; 10 (4)

  Abstract

  The detection of biomarker-targeting surface-enhanced Raman scattering (SERS) nanoparticles (NPs) in the human gastrointestinal tract has the potential to improve early cancer detection; however, a clinically relevant device with rapid Raman-imaging capability has not been described. Here we report the design and in vivo demonstration of a miniature, non-contact, opto-electro-mechanical Raman device as an accessory to clinical endoscopes that can provide multiplexed molecular data via a panel of SERS NPs. This device enables rapid circumferential scanning of topologically complex luminal surfaces of hollow organs (e.g., colon and esophagus) and produces quantitative images of the relative concentrations of SERS NPs that are present. Human and swine studies have demonstrated the speed and simplicity of this technique. This approach also offers unparalleled multiplexing capabilities by simultaneously detecting the unique spectral fingerprints of multiple SERS NPs. Therefore, this new screening strategy has the potential to improve diagnosis and to guide therapy by enabling sensitive quantitative molecular detection of small and otherwise hard-to-detect lesions in the context of white-light endoscopy.

  View details for DOI 10.1371/journal.pone.0123185

  View details for PubMedID 25923788

  View details for PubMedCentralID PMC4414592

• The tumor suppressive reagent taurolidine inhibits growth of malignant melanoma - a mouse model JOURNAL OF SURGICAL RESEARCH Braumann, C., Jacobi, C. A., Rogalla, S., Menenakos, C., Fuehrer, K., Trefzer, U., Hofmann, M. 2007; 143 (2): 372-378

  Abstract

  The tumor suppressive agent taurolidine (TRD) inhibits tumor growth of more than 30 cell lines in vitro and reduces tumor load in early and advanced stages of neoplastic disease in animals. TRD has been shown to induce apoptosis of melanoma cells in vitro. Therefore, the effects of TRD on disseminated melanoma were evaluated in a mice model.After general anesthesia, a midline laparotomy was performed and 1.5 million malignant melanoma cells (B78-D14) were applied in the spleen and 1 million cells at the back (C57BL/6). Animals were randomized and either treated intraperitoneally (i.p., n = 40, 7 days, 12 hourly) or intravenously (i.v., n = 40, 2 days, 12 hourly) with 1%, 2%, or 3% TRD or with Ringer's solution (control group). On day 28, all animals were sacrificed and the total tumor weight and the number of metastatic lesions were determined by two investigators blinded for randomization.The i.p. therapy caused a dose-dependent inhibition of total tumor growth (P = 0.003) and i.p. tumor growth (P = < 0.001), whereas subcutaneous (s.c.) tumor growth was not affected (P = 0.132) compared with the i.p. control group. The i.v. therapy reduced the total tumor growth (P = 0.013) and the s.c. tumor growth (P = 0.016), whereas the i.p. tumor load was not reduced (P = 0.122) compared with the control group. Both i.p. and i.v. therapy with 3% TRD significantly decreased the total number of metastatic lesions. The animal weight was not affected.The i.p. and i.v. therapies reduce total tumor weight and number of metastatic lesions of disseminated malignant melanoma in a dose-dependent fashion in mice. Our encouraging findings should be further confirmed in clinical studies examining the influence of TRD in patients with disseminated malignant melanoma for whom prognosis still remains dismal.

  View details for DOI 10.1016/j.jss.2007.01.041

  View details for Web of Science ID 000251281200027

  View details for PubMedID 17612567

• High doses of taurolidine inhibit advanced intraperitoneal tumor growth in rats JOURNAL OF SURGICAL RESEARCH Braumann, C., Stuhldreier, B., Bobrich, E., Menenakos, C., Rogalla, S., Jacobi, C. A. 2005; 129 (1): 129-135

  Abstract

  The antitumor agent taurolidine (TRD) affects tumor growth in animals. Thus far, no animal studies have been published concerning the systemic or local toxicity and the effectiveness of long-term intraperitoneal (i.p.) and intravenous (i.v.) administration on advanced tumor growths.In a first experiment (A) the systemic toxicity of the liver and kidneys was examined only after i.v. treatment in 40 rats (BD IX). For local toxicity the superior vena cava (SVC) was histologically analyzed. In a second study (B) 20,000 colon adenocarcinoma cells (DHD/K12/TRb) were initially applied i.p. after laparotomy in 80 rats (BD IX). After 28 days a port catheter system was placed in the SVC and left for 1 week. The animals were randomized into eight groups (n = 10) and received a 7-day treatment (eight hourly, 1 ml): 1, 2, 3% TRD or Ringer's solution (control group) either i.p. or i.v. Total i.p. tumor weight was measured 4 weeks after the end of the therapy. Side effects on differential blood counts and animal weight changes were examined.No organ lesions were detected in liver, kidneys, and SVC in experiment A. The i.v. administration of 2% TRD (P = 0.034) and 3% TRD (P = 0.05) as well the i.p. application of 2% TRD (P = 0.05) decreased the development of advanced i.p. tumor lesions. No changes of differential blood count nor relevant animal weight changes resulted. Three port catheter-related infections were examined.TRD does not impair the liver tissue, kidneys, SVC, and leucopoiesis. The intravenous therapy of 2% TRD is safe and anti-tumorigenic in advanced local tumor growth in rats.

  View details for DOI 10.1016/j.jss.2005.03.012

  View details for Web of Science ID 000233206500020

  View details for PubMedID 15916768