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1.  Fluorescence-guided Surgery with a Fluorophore-conjugated Antibody to Carcinoembryonic Antigen (CEA), that Highlights the Tumor, Improves Surgical Resection and Increases Survival in Orthotopic Mouse Models of Human Pancreatic Cancer 
Annals of surgical oncology  2014;21(4):1405-1411.
We have developed a method of distinguishing normal tissue from pancreatic cancer in vivo using fluorophore-conjugated antibody to carcinoembryonic antigen (CEA). The objective of this study was to evaluate whether fluorescence-guided surgery (FGS) with a fluorophore-conjugated antibody to CEA, to highlight the tumor, can improve surgical resection and increase disease free survival (DFS) and overall survival (OS) in orthotopic mouse models of human pancreatic cancer.
We established nude-mouse models of human pancreatic cancer with surgical orthotopic implantation of the human BxPC-3 pancreatic cancer. Orthotopic tumors were allowed to develop for 2 weeks. Mice then underwent bright-light surgery (BLS) or FGS 24 h after intravenous injection of anti-CEA-Alexa Fluor 488. Completeness of resection was assessed from postoperative imaging. Mice were followed postoperatively until premorbid to determine DFS and OS.
Complete resection was achieved in 92 % of mice in the FGS group compared to 45.5 % in the BLS group (p = 0.001). FGS resulted in a smaller postoperative tumor burden (p = 0.01). Cure rates with FGS compared to BLS improved from 4.5 to 40 %, respectively (p = 0.01), and 1-year postoperative survival rates increased from 0 % with BLS to 28 % with FGS (p = 0.01). Median DFS increased from 5 weeks with BLS to 11 weeks with FGS (p = 0.0003). Median OS increased from 13.5 weeks with BLS to 22 weeks with FGS (p = 0.001).
FGS resulted in greater cure rates and longer DFS and OS using a fluorophore-conjugated anti-CEA antibody. FGS has potential to improve the surgical treatment of pancreatic cancer.
PMCID: PMC4334378  PMID: 24499827
Cell proliferation  2009;43(1):41-48.
Spinal cord tumors are highly malignant and often lead to paralysis and death mainly due to their infiltrative nature, high recurrence rate, and limited treatment options. In this study, we measured the antitumor efficacy of the Salmonella typhimurium A1-R tumor-targeting strain, administered systemically or intrathecally, to spinal cord cancer in orthotopic mouse models.
Materials and Methods
Tumor fragments of U87-RFP were implanted by surgical orthotopic implantation into the dorsal site of the spinal cord. Five and ten days after transplantation, 8 mice in each group were treated with A1-R (2 × 107 cfu / 200μl i.v. injection or 2 × 106 cfu / 10μl intrathecal injection).
The untreated mice showed progressive paralysis beginning at 6 days after tumor transplantation and developed complete paralysis between 18 to 25 days. The mice treated i.v. with A1-R had an onset of paralysis at approximately 11 days and at day-30, 5 mice developed complete paralysis, while other 3 mice had partial paralysis. Mice treated via intrathecal injection of A1-R had an onset of paralysis at approximately 18 days and one mouse was still not paralyzed at day-30. Only one mouse developed complete paralysis at day 30 in this group. The intrathecally-treated animals had a significant increase in survival over the i.v.-treated group as well as the control group.
These results suggest that S. typhimurium A1-R monotherapy can effectively treat spinal cord glioma.
PMCID: PMC4299869  PMID: 19922490
Salmonella typhimurium A1-R; auxotroph; GFP; RFP; spinal cord tumor; targeted therapy imaging
3.  Comparison of a chimeric anti-carcinoembryonic antigen antibody conjugated with visible or near-infrared fluorescent dyes for imaging pancreatic cancer in orthotopic nude mouse models 
Journal of Biomedical Optics  2013;18(12):126016.
The aim of this study was to evaluate a set of visible and near-infrared dyes conjugated to a tumor-specific chimeric antibody for high-resolution tumor imaging in orthotopic models of pancreatic cancer. BxPC-3 human pancreatic cancer was orthotopically implanted into pancreata of nude mice. Mice received a single intravenous injection of a chimeric anti-carcinoembryonic antigen antibody conjugated to one of the following fluorophores: 488-nm group (Alexa Fluor 488 or DyLight 488); 550-nm group (Alexa Fluor 555 or DyLight 550); 650-nm group (Alexa Fluor 660 or DyLight 650), or the 750-nm group (Alexa Fluor 750 or DyLight 755). After 24 h, the Olympus OV100 small-animal imaging system was used for noninvasive and intravital fluorescence imaging of mice. Dyes were compared with respect to depth of imaging, resolution, tumor-to-background ratio (TBR), photobleaching, and hemoglobin quenching. The longer wavelength dyes had increased depth of penetration and ability to detect the smallest tumor deposits and provided the highest TBRs, resistance to hemoglobin quenching, and specificity. The shorter wavelength dyes were more photostable. This study showed unique advantages of each dye for specific cancer imaging in a clinically relevant orthotopic model.
PMCID: PMC3868446  PMID: 24356647
dyes; fluorescence; antibody; CEA; imaging pancreatic cancer; cell-line; orthotopic; nude mice
4.  Metastatic Recurrence in a Pancreatic Cancer Patient Derived Orthotopic Xenograft (PDOX) Nude Mouse Model Is Inhibited by Neoadjuvant Chemotherapy in Combination with Fluorescence-Guided Surgery with an Anti-CA 19-9-Conjugated Fluorophore 
PLoS ONE  2014;9(12):e114310.
The aim of this study is to determine the efficacy of neoadjuvant chemotherapy (NAC) with gemcitabine (GEM) in combination with fluorescence-guided surgery (FGS) on a pancreatic cancer patient derived orthotopic xenograft (PDOX) model. A PDOX model was established from a CA19-9-positive, CEA-negative tumor from a patient who had undergone a pancreaticoduodenectomy for pancreatic adenocarcinoma. Mice were randomized to 4 groups: bright light surgery (BLS) only; BLS+NAC; FGS only; and FGS+NAC. An anti-CA19-9 or anti-CEA antibody conjugated to DyLight 650 was administered intravenously via the tail vein of mice with the pancreatic cancer PDOX 24 hours before surgery. The PDOX was brightly labeled with fluorophore-conjugated anti-CA19-9, but not with a fluorophore-conjugated anti-CEA antibody. FGS was performed using the fluorophore-conjugated anti-CA19-9 antibody. FGS had no benefit over BLS to prevent metastatic recurrence. NAC in combination with BLS did not convey an advantage over BLS to prevent metastatic recurrence. However, FGS+NAC significantly reduced the metastatic recurrence frequency to one of 8 mice, compared to FGS only after which metastasis recurred in 6 out of 8 mice, and BLS+NAC with metastatic recurrence in 7 out of 8 mice (p = 0.041). Thus NAC in combination with FGS can reduce or even eliminate metastatic recurrence of pancreatic cancer sensitive to NAC. The present study further emphasizes the power of the PDOX model which enables metastasis to occur and thereby identify the efficacy of NAC in combination with FGS on metastatic recurrence.
PMCID: PMC4252107  PMID: 25463150
6.  Efficacy of tumor-targeting Salmonella typhimurium A1-R in combination with anti-angiogenesis therapy on a pancreatic cancer patient-derived orthotopic xenograft (PDOX) and cell line mouse models 
Oncotarget  2014;5(23):12346-12357.
The aim of the present study was to examine the efficacy of tumor-targeting Salmonella typhimurium A1-R treatment following anti-vascular endothelial growth factor (VEGF) therapy on VEGF-positive human pancreatic cancer. A pancreatic cancer patient-derived orthotopic xenograft (PDOX) that was VEGF-positive and an orthotopic VEGF-positive human pancreatic cancer cell line (MiaPaCa-2-GFP) as well as a VEGF-negative cell line (Panc-1) were tested. Nude mice with these tumors were treated with gemcitabine (GEM), bevacizumab (BEV), and S. typhimurium A1-R. BEV/GEM followed by S. typhimurium A1-R significantly reduced tumor weight compared to BEV/GEM treatment alone in the PDOX and MiaPaCa-2 models. Neither treatment was as effective in the VEGF-negative model as in the VEGF-positive models. These results demonstrate that S. typhimurium A1-R following anti-angiogenic therapy is effective on pancreatic cancer including the PDOX model, suggesting its clinical potential.
PMCID: PMC4322966  PMID: 25402324
Pancreatic cancer; Salmonella typhimurium A1-R; patient-derived orthotopic xenograft (PDOX); orthotopic; nude mice; GFP; VEGF; anti-angiogenic therapy; bevacizumab; gemcitabine
7.  Hospitalized but not Admitted: Characteristics of Patients with “Observation Status” at an Academic Medical Center 
JAMA internal medicine  2013;173(21):1991-1998.
The Centers for Medicare and Medicaid Services (CMS)defines observation status for hospitalized patients as a “well-defined set of specific, clinically appropriate services,” usually lasting <24 hours, and that in “only rare and exceptional cases” should last > 48 hours. Although an increasing proportion of observation care occurs on hospital wards, studies of patients with observation status have focused on the efficiency of dedicated units.
To describe inpatient and observation care.
Design and Setting
Descriptive study of all inpatient and observation stays between July 1, 2010 and December 31, 2011 at the University of Wisconsin Hospital and Clinics, a 566 bed tertiary academic medical center.
All patients with observation or inpatient stays during the study period.
Main Outcome and Measures
Patient demographics, length of stay, difference between cost and reimbursement per stay, and percent of patients discharged to skilled nursing facilities.
Of 43,853 stays, 4,578 (10.4%) were observation, with 1,141 distinct diagnosis codes. Average observation length of stay was 33.3 hours, with 44.4% of stays <24 hours, and 16.5% >48 hours. Observation care had a negative margin per stay (-$331); the inpatient margin per stay was positive (+$2,163). Adult General Medicine patients accounted for 2,404 (52.5%) of all observation stays; 25.4% of the 9,453 Adult General Medicine stays were observation. The mean length of stay for general medicine observation patients was 41.1 hours, with 32.6% of stays < 24 hours, and 26.4% >48 hours. As compared to observation patients on other clinical services, Adult General Medicine had the highest percent >65 years (40.9%), highest percent female (57.9%), highest percent discharged to skilled nursing facilities (11.6%) and the most negative margin per stay (-$1,378).
Conclusions and Relevance
In an academic medical center, observation status for hospitalized patients differed markedly from the CMS definition. Patients had a wide variety of diagnoses; lengths of stay were typically > 24 hours and often > 48 hours. The hospital lost money, primarily because reimbursement for general medicine patients was inadequate to cover the costs. It is uncertain what role, if any, observation status for hospitalized patients should have in the era of health care reform.
PMCID: PMC3942156  PMID: 23835927
8.  Orthotopic Mouse Models of Tumor Metastasis Expressing Fluorescent Reporters Produce Imageable Circulating Tumor Cells 
Cancer Microenvironment  2014;7(3):133-138.
Circulating tumor cells (CTC) are of high importance, since they are potential metastatic precursors and are readily available for prognostic analysis and treatment testing. In this review, we demonstrate the great power that green fluorescent protein (GFP) labeling and orthotopic mouse models of cancer confer to the study of CTCs for isolation and characterization, including metastatic testing in mice and the chick embryo as well as drug response testing in vitro. We also describe a facile method to label patient CTCs ex vivo using a telomerase-expressing GFP-containing adenovirus that will allow the CTC studies described in this review to be translated clinically.
PMCID: PMC4275499  PMID: 25417145
CTC; Mice; Orthotopic; GFP; RFP; Isolation; Metastasis; Drug response
9.  Comparison of Efficacy and Toxicity of Traditional Chinese Medicine (TCM) Herbal Mixture LQ and Conventional Chemotherapy on Lung Cancer Metastasis and Survival in Mouse Models 
PLoS ONE  2014;9(10):e109814.
Unlike Western medicine that generally uses purified compounds and aims to target a single molecule or pathway, traditional Chinese medicine (TCM) compositions usually comprise multiple herbs and components that are necessary for efficacy. Despite the very long-time and wide-spread use of TCM, there are very few direct comparisons of TCM and standard cytotoxic chemotherapy. In the present report, we compared the efficacy of the TCM herbal mixture LQ against lung cancer in mouse models with doxorubicin (DOX) and cyclophosphamide (CTX). LQ inhibited tumor size and weight measured directly as well as by fluorescent-protein imaging in subcutaneous, orthotopic, spontaneous experimental metastasis and angiogenesis mouse models of lung cancer. LQ was efficacious against primary and metastatic lung cancer without weight loss and organ toxicity. In contrast, CTX and DOX, although efficacious in the lung cancer models caused significant weight loss, and organ toxicity. LQ also had anti-angiogenic activity as observed in lung tumors growing in nestin-driven green fluorescent protein (ND-GFP) transgenic nude mice, which selectively express GFP in nascent blood vessels. Survival of tumor-bearing mice was also prolonged by LQ, comparable to DOX. In vitro, lung cancer cells were killed by LQ as observed by time-lapse imaging, comparable to cisplatinum. LQ was more potent to induce cell death on cancer cell lines than normal cell lines unlike cytotoxic chemotherapy. The results indicate that LQ has non-toxic efficacy against metastatic lung cancer.
PMCID: PMC4186882  PMID: 25286158
10.  Gene Copy-Number Variations (CNVs) and Protein Levels of Complement C4A and C4B as Novel Biomarkers for Partial Disease Remissions in New-Onset Type 1 Diabetes Patients 
Pediatric diabetes  2011;13(5):408-418.
To determine the roles of complement C4A and C4B gene CNVs and their plasma protein concentrations in residual insulin secretion and loss of pancreatic beta-cell function in new-onset type 1 diabetes patients.
We studied 34 patients of European ancestry with new-onset type 1 diabetes, aged between 3 and 17 years (10.7±3.45), at Nationwide Children's Hospital in Columbus, Ohio. Gene copy-number and size variations of complement C4A and C4B were determined by genomic Southern blot analyses. C4A and C4B protein phenotypes were elucidated by immunofixation and radial immunodiffusion. Two-digit HLA-DRB1 genotypes were determined by sequence-specific PCR. At 1 month and 9-month post diagnosis, stimulated C-peptide levels were measured after a standardized mixed-meal tolerance test.
The diploid gene copy-numbers of C4A varied from 0 to 4, and those of C4B from 0 to 3. Patients with higher copy-number of C4A or higher C4A plasma protein concentrations at diagnosis had higher C-peptide levels at 1 month post diagnosis (p=0.008; p=0.008). When controlled by the Z-score of body-mass index, C4A copy-numbers, C4A protein concentrations, the age of disease-onset, the number of HLA-DR3 but not DR4 alleles were significant parameters in determining C-peptide levels. At 9-month post diagnosis, 42.3% of patients remained in partial remission, and these patients were characterized by lower total C4B copy-numbers or lower C4B protein concentrations (p=0.02, p=0.0004).
C4A appears to associate with the protection of residual beta-cell function in new-onset type 1 diabetes; C4B is correlated with the end of disease remission at 9-month post diagnosis.
PMCID: PMC4178531  PMID: 22151770
Type 1 Diabetes; Partial disease remission; C-Peptide; Complement C4; CNV; HLA-DRB1
11.  In Vivo Serial Selection of Human Pancreatic Cancer in Orthotopic Mouse Models Produces High Metastatic Variants Irrespective of Kras Status 
The Journal of surgical research  2013;184(1):290-298.
Kras mutations have been thought to play an important role in pancreatic cancer progression. In this study, we evaluated how serially passaging primary pancreatic tumors with and without Kras mutations, in nude mice, can generate more aggressive variants of human pancreatic cancer.
Materials & Methods
Orthotopic mouse models of human pancreatic cancer were established by injecting 1×106 cells of the Kras wildtype BxPC-3 cell line, expressing red fluorescent protein (RFP) or the Kras mutant Panc-1 cell line expressing green fluorescent protein (GFP), into the pancreas. Pancreatic tumors were harvested from premorbid mice to establish cell lines. One million passaged cells were then orthotopically injected into another set of mice. Serial passaging continued until decreasing lifespan of the implanted mice stabilized, which occurred by 6 passages. Mice harboring serially-passaged cell lines were followed with weekly imaging.
Serially passaging generated more aggressive variants of both human pancreatic cancer cell lines, one which was Kras wild-type (BXPC-3) and the other Kras mutant, Panc-1, which displayed faster tumor growth and shortened survival time. Overall survival decreased from 18 weeks in mice with the parental cell line (P0) tumor to ~6 weeks in mice by the sixth passage (P6). Average time to metastasis was shortened from 14 weeks to ~3 weeks or less. At termination, mice with the passaged tumor demonstrated a greater extent of distant metastasis.
Serial passaging of tumor creates more aggressive variants of human pancreatic cancer cell lines regardless of Kras mutation. The aggressive variants can be used to study the molecular basis of highly malignant pancreatic cancer and to screen for effective agents against this disease.
PMCID: PMC3724759  PMID: 23590868
pancreatic cancer; orthotopic mouse models; in vivo selection; Kras; metastasis; variants; survival
12.  Methylene Blue for Distributive Shock: A Potential New Use of an Old Antidote 
Journal of Medical Toxicology  2013;9(3):242-249.
Methylene blue is used primarily in the treatment of patients with methemoglobinemia. Most recently, methylene blue has been used as a treatment for refractory distributive shock from a variety of causes such as sepsis and anaphylaxis. Many studies suggest that the nitric oxide–cyclic guanosine monophosphate (NO–cGMP) pathway plays a significant role in the pathophysiology of distributive shock. There are some experimental and clinical experiences with the use of methylene blue as a selective inhibitor of the NO–cGMP pathway. Methylene blue may play a role in the treatment of distributive shock when standard treatment fails.
PMCID: PMC3770994  PMID: 23580172
Methylene blue; Refractory shock; Cardiovascular drug overdoses; Calcium channel blockers
13.  Comparison of efficacy of Salmonella typhimurium A1-R and chemotherapy on stem-like and non-stem human pancreatic cancer cells 
Cell Cycle  2013;12(17):2774-2780.
The XPA1 human pancreatic cancer cell line is dimorphic, with spindle stem-like cells and round non-stem cells. We report here the in vitro IC50 values of stem-like and non-stem XPA1 human pancreatic cells cells for: (1) 5-fluorouracil (5-FU), (2) cisplatinum (CDDP), (3) gemcitabine (GEM), and (4) tumor-targeting Salmonella typhimurium A1-R (A1-R). IC50 values of stem-like XPA1 cells were significantly higher than those of non-stem XPA1 cells for 5-FU (P = 0.007) and CDDP (P = 0.012). In contrast, there was no difference between the efficacy of A1-R on stem-like and non-stem XPA1 cells. In vivo, 5-FU and A1-R significantly reduced the tumor weight of non-stem XPA1 cells (5-FU; P = 0.028; A1-R; P = 0.011). In contrast, only A1-R significantly reduced tumor weight of stem-like XPA1 cells (P = 0.012). The combination A1-R with 5-FU improved the antitumor efficacy compared with 5-FU monotherapy on the stem-like cells (P = 0.004). The results of the present report indicate A1-R is a promising therapy for chemo-resistant pancreatic cancer stem-like cells.
PMCID: PMC3899191  PMID: 23966167
Salmonella typhimurium; amino acid auxotrophy; selective tumor targeting; pancreatic cancer; stem cell; chemoresistance; RFP; GFP; fluorescence imaging; confocal microscopy
14.  Selective methioninase-induced trap of cancer cells in S/G2 phase visualized by FUCCI imaging confers chemosensitivity 
Oncotarget  2014;5(18):8729-8736.
A major impediment to the response of tumors to chemotherapy is that the large majority of cancer cells within a tumor are quiescent in G0/G1, where cancer cells are resistant to chemotherapy. To attempt to solve this problem of quiescent cells in a tumor, cancer cells were treated with recombinant methioninase (rMETase), which selectively traps cancer cells in S/G2. The cell cycle phase of the cancer cells was visualized with the fluorescence ubiquitination cell cycle indicator (FUCCI). At the time of rMETase-induced S/G2-phase blockage, identified by the cancer cells' green fluorescence by FUCCI imaging, the cancer cells were administered S/G2-dependent chemotherapy drugs, which interact with DNA or block DNA synthesis such as doxorubicin, cisplatin, or 5-fluorouracil. Treatment of cancer cells with drugs only, without rMETase-induced S/G2 phase blockage, led to the majority of the cancer-cell population being blocked in G0/G1 phase, identified by the cancer cells becoming red fluorescent in the FUCCI system. The G0/G1 blocked cells were resistant to the chemotherapy. In contrast, trapping of cancer cells in S/G2 phase by rMETase treatment followed by FUCCI-imaging-guided chemotherapy was highly effective in killing the cancer cells.
PMCID: PMC4226717  PMID: 25238266
cell cycle; FUCCI; imaging; S/G2 phase block; recombinant methioninase; rMETase; chemotherapy; HeLa cells; MCF-7 cells
15.  In Vitro Culture and Characterization of Human Lung Cancer Circulating Tumor Cells Isolated by Size Exclusion from an Orthotopic Nude-Mouse Model Expressing Fluorescent Protein 
Journal of Fluorescence  2014;24(5):1531-1536.
In the present study, we demonstrate an animal model and recently introduced size–based exclusion method for circulating tumor cells (CTCs) isolation. The methodology enables subsequent in vitro CTC-culture and characterization. Human lung cancer cell line H460, expressing red fluorescent protein (H460-RFP), was orthotopically implanted in nude mice. CTCs were isolated by a size-based filtration method and successfully cultured in vitro on the separating membrane (MetaCell®), analyzed by means of time-lapse imaging. The cultured CTCs were heterogeneous in size and morphology even though they originated from a single tumor. The outer CTC-membranes were blebbing in general. Abnormal mitosis resulting in three daughter cells was frequently observed. The expression of RFP ensured that the CTCs originated from lung tumor. These readily isolatable, identifiable and cultivable CTCs can be used to characterize individual patient cancers and for screening of more effective treatment.
PMCID: PMC4159578  PMID: 25141982
Lung cancer; Orthotopic; Circulating tumor cells; CTC; In vitro culture; CTC; MetaCell; Filtration; Size; Fluorescence; RFP
16.  The Tumor-Educated-Macrophage Increase of Malignancy of Human Pancreatic Cancer Is Prevented by Zoledronic Acid 
PLoS ONE  2014;9(8):e103382.
We previously defined macrophages harvested from the peritoneal cavity of nude mice with subcutaneous human pancreatic tumors as “tumor-educated-macrophages” (Edu) and macrophages harvested from mice without tumors as “naïve-macrophages” (Naïve), and demonstrated that Edu-macrophages promoted tumor growth and metastasis. In this study, Edu- and Naïve-macrophages were compared for their ability to enhance pancreatic cancer malignancy at the cellular level in vitro and in vivo. The inhibitory efficacy of Zoledronic acid (ZA) on Edu-macrophage-enhanced metastasis was also determined. XPA1 human pancreatic cancer cells in Gelfoam co-cultured with Edu-macrophages proliferated to a greater extent compared to XPA1 cells cultured with Naïve-macrophages (P = 0.014). XPA1 cells exposed to conditioned medium harvested from Edu culture significantly increased proliferation (P = 0.016) and had more migration stimulation capability (P<0.001) compared to cultured cancer cells treated with the conditioned medium from Naïve. The mitotic index of the XPA1 cells, expressing GFP in the nucleus and RFP in the cytoplasm, significantly increased in vivo in the presence of Edu- compared to Naïve-macrophages (P = 0.001). Zoledronic acid (ZA) killed both Edu and Naïve in vitro. Edu promoted tumor growth and metastasis in an orthotopic mouse model of the XPA1 human pancreatic cancer cell line. ZA reduced primary tumor growth (P = 0.006) and prevented metastasis (P = 0.025) promoted by Edu-macrophages. These results indicate that ZA inhibits enhanced primary tumor growth and metastasis of human pancreatic cancer induced by Edu-macrophages.
PMCID: PMC4130525  PMID: 25116261
17.  In Vivo Fluorescence Imaging of Gastrointestinal Stromal Tumors Using Fluorophore-Conjugated Anti-KIT Antibody 
Annals of surgical oncology  2013;20(0 3):S693-S700.
Gastrointestinal stromal tumors (GISTs) are frequently characterized by KIT overexpression. Tumor-free margins and complete cytoreduction of disease are mainstays of treatment. We hypothesized that fluorescently labeled anti-KIT antibodies can label GIST in vivo.
KIT K641E+/− transgenic mice that spontaneously develop cecal GISTs were used in this study, with C57BL/6 mice serving as controls. Alexa 488 fluorophore-conjugated anti-KIT antibodies were delivered via the tail vein 24 h prior to fluorescence imaging. Following fluorescence laparoscopy, mice were sacrificed. The gastrointestinal tracts were grossly examined for tumors followed by fluorescence imaging. Tumors were harvested for histologic confirmation.
KIT K641E+/− mice and C57BL/6 control mice received anti-KIT antibody or isotope control antibody. Fluorescence laparoscopy had a high tumor signal-to-background noise ratio. Upon blinded review of intravital fluorescence and bright light images, there were 2 false-positive and 0 false-negative results. The accuracy was 92 %. The sensitivity, specificity, positive and negative predictive values were 100, 87, 85, and 100 %, respectively, for the combined modalities.
In this study, we present a method for in vivo fluorescence labeling of GIST in a murine model. Several translatable applications include: laparoscopic staging; visualization of peritoneal metastases; assessment of margin status; endoscopic differentiation of GISTs from other benign submucosal tumors; and longitudinal surveillance of disease response. This novel approach has clear clinical applications that warrant further research and development.
PMCID: PMC4127988  PMID: 23943029
18.  Antigen-specific bacterial vaccine combined with anti-PD-L1 rescues dysfunctional endogenous T cells to reject long-established cancer 
Cancer immunology research  2013;1:123-133.
Immunogenic tumors grow progressively even when heavily infiltrated by CD8+ T cells. We investigated how to rescue CD8+ T cell function in long-established immunogenic melanomas that contained a high percentage of endogenous PD-1+ tumor-specific CD8+ T cells that were dysfunctional. Treatment with αPD-L1 and αCTLA-4 blocking antibodies did not prevent tumors from progressing rapidly. We then tested exogenous tumor-specific antigen delivery into tumors using Salmonella Typhimurium A1-R to increase antigen levels and generate a proinflammatory tumor microenvironment. Antigen-producing A1-R rescued the endogenous tumor-specific CD8+ T cell response: proliferation was induced in the lymphoid organs and effector function was recovered in the tumor. Treatment with antigen-producing A1-R led to improved mouse survival and resulted in 32% rejection of long-established immunogenic melanomas. Following treatment with antigen-producing A1-R, the majority of tumor-specific CD8+ T cells still expressed a high level of PD-1 in the tumor. Combining antigen-producing A1-R with αPD-L1 blocking antibody enhanced the expansion of tumor-specific CD8+ T cells and resulted in 80% tumor rejection. Collectively, these data demonstrate a powerful new therapeutic approach to rescue dysfunctional endogenous tumor-specific CD8+ T cells and eradicate advanced immunogenic tumors.
PMCID: PMC3895468  PMID: 24455752
Tumor rejection; CD8+ T cell rescue; S. Typhimurium; PD-L1; vaccine
19.  A dual color, genetically engineered mouse model for multi-spectral imaging of the pancreatic microenvironment 
Pancreas  2013;42(6):952-958.
To develop a mouse model for multi-spectral fluorescence imaging of the pancreas and pancreatic microenvironment.
Cre/loxP technology was used to develop this model. We crossed mT/mG indicator mice, engineered to constitutively express a conditional tdTomato transgene that converts to green fluorescent protein (GFP) expression following exposure to Cre recombinase, with Pdx1-Cre transgenic mice. To characterize this model for studies of pancreas biology, we performed bright light and fluorescence imaging of body cavities and intact organs and confocal microscopy of pancreata from offspring of Pdx1-Cre and mT/mG crosses.
Pdx1-Cre - mT/mG mice demonstrated bright GFP expression within the pancreas and duodenum and intense tdTomato expression in all other organs. GFP expression was mosaic in Pdx1-Cre - mT/mG pancreata, with most showing extensive conversion from tdTomato to GFP expression within the epithelial-derived elements of the pancreatic parenchyma. Because both GFP and tdTomato are membrane-targeted, individual cell borders were clearly outlined in confocal images of mT/mG pancreata.
This mouse model enables multi-spectral fluorescence imaging of individual cells and cell processes at the microscopic level of the pancreatic microenvironment; it should prove valuable for a variety of fluorescence imaging studies, ranging from pancreatic development to pancreatic cancer biology.
PMCID: PMC3713119  PMID: 23648841
Pancreas; Cre recombinase; pdx1; conditional gene targeting; fluorescence imaging
20.  Tumor-targeting Salmonella typhimurium A1-R prevents experimental human breast cancer bone metastasis in nude mice 
Oncotarget  2014;5(16):7119-7125.
Bone metastasis is a lethal and morbid late stage of breast cancer that is currently treatment resistant. More effective mouse models and treatment are necessary. High bone-metastatic variants of human breast cancer cells were selected in nude mice by cardiac injection. After cardiac injection of a high bone-metastatic variant of breast cancer, all untreated mice had bone metastases compared to only 20% with parental cells. Treatment with tumor-targeting Salmonella typhimurium A1-R completely prevented the appearance of bone metastasis of the high metastatic variant in nude mice (P < 0.001). After injection of the highly bone-metastatic breast cancer variant to the tibia of nude mice, S. typhimurium A1-R treatment significantly reduced tumor growth in the bone (P < 0.001). These data indicated that S. typhimurium A1-R is useful to prevent and inhibit breast cancer bone metastasis and should be of future clinical use for breast cancer in the adjuvant setting.
PMCID: PMC4196188  PMID: 25216526
breast cancer; bone metastasis; GFP; RFP; bacterial therapy; Salmonella typhimurium A1-R
21.  High-throughput screening for Salmonella avirulent mutants that retain targeting of solid tumors 
Cancer research  2010;70(6):2165-2170.
Salmonella has a natural ability to target a wide range of tumors in animal models. However, strains used for cancer therapy have generally been selected only for their avirulence rather than tumor-targeting ability. To select Salmonella strains that are avirulent and yet efficient in tumor-targeting, a necessary criteria for clinical applications, we measured the relative fitness of 41,000 Salmonella transposon insertion mutants growing in mouse models of human prostate cancer and melanoma. Two classes of potentially safe mutants were identified. Class 1 mutants showed reduced fitness in normal tissues and unchanged fitness in tumors (e.g., mutants in htrA, SPI-2, and STM3120). Class 2 mutants showed reduced fitness in tumors and normal tissues (e.g., mutants in aroA and aroD). In a competitive fitness assay in human PC3 tumors growing in mice, class 1 mutant STM3120 had a fitness advantage over class 2 mutants aroA and aroD, validating the findings of the initial screening of a large pool of transposon mutants and indicating a potential advantage of class 1 mutants for delivery of cancer therapeutics. In addition, an STM3120 mutant successfully targeted tumors after intragastric delivery, opening up the oral route as an option for therapy administration.
PMCID: PMC4103738  PMID: 20231149
22.  Tumor-Specific Fluorescent Antibody Imaging Enables Accurate Staging Laparoscopy in an Orthotopic Model of Pancreatic Cancer 
Hepato-gastroenterology  2012;59(118):1994-1999.
Laparoscopy is important in staging pancreatic cancer, but false negatives remain problematic. Making tumors fluorescent has the potential to improve the accuracy of staging laparoscopy.
Orthotopic and carcinomatosis models of pancreatic cancer were established with BxPC-3 human pancreatic cancer cells in nude mice. Alexa488-anti-CEA conjugates were injected via tail vein 24 hours prior to laparoscopy. Mice were examined under bright field laparoscopic (BL) and fluorescence laparoscopic (FL) modes. Outcomes measured included time to identification of primary tumor for the orthotopic model and number of metastases identified within 2 minutes for the carcinomatosis model.
FL enabled more rapid and accurate identification and localization of primary tumors and metastases than BL. Using BL took statistically significantly longer time than FL. More metastatic lesions were detected and localized under FL compared to BL and with greater accuracy, with sensitivities of 96% vs. 40%, respectively, when compared to control. FL was sensitive enough to detect metastatic lesions <1mm.
The use of fluorescence laparoscopy with tumors labeled with fluorophore-conjugated anti-CEA antibody permits rapid detection and accurate localization of primary and metastatic pancreatic cancer in an orthotopic model. The results of the present report demonstrate the future clinical potential of fluorescence laparoscopy.
PMCID: PMC4096574  PMID: 22369743
Pancreatic Cancer; CEA; Staging; Laparoscopy; Fluorescence
23.  Medical examiner and medical toxicologist agreement on cause of death 
Forensic science international  2010;206(0):71-76.
Poisoning is a significant public health threat as the second leading cause of injury-related death in the US. Disagreements on cause of death determination may have widespread implications across several realms of public health including policy and prevention efforts, interpretation of the poisoning literature, epidemiologic data analysis, medical-legal case outcomes, and individualized autopsy interpretation. We aimed to test agreement between the cause of death determined by the medical examiner (ME) and a medical toxicologist (MT) adjudication panel (MTAP) in cases of poisoning. This retrospective 7-year study evaluated all deaths attributed to poisoning in one large urban catchment area. Cross-matched data were obtained from Department of Vital Statistics and the Poison Control Center (PCC). Out of >380,000 deaths in the catchment area over the study period, there were 7050 poisonings in the Vital Statistics database and 414 deaths reported to PCC. Cross-matching yielded 321 cases for analysis. The ME and MTAP concurred on cause of death in 66%, which was only fair agreement (κ 0.25, CI 0.14–0.38). Factors associated with the likelihood of agreement were peri-mortem fire exposures, prehospital cardiac arrest, and timing of drug toxicity (chronic versus acute). In conclusion, agreement for poisoning cause of death between specialties was much lower than expected. We recommend an improved formal process of information sharing and consultation between specialties to assure that all existing information is analyzed thoroughly to enhance cause of death certainty.
PMCID: PMC4094358  PMID: 20655675
Poisoning; Medical examiner; Toxicology; Cause of death
24.  Utility of serum lactate to predict drug-overdose fatality 
Poisoning is the second leading cause of injury-related fatality in the United States. An elevated serum lactate concentration identifies medical and surgical patients at risk for death; however, its utility in predicting death in drug overdose is controversial and unclear.
We aimed to evaluate the prognostic utility of serum lactate concentration for fatality in emergency department (ED) patients with acute drug overdose.
Materials and Methods
This was a case–control study at two urban university teaching hospitals affiliated with a regional poison control center. Data were obtained from electronic medical records, poison center data, and the office of the chief medical examiner. Controls were consecutive acute drug overdoses over a 1-year period surviving to hospital discharge. Cases were subjects over a 7-year period with fatality because of drug overdose. Serum lactate concentration was obtained from the initial blood draw in the ED and correlated with fatality.
During the study period, 873 subjects were screened with 50 cases and 100 controls included. Drug exposures and baseline characteristics were similar between groups. Mean lactate concentration (mmol/L) was 9.88 ± 6.7 for cases and 2.76 ± 2.9 for controls (p < 0.001). The receiver operating characteristic area under the curve for prediction of fatality was 0.87 (95% CI: 0.81–0.94). The optimal lactate cutpoint was 3.0 mmol/L (84% sensitivity, 75% specificity), which conferred a 15.8-fold increase in odds of fatality (p < 0.001).
In this derivation study, serum lactate concentration had excellent prognostic utility to predict drug-overdose fatality. Prospective validation in the ED evaluation of drug overdoses is warranted.
PMCID: PMC4091774  PMID: 20704455
Overdose; Fatality; Acute poisoning
25.  Status epilepticus and wide-complex tachycardia secondary to diphenhydramine overdose 
Diphenhydramine is an H1 histamine antagonist that is commonly used for allergic reactions, colds and cough, and as a sleep aid. In addition to anticholinergic and antihistaminergic effects, sodium channel blockade becomes evident following diphenhydramine overdose. While seizures may occur following overdose of a diphenhydramine, status epilepticus is distinctly uncommon. We report a case with both status epilepticus and wide-complex dysrhythmias following an intentional diphenhydramine overdose.
Case report
A 36-year-old woman with a medical history of hypothyroidism on levothyroxine was brought to the emergency department with active seizures by emergency medical services after what was later determined to be a diphenhydramine overdose. One hour after an argument with her husband he found her lethargic in a locked room. Initial vital signs were: blood pressure, 90/55 mmHg; heart rate, 160 beats/min; respiratory rate 18 breaths/min; room air oxygen saturation, 99%; temperature, 99.8°F; rapid point-of-care glucose, 130 mg/dL. The generalized seizures continued for duration of 30 min, despite the intravenous administration of 8 mg of lorazepam. The patient underwent endotracheal intubation and a propofol infusion terminated her seizures. An electrocardiogram after the status was terminated which revealed a wide-complex tachycardia with QRS duration of 127 ms. The QRS narrowed after 200 mEq of intravenous sodium bicarbonate was administrated. The patient was neurologically intact upon extubation on hospital day 2. The serum diphenhydramine concentration drawn on arrival to the ED was 1200 ng/mL (9–120 ng/ mL); a tricyclic screen was negative.
While seizures and sodium channel blockade are recognized complications of diphenhydramine toxicity, reported cases of status epilepticus from diphenhydramine overdose are rare. Elements of the patient’s presentation were similar to a tricyclic overdose and management required aggressive control of her seizures, sodium bicarbonate therapy, and recognizing that physostigmine was contraindicated due to wide complex tachycardia.
Diphenhydramine overdose may cause status epilepticus and wide-complex tachycardia. Management should focus on antidotal therapy with sodium bicarbonate and supportive neurological management with appropriate anticonvulsants and airway protection if clinically indicated.
PMCID: PMC4091778  PMID: 21171853
Heart; CNS/psychological; Other

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