Contrast-enhanced computed tomography for ex vivo assessment of human kidneys: A proof-of-concept study.

BACKGROUND: Ex vivo perfusion of transplant-declined human organs has emerged as a promising platform to study the response of an organ to novel therapeutic strategies. However, to fully realize the capability of this platform for performing translational research in human organ pathophysiology, there is a need for robust assays to assess organ function and disease. State-of-the-art research methods rely on analyses of biopsies taken during perfusion, which both damages the organ and only provides localized information. Developing non-invasive, whole organ methods of assessment is critical to the further development of this research platform. METHODS: We use ex vivo cold infusion scanning (EXCIS) with contrast-enhanced computed tomography (CT) to quantify perfusion in kidneys preserved ex vivo. EXCIS-CT computes three complementary metrics for whole organ assessment: a dynamic assessment of contrast filling, a measure of vascular network anatomical structure, and a static assessment of perfusion heterogeneity. RESULTS: These metrics were applied to a series of six transplant-declined human kidneys, which demonstrated a range of anatomies and perfusion. Lastly, two transplant-declined human kidneys were imaged before and after a 1-h period of ex vivo normothermic perfusion (NMP). We found variable responses to NMP, with one kidney maintaining the vascular network and hemodynamics and the other showing significant changes in vessel size and spatial perfusion profile. CONCLUSIONS: EXCIS-CT provides metrics that can be used to characterize whole organ perfusion and vascular function.

Lysis of cold-storage-induced microvascular obstructions for ex vivo revitalization of marginal human kidneys.

Thousands of kidneys from higher-risk donors are discarded annually because of the increased likelihood of complications posttransplant. Given the severe organ shortage, there is a critical need to improve utilization of these organs. To this end, normothermic machine perfusion (NMP) has emerged as a platform for ex vivo assessment and potential repair of marginal organs. In a recent study of 8 transplant-declined human kidneys on NMP, we discovered microvascular obstructions that impaired microvascular blood flow. However, the nature and physiologic impact of these lesions were unknown. Here, in a study of 39 human kidneys, we have identified that prolonged cold storage of human kidneys induces accumulation of fibrinogen within tubular epithelium. Restoration of normoxic conditions-either ex vivo during NMP or in vivo following transplant-triggered intravascular release of fibrinogen correlating with red blood cell aggregation and microvascular plugging. Combined delivery of plasminogen and tissue plasminogen activator during NMP lysed the plugs leading to a significant reduction in markers of renal injury, improvement in indicators of renal function, and improved delivery of vascular-targeted nanoparticles. Our study suggests a new mechanism of cold storage injury in marginal organs and provides a simple treatment with immediate translational potential.

Prognostic factors in kidney transplantation in the septuagenarian: a multicenter analysis.

Kidney transplant (KT) programs have extended recipient eligibility to those who were previously excluded due to advanced age. We aimed to determine the outcomes of the patients ≥70 years undergoing KT and investigate factors predicting survival. Two thousand six hundred and twenty-four KT patients between 2003 and 2013 at two institutions were divided into two groups; those ≥70 years (n=300) and those <70 years (n=2324) at the time of KT. Patient survival at 1, 3, and 5 years was 95%, 86%, and 77% in ≥70 years of age group and 98%, 95%, and 90% in the <70 years group (P<.001). When graft loss due to death was censored, graft survival was not significantly different between the two groups (P=.18). On multivariable analysis, the significant predictors of inferior survival in patients ≥70 years included: body mass index (BMI)>30 kg/m(2) (hazard ratio [HR] 1.07; P=.01), panel reactive antibody (PRA)>20% (HR 2.38; P=.01), previous coronary artery bypass grafting (CABG; HR 1.95; P=.03) and peripheral vascular disease (PVD; HR 2.60; P=.04). Acceptable outcomes can be achieved in KT recipients ≥70 years. Caution should be used when listing these patients if they have BMI>30 kg/m(2) , PRA>20%, CABG or PVD.

The impact of obesity on breast cancer: a retrospective review.

BACKGROUND: Obesity has been linked to many adverse health consequences, including breast cancer; however, the impact on clinical presentation, tumor characteristics, and survival outcomes has yet to be clearly defined. METHODS: Retrospective review of a prospectively collected database of patients treated at a single institution for invasive breast cancer from 2000-2008 comparing two groups: nonobese (body mass index of <30) and obese (body mass index of ≥ 30) patients. Continuous variables, categorical variables, and survival data were analyzed. RESULTS: Of 1352 total patients, 76% were classified as nonobese and 24% were obese. When comparing age, obese patients presented less frequently than nonobese patients <50 years old (10% vs. 90%), and when comparing patients >50 years old (18% vs. 82%, P = 0.0019). Obese patients were more likely to present with disease detected by imaging when compared to nonobese patients (67% vs. 56%, P = 0.0006). Obese patients had larger tumors (1.7 cm vs. 1.4 cm, P < 0.001) and higher rates of lymph node (LN) metastases (31% vs. 25%, P = 0.026). On multivariate analysis, obesity was associated with nonpalpable tumors, larger tumors, a higher incidence of LN metastasis, lower incidence of Her2 positivity, lower incidence of multifocality, and less likely to undergo reconstruction after mastectomy. CONCLUSIONS: Obese patients clinically present at older ages with mammographically detected breast cancer at more advanced stages than nonobese patients. Strategies to encourage screening among the obese patient population are important.

A digital pathology tool for quantification of color features in histologic specimens.

In preclinical research, histological analysis of tissue samples is often limited to qualitative or semiquantitative scoring assessments. The reliability of this analysis can be impaired by the subjectivity of these approaches, even when read by experienced pathologists. Furthermore, the laborious nature of manual image assessments often leads to the analysis being restricted to a relatively small number of images that may not accurately represent the whole sample. Thus, there is a clear need for automated image analysis tools that can provide robust and rapid quantification of histologic samples from paraffin-embedded or cryopreserved tissues. To address this need, we have developed a color image analysis algorithm (DigiPath) to quantify distinct color features in histologic sections. We demonstrate the utility of this tool across multiple types of tissue samples and pathologic features, and compare results from our program to other quantitative approaches such as color thresholding and hand tracing. We believe this tool will enable more thorough and reliable characterization of histological samples to facilitate better rigor and reproducibility in tissue-based analyses.

The proliferative status of thyrotropes is dependent on modulation of specific cell cycle regulators by thyroid hormone.

In this report we have examined changes in cell growth parameters, cell cycle effectors, and signaling pathways that accompany thyrotrope growth arrest by thyroid hormone (TH) and growth resumption after its withdrawal. Flow cytometry and immunohistochemistry of proliferation markers demonstrated that TH treatment of thyrotrope tumors resulted in a reduction in the fraction of cells in S-phase that is restored upon TH withdrawal. This is accompanied by dephosphorylation and rephosphorylation of retinoblastoma (Rb) protein. The expression levels of cyclin-dependent kinase 2 and cyclin A, as well as cyclin-dependent kinase 1 and cyclin B, were decreased by TH, and after withdrawal not only did these regulators of Rb phosphorylation and mitosis increase in their expression but so too did the D1 and D3 cyclins. We also noted a rapid induction and subsequent disappearance of the type 5 receptor for the growth inhibitor somatostatin with TH treatment and withdrawal, respectively. Because somatostatin can arrest growth by activating MAPK pathways, we examined these pathways in TtT-97 tumors and found that the ERK pathway and several of its upstream and downstream effectors, including cAMP response element binding protein, were activated with TH treatment and deactivated after its withdrawal. This led to the hypothesis that TH, acting through increased type 5 somatostatin receptor, could activate the ERK pathway leading to cAMP response element binding protein-dependent decreased expression of critical cell cycle proteins, specifically cyclin A, resulting in hypophosphorylation of Rb and its subsequent arrest of S-phase progression. These processes are reversed when TH is withdrawn, resulting in an increase in the fraction of S-phase cells.

Hepatoid Carcinoma of the Pancreas: Case Report, Next-Generation Tumor Profiling, and Literature Review.

Fewer than 25 cases of hepatoid carcinoma of the pancreas have been reported in the literature. We present a case in a 61-year-old male with a remote history of Hodgkin's lymphoma and gastric neuroendocrine cell hyperplasia. On surveillance endoscopic ultrasound, an 8 × 5 mm cystic lesion was seen in the tail of the pancreas. MRI showed a focal pancreatic duct cut-off with mild ductal dilation. Fine needle aspiration was performed, which was concerning for acinar cell carcinoma. The patient underwent distal pancreatectomy and recovered uneventfully. Final pathology demonstrated a 1.3-cm hepatoid carcinoma of the pancreas, with a final clinicopathological stage of T1N0M0. Next-generation nucleic acid sequencing of the tumor did not suggest a viable adjuvant chemotherapeutic agent, and no adjuvant therapy was administered. The patient has no evidence of disease 6 months following resection. A further characterization and description of the outcomes of these rare tumors is warranted to help guide providers and counsel patients.

Role of ERCP in pediatric blunt abdominal trauma: a case series at a level one pediatric trauma center.

BACKGROUND: There is no consensus regarding the appropriate use of endoscopic retrograde cholangiopancreatography (ERCP) in pediatric trauma. We report our experience with ERCP for management of pediatric pancreatic and biliary injury following blunt abdominal trauma. METHODS: A retrospective chart review was performed for pediatric patients with blunt abdominal trauma from July 2008 through December 2012 at our pediatric trauma center. For patients who underwent ERCP, demographics, injury characteristics, diagnostic details, procedures performed, length of stay, total parenteral nutrition use, and complications were reviewed. RESULTS: There were 532 patients identified: 115 hepatic injuries, 25 pancreatic injuries and one gall bladder injury. Nine patients (mean age 7.8 years) underwent ERCP. Seven (78%) had pancreatic injuries, while two (22%) had bilateral hepatic duct injuries. The median time to diagnosis was one day (range, 0-12). Diagnostic ERCP only was performed in three patients, two of which proceeded to distal pancreatectomy. Five patients had stents placed (two biliary and three pancreatic) and four sphincterotomies were performed. Despite pancreatic stenting, one patient required distal pancreatectomy for persistent leak. Median length of stay was 11 days. CONCLUSIONS: Pediatric pancreatic and biliary ductal injuries following blunt abdominal trauma are uncommon. ERCP can safely provide definitive treatment for some patients.

Metastatic Fibrolamellar Hepatocellular Carcinoma to the Pancreas.

Fibrolamellar hepatocellular carcinoma (FL-HCC) is a rare variant of hepatocellular carcinoma, usually presenting in the younger population (<40 years) without underlying liver disease. Although it has a better prognosis than hepatocellular carcinoma, it has a high rate of recurrence months to years after primary resection. While sites of recurrence usually involve the liver, regional lymph nodes, peritoneum, and lung, metastasis to the pancreas is extremely rare, with only 2 other cases reported in the literature. We present the case of a 46-year-old patient with metastatic FL-HCC to the pancreas 30 years after diagnosis and 26 years since his last resected liver recurrence.

Early gene expression changes preceding thyroid hormone-induced involution of a thyrotrope tumor.

Treatment with thyroid hormone (TH) results in shrinkage of a thyrotropic tumor grown in a hypothyroid host. We used microarray and Northern analysis to assess the changes in gene expression that preceded tumor involution. Of the 1,176 genes on the microarray, 7 were up-regulated, whereas 40 were decreased by TH. Many of these were neuroendocrine in nature and related to growth or apoptosis. When we examined transcripts for cell cycle regulators only cyclin-dependent kinase 2, cyclin A and p57 were down-regulated, whereas p15 was induced by TH. Retinoblastoma protein, c-myc, and mdm2 were unchanged, but E2F1 was down-regulated. TH also decreased expression of brain-derived neurotrophic factor, its receptor trkB, and the receptor for TRH. These, in addition to two other genes, neuronatin and PB cadherin, which were up- and down-regulated, respectively, showed a more rapid response to TH than the cell cycle regulators and may represent direct targets of TH. Finally, p19ARF was dramatically induced by TH, and although this protein can stabilize p53 by sequestering mdm2, we found no increase in p53 protein up to 48 h of treatment. In summary, we have described early changes in the expression of genes that may play a role in TH-induced growth arrest of a thyrotropic tumor. These include repression of specific growth factor and receptors and cell cycle genes as well as induction of other factors associated with growth arrest and apoptosis.

Perioperative management of patients with diabetes undergoing ambulatory elective surgery.

OBJECTIVE: The objective was to assess processes of care for patients with diabetes undergoing elective surgery. METHODS: A retrospective review of medical records was conducted to determine frequency of perioperative glucose monitoring, changes in glucose control, and treatment of intraoperative hyperglycemia. RESULTS: A total of 268 patients underwent 287 elective procedures. Mean age was 67 years, 63% were men, 97% had type 2 diabetes, and most (57%) were treated with oral hypoglycemic agents. Average perioperative time was approximately 8 h. Mean preoperative hemoglobin A1c was 7.0%; however, this value was checked in only 52% of cases. A glucose measurement was obtained in 89% of cases in the preoperative area and in 87% in the postanesthesia care unit, but in only 33% of cases did a value get checked intraoperatively. Average glucose was 139 mg/dl preoperatively, increasing to 166 mg/dl postoperatively (p <.001). Glucose levels increased regardless of type of outpatient medical therapy used to treat hyperglycemia, except for those on combination oral agents plus insulin (p =.06). CONCLUSIONS: These data indicate suboptimal documentation of outpatient hemoglobin A1c. Intraoperative glucose monitoring seldom occurred, despite prolonged periods under anesthesia and perioperative deterioration of glycemic control. Standards need to be developed and interventions are needed to enhance management of diabetes patients undergoing elective procedures.

Clinical inertia during postoperative management of diabetes mellitus: relationship between hyperglycemia and insulin therapy intensification.

OBJECTIVE: Our objective was to assess the application of insulin regimens in surgical postoperative patients with diabetes. METHODS: A chart review was conducted of patients with diabetes who were hospitalized postoperatively between January 1 and April 30, 2011. Analysis was restricted to patients hospitalized for ≥3 days and excluded cases with an endocrinology consult. Insulin regimens were categorized as "basal plus short acting," "short acting only," or "none," and the pattern of use was evaluated by hyperglycemia severity according to tertiles of both mean glucose and the number of glucose measurements >180 mg/dl. RESULTS: Among cases selected for analysis (n = 119), examination of changes in insulin use based on tertiles of mean glucose showed that use of basal plus short-acting insulin increased from 10% in the lowest tertile (mean glucose, 120 mg/dl) to 18% in the highest tertile (mean glucose, 198 mg/dl; p < .01); however, 70% of patients in the highest tertile continued to receive short-acting insulin only, with 12% receiving no insulin. Intensification of insulin to a basal plus short-acting regimen was also seen when changes were evaluated by the number of measurements >180 mg/dl (p < .01), but 70% and 12% of patients in the highest tertile still remained only on short-acting insulin or received no insulin, respectively. CONCLUSIONS: Use of basal plus short-acting insulin therapy increased with worsening hyperglycemia, but many cases did not have therapy intensified to the recommended insulin regimen--evidence of clinical inertia. Strategies should be devised to overcome inpatient clinical inertia in the treatment of postoperative patients with diabetes.

A significant number of women present with palpable breast cancer even with a normal mammogram within 1 year.

BACKGROUND: Although mammography is the gold standard for breast cancer screening, clinical breast examination (CBE) and self breast examination (SBE) are important adjuncts whose utility has been questioned. METHODS: A retrospective review of invasive breast cancer patients from 2000 to 2008 was performed. We compared 3 groups: breast cancer detected by (1) imaging only (nonpalpable) or palpable mass with a normal mammogram (2) ≥ 1 year (mammogram ≥ 1 year) or (3) <1 year (mammogram <1 year). RESULTS: Of 1,222 women, presentation included 67% nonpalpable, 21% mammogram ≥ 1 year, and 13% mammogram <1 year. Patients presenting with palpable masses on SBE or CBE even with a normal mammogram within 1 year tended to have more aggressive tumors (larger size, lymph node positive, and triple-negative disease) resulting in more aggressive therapy (a higher mastectomy rate and a greater likelihood of chemotherapy). CONCLUSION: A significant number of women present with palpable breast cancer within 1 year of a normal mammogram, many with an aggressive cancer. Therefore, we continue to advocate SBE and CBE for breast cancer screening.

Pituitary tumors arising from glycoprotein hormone alpha-subunit-deficient mice contain transcription factors and receptors present in thyrotropes.

Glycoprotein-hormone alpha-subunit deficient (alphaSUnull) mice are hypothyroid and hypogonadal due to the absence of functional TSH, LH and FSH, despite normal production of the corresponding beta subunits. Pituitary tumors spontaneously developing in alphaSUnull mice were propagated in hypothyroid mice. The purpose of the current studies was to compare the gene expression profile of these alphaSUnull tumors with previously characterized TtT-97 thyrotropic tumors. A group of animals bearing each tumor type was treated with thyroid hormone (T4) prior to tumor removal. Both tumor types equally expressed TSHbeta mRNA, which significantly decreased when exposed to T4, whereas alpha-subunit mRNA was absent in alphaSUnull tumors. Northern blot analysis was performed using cDNA probes for the following transcription factors: Pit1, GATA2, pLIM, Msx1, Ptx1 and Ptx2. Both tumors were found to contain identical transcripts with similar responses to T4, with the exception of Pit1. In contrast to the signal pattern seen in TtT-97, only two bands were seen in alphaSUnull tumors, which were similar in size to those in alphaTSH cells, a thyrotropic cell line that lacks TSHbeta-subunit expression and Pit1 protein. However, western blot analysis revealed a protein band in the alphaSUnull tumors consistent with Pit1, while this signal was absent in alphaTSH cells. Northern blot analysis was also performed with specific cDNA probes for the following receptors: TRbeta1, TRbeta2, TRalpha1, non-T3 binding alpha2, RXRgamma and Sst5. Similarly-sized transcripts were found in both types of tumor, although the signal for Sst5 was seen in T4-treated alphaSUnull tumors only with a more sensitive RT-PCR analysis. The overall similarity between the two tumor types renders the alphaSUnull tumor as a suitable thyrotropic tumor model.