Rajendra Ramsamooj

De novo nonalcoholic fatty liver disease after liver transplantation

Hepatic steatosis is a recognized problem in patients after orthotopic liver transplant (OLT). However, de novo development of nonalcoholic fatty liver disease (NAFLD) has not been well described. The aim of this study was to determine the prevalence and predictors of de novo NAFLD after OLT. A retrospective analysis was performed on 68 OLT patients with donor liver biopsies and posttransplantation liver biopsies. Individual medical charts were reviewed for demographics, indication for OLT, serial histology reports, genotypes for hepatitis C, comorbid conditions, and medications. Liver biopsies were reviewed blindly and graded according to the Brunt Scoring System. Multivariate logistic regression analysis was used to study the risk factors for developing NAFLD. The interval time from OLT to subsequent follow‐up liver biopsy was 28 ± 18 months. A total of 12 patients (18%) developed de novo NAFLD, and 6 (9%) developed de novo NASH. The regression model indicated that the use of angiotensin‐converting enzyme inhibitors (ACE‐I) was associated with a reduced risk of developing NAFLD after OLT (odds ratio, 0.09; 95% confidence interval, 0.010‐0.92; P = 0.042). Increase in body mass index (BMI) of greater than 10% after OLT was associated with a higher risk of developing NAFLD (odds ratio, 19.38; 95% confidence interval, 3.50‐107.40; P = 0.001). In conclusion, de novo NAFLD is common in the post‐OLT setting, with a significant association with weight gain after transplant. The use of an ACE‐I may reduce the risk of developing post‐OLT NAFLD. Liver Transpl, 2006.

Knockout of Toll-Like Receptor-2 Attenuates Both the Proinflammatory State of Diabetes and Incipient Diabetic Nephropathy

Objective—Type 1 diabetes (T1DM) is a proinflammatory state and confers an increased risk for vascular complications. Toll-like receptors (TLR) could participate in diabetic vasculopathies. Whether TLR activation contributes to the proinflammatory state of T1DM and the pathogenesis of diabetic nephropathy remains unknown. Methods and Results—We induced T1DM in TLR2 knockout mice (TLR2−/−) and wild-type littermates (C57BL/6J-WT) using streptozotocin (STZ). Fasting blood, peritoneal macrophages, and kidneys were obtained for flow cytometry, Western blot, microscopy, and cytokine assays at 6 and 14 weeks after induction of diabetes. Macrophage TLR2 expression and MyD88-dependent signaling were increased in diabetic mice (WT+STZ) compared with nondiabetic WT mice. These biomarkers were attenuated in diabetic TLR2−/− macrophages. WT+STZ mice showed increased kidney:body weight ratio due to cell hypertrophy, increased albuminuria, decreased kidney nephrin, podocin, and podocyte number and increased transforming growth factor-&bgr; and laminin compared with WT mice. Nephrin, podocin, and podocyte number and effacement were restored, and transforming growth factor-&bgr; and laminin levels were decreased in TLR2−/−+ STZ mice kidneys versus WT+STZ. Peritoneal and kidney macrophages were predominantly M1 phenotype in WT+STZ mice; this was attenuated in TLR2−/−+STZ mice. Conclusion—These data support a role for TLR2 in promoting inflammation and early changes of incipient diabetic nephropathy, in addition to albuminuria and podocyte loss.

Dermatofibrosarcoma protuberans: reappraisal of wide local excision and impact of inadequate initial treatment.

Background: The extent of local invasion in dermatofibrosarcoma protuberans (DFSP) is often clinically difficult to appreciate, and this leads to inadequate resections. We examined the effect of inadequate initial treatment and the efficacy of wide resection.Methods: We performed a retrospective analysis of the records of 35 patients with DFSP treated at our institution (1985 and 2001). Data were analyzed with Wilcoxon’s ranked sum test and Fisher’s exact test.Results: Of the 24 patients eligible for analysis, 11 had definitive wide resection after diagnostic excisions elsewhere (primary group), and 13 had recurrent tumors after previous surgical treatment elsewhere (recurrent group). Twenty-three patients were treated with wide resection only, and adjuvant radiation was administered to one patient who had a fibrosarcoma. At a median follow-up of 54 months, patients definitively treated at our institution had a 100% local recurrence–free survival. In comparison to the primary group, recurrent DFSPs were significantly larger and deeper and occurred in the head and neck region. Five cases had bone involvement, and of these, 80% occurred in the recurrent group.Conclusions: Inadequate initial treatment results in larger, deeper recurrent lesions, but these can be managed by appropriate wide excision. Wide resection of DFSP (whether recurrent or primary) with negative histological margins predicts a superior local recurrence–free survival.

Increased Soluble Leptin Receptor Levels in Morbidly Obese Patients With Insulin Resistance and Nonalcoholic Fatty Liver Disease

The adipocyte hormone, leptin has been demonstrated to have profibrogenic actions in vitro and in animal models. However, no correlation was found between plasma leptin levels and fibrosis stage in humans. Thus, our aim was to study whether soluble leptin receptor (SLR) or free leptin index (FLI; calculated as the ratio of leptin to SLR), may correlate better with the features of metabolic syndrome and with the histological grade and stage of nonalcoholic fatty liver disease (NAFLD)/nonalcoholic steatohepatitis (NASH). We studied a population (n = 104) of morbidly obese patients undergoing bariatric surgery. Data including BMI, type 2 diabetes mellitus, hypertension, and hyperlipidemia were obtained. Plasma fasting leptin and SLR, fasting glucose and insulin were measured, and homeostasis model of assessment insulin resistance (HOMAIR) index and FLI were calculated. All patients had intraoperative liver biopsies. Leptin levels correlated with the BMI. The multiple regression analysis indicated that increasing HOMA and decreasing FLI were predictors of steatosis in the liver (P < 0.0003). SLR levels were positively correlated with the presence of diabetes mellitus and the stage of fibrosis. In conclusion, increased SLR levels in morbidly obese patients with diabetes are correlated with the stage of liver fibrosis, and may reflect progressive liver disease.

Investigation of near-infrared autofluorescence imaging for the detection of breast cancer

Detection of breast cancer in fresh tissue obtained from surgery is investigated using near-infrared autofluorescence imaging under laser excitation at 532 and 632.8 nm. The differences in intensity between the three main components of breast tissue (cancer, fibrous, and adipose) are estimated and compared to those obtained from cross-polarized light scattering images recorded under polarized illumination at 700 nm. The optical spectroscopic images for each tissue sample were subsequently compared with the histopathology slides. The experimental results indicate that the intensity of the near-infrared emission is considerably different in breast cancer compared to that of the adjacent nonneoplastic tissues (adipose and fibrous tissue). The experimental results suggest that 632.8-nm excitation offers key advantages compared to 532 nm excitation.

Imaging of tissue microstructures using a multimodal microscope design

We investigate a microscope design that offers high signal sensitivity and hyperspectral imaging capabilities and allows for implementation of various optical imaging approaches while its operational complexity is minimized. This system uses long working distance microscope objectives that enable for off-axis illumination of the tissue, thereby allowing for excitation at any optical wavelength and nearly eliminating spectral noise from the optical elements. Preliminary studies using human and animal tissues demonstrate the feasibility of this approach for real-time imaging of intact tissue microstructures using autofluorescence and light scattering imaging methods.

Factors influencing rat survival in a warm renal ischemia model: time to adapt the protocols.

INTRODUCTION Survival in warm renal ischemia models is not only dependent on the treatment or surgical technique being evaluated, but also on factors inherent to the model itself. Use of rats of various strains in previous studies makes interstudy comparison difficult when trying to design an appropriate model control that would yield intermediate survival. In this study, impact of rat strain on survival after prolonged warm renal ischemia in the setting of delivery-controlled inhalational anesthesia was evaluated. MATERIALS AND METHODS Under general delivery-controlled inhalation anesthesia with isoflurane, Dahl salt-sensitive, Wistar-Furth, Sprague-Dawley, and spontaneously hypertensive rats (n = 66 rats) were subjected to 150 minutes of unilateral renal warm ischemia time, subsequent reperfusion, and contralateral nephrectomy. Animals were followed up for 1 month, after which survivors were euthanized and morphologic changes in kidneys were scored. RESULTS Thirty-day survival was: Dahl salt sensitive, 78%; Wistar-Furth, 67%; Sprague-Dawley, 55%; and spontaneously hypertensive rats, 0% (P < .0001). Histologic acute injury scores were higher for non-survivors versus 30-day survivors (P < .0001). CONCLUSION Our data strongly suggest that rat strain is a major factor influencing survival and that strain and warm ischemia time selections must be considered together when designing a model control yielding intermediate survival. Further study is warranted in order to compare the effect of delivery-controlled inhalational versus historical anesthesia methods on animal survival.

Identification of Cancer Stem Cell Subpopulations of CD34+ PLC/PRF/5 That Result in Three Types of Human Liver Carcinomas

CD34(+) stem cells play an important role during liver development and regeneration. Thus, we hypothesized that some human liver carcinomas (HLCs) might be derived from transformed CD34(+) stem cells. Here, we determined that a population of CD34(+) cells isolated from PLC/PRF/5 hepatoma cells (PLC) appears to function as liver cancer stem cells (LCSCs) by forming HLCs in immunodeficient mice with as few as 100 cells. Moreover, the CD34(+) PLC subpopulation cells had an advantage over CD34(-) PLCs at initiating tumors. Three types of HLCs were generated from CD34(+) PLC: hepatocellular carcinomas (HCCs); cholangiocarcinomas (CC); and combined hepatocellular cholangiocarcinomas (CHCs). Tumors formed in mice transplanted with 12 subpopulations and 6 progeny subpopulations of CD34(+) PLC cells. Interestingly, progenies with certain surface antigens (CD133, CD44, CD90, or EPCAM) predominantly yielded HCCs. CD34(+) PLCs that also expressed OV6 and their progeny OV6(+) cells primarily produced CHC and CC. This represents the first experiment to demonstrate that the OV6(+) antigen is associated with human CHC and CC. CD34(+) PLCs that also expressed CD31 and their progeny CD31(+) cells formed CHCs. Gene expression patterns and tumor cell populations from all xenografts exhibited diverse patterns, indicating that tumor-initiating cells (TICs) with distinct antigenic profiles contribute to cancer cell heterogeneity. Therefore, we identified CD34(+) PLC cells functioning as LCSCs generating three types of HLCs. Eighteen subpopulations from one origin had the capacity independently to initiate tumors, thus functioning as TICs. This finding has broad implications for better understanding of the multistep model of tumor initiation and progression. Our finding also indicates that CD34(+) PLCs that also express OV6 or CD31 result in types of HLCs. This is the first report that PLC/PRF/5 subpopulations expressing CD34 in combination with particular antigens defines categories of HLCs, implicating a diversity of origins for HLC.

Clonogenically Culturing and Expanding CD34+ Liver Cancer Stem Cells in Vitro

A large number of cancer stem cells (CSCs) have been isolated and identified; however, none has been cultured in an unlimited manner in vitro without losing tumorigenicity and multipotency. In this study, we successfully clonogenically cultured a newly identified CD34+ liver CSC (LCSC) on feeder cells up to 22 passages (to date) without losing CSC property. Cloned CD34+ LCSC formed a round packed morphology and it could also be cryopreserved and recultured. Stem cell markers, CD34, CD117, and SOX2; normal liver stem cell markers, alpha fetoprotein, CK19, CK18, and OV6; putative CSC markers, CD44, CD133, EpCAM, and CD90; as well as CD31 were expressed in cloned CD34+ LCSC. SOX2 was the major factor in maintaining this LCSC before colonization, and interestingly, OCT4, SOX2, NAONG, Klf4, c-Myc, and Lin28 were upregulated in association with symmetric self-renewal for colony growth of CD34+ LCSC on feeder cells. Gene expression patterns of in vitro differentiation were consistent with our in vivo finding; furthermore, the tumorigenicity of cloned CD34+ LCSC was not different from uncloned CD34+ LCSC sorted from parental PLC. These results show that our cloned CD34+ LCSC maintained CSC property, including self-renewal, bipotency, and tumorigenicity after long-term culture, demonstrating that this LCSC can be cultured in an unlimited manner in vitro. Thus, establishing pure population of CSCs isolated from the patients will provide an opportunity to explore the mechanisms of tumorigenesis and cancer development, and to identify unique biomarkers presenting potential indicators of drug efficacy against CSCs for establishment of a novel strategy for cancer therapy.

Multi-Color Autofluorescence and Scattering Spectroscopy Provides Rapid Assessment of Kidney Function Following Ischemic Injury

A major source of kidneys for transplant comes from deceased donors whose tissues have suffered an unknown amount of warm ischemia prior to retrieval, with no quantitative means to assess function before transplant. Toward addressing this need, non-contact monitoring of optical signatures in rat kidneys was performed in vivo during ischemia and reperfusion. Kidney autofluorescence images were captured under ultraviolet illumination (355 nm, 325 nm, and 266 nm) in order to provide information on related metabolic and non-metabolic response. In addition, light scattering images under 355 nm, 325 nm, and 266 nm, 500 nm illumination were monitored to report on changes in kidney optical properties giving rise to the observed autofluorescence signals during these processes. During reperfusion, various signal ratios were generated from the recorded signals and then parametrized. Time-dependent parameters derived from the ratio of autofluorescence under 355 nm excitation to that under 266 nm excitation, as well as from 500 nm scattered signal, were found capable of discriminating dysfunctional kidneys from those that were functional (p < 0.01) within hours of reperfusion. Kidney dysfunction was confirmed by subsequent survival study and histology following autopsy up to a week later. Physiologic changes potentially giving rise to the observed signals, including those in cellular metabolism, vascular response, tissue microstructure, and microenvironment chemistry, are discussed.