Improved glucose regulation on a low carbohydrate diet in diabetic rats transplanted with macroencapsulated porcine islets.

Islet xenografts from porcine donors can reverse diabetes in experimental animal models and may be an alternative to human islet transplantation. We have recently reported the ability of porcine islets encapsulated in a double layer of hydrophilic agarose to maintain in vitro functional ability for >6 months. Although beta-cells are capable of adapting their secretory capacity in response to glucose levels, evidence has shown that prolonged hyperglycemia can compromise this ability. The aim of the present study was to determine the effects of diet manipulation on the long-term regulation of blood glucose levels, and the preservation of functional islet in the macrobeads. Twenty-one streptozotocin-induced diabetic Wistar-Furth male rats were randomly assigned to two diets containing 64% carbohydrate (CHO) or 20% CHO. Groups of five to six animals assigned to either diet were implanted with either empty (EM) or porcine islet-containing macrobeads (PIM) and followed for 333 days. Observations included general condition, body weight, blood glucose, and food and water intakes. Monthly blood samples were collected for insulin and C-peptide analysis. The 20% CHO diet significantly lowered blood glucose values when compared with those of the 64% CHO groups for both the empty (14.94 +/- 0.41 vs. 16.26 +/- 0.42 mmol/L, respectively, p < 0.001) and islet macrobead recipients (12.88 +/- 0.39 vs. 15.57 +/-0.85 mmol/L, respectively, p <0.001). The different diets, however, had no statistically significant effects on the preservation of islet mass in the macrobead. Serum porcine C-peptide was detected throughout the experiment in animals receiving porcine islet macrobeads, regardless of diet. Diabetic rats fed a low carbohydrate level diet and transplanted with porcine islet macrobeads had improved total tissue glucose disposal and improved clinical parameters associated with diabetes.

Encapsulation of porcine islets permits extended culture time and insulin independence in spontaneously diabetic BB rats.

The ability to culture porcine islets for extended times allows for both their functional assessment and the assurance of their microbiological safety prior to transplantation. We have previously shown that agarose-encapsulated porcine islets can be cultured for at least 24 weeks. In the current study, porcine islet agarose macrobeads cultured for up to 67 weeks were assessed for their ability to restore normoglycemia, respond to an intraperitoneal glucose challenge, maintain spontaneously diabetic BB rats free of insulin therapy for more than 6 months, and for their biocompatibility. Porcine islets were encapsulated in agarose macrobeads and subjected to weekly static perifusion assays for the assessment of insulin production. After in vitro culture for either 9, 40, or 67 weeks, 56-60 macrobeads were transplanted to each spontaneously diabetic BB rat. Transplanted rats were monitored daily for blood glucose levels. Glucose tolerance tests and assessments for porcine C-peptide were conducted at various intervals throughout the study. Normoglycemia (100-200 mg/dl) was initially restored in all islet transplanted rats. Moderate hyperglycemia (200-400 mg/dl) developed at around 30 days posttransplantation and continued throughout the study period of 201-202 days. Importantly, all rats that received encapsulated porcine islets continued to gain weight and were free of exogenous insulin therapy for the entire study. Porcine C-peptide (0.2-0.9 ng/ml) was detected in the serum of islet recipients throughout the study period. No differences were detected between recipient animals receiving islet macrobeads of various ages. These results demonstrate that the encapsulation of porcine islets in agarose macrobeads allows for extended culture periods and is an appropriate strategy for functional and microbiological assessment prior to clinical use.

The use of pancreas biopsy scoring provides reliable porcine islet yields while encapsulation permits the determination of microbiological safety.

For clinical xenogenic islet transplantation to be successful, several requirements must be met. Among them is a sizeable and reliable source of fully functional and microbiologically safe islets. The inherent variability among porcine pancreases, with respect to islet yield, prompted us to develop a Biopsy Score technique to determine the suitability of each pancreas for islet isolation processing. The Biopsy Score consists of an assessment of five variables: warm ischemia time, pancreas color, fat content, islet size, and islet demarcation, each of which is assigned a value of -1 or +1, depending on whether or not the established criteria is met. For determination of islet size and demarcation, fresh biopsies of porcine pancreases are stained with dithizone (DTZ) solution and examined under a dissecting microscope. Based on the scoring of such biopsies in pancreases from 26-56-month-old sows, we report here that the presence of large (>100 microm diameter), well-demarcated islets in the pancreas biopsy is a reliable predictor of isolation success. Encapsulation of the isolated porcine islets within the inner layer of a 1.5% agarose and an outer layer of 5.0% agarose macrobead, containing 500 equivalent islet number (EIN), provides for extended in vitro functional viability (>6 months of insulin production in response to glucose), as well as for comprehensive microbiological testing and at least partial isolation of the xenogeneic islets from the host immune system. All microbiological testing to date has been negative, except for the presence of porcine endogenous retrovirus (PERV). Taken together, we believe that the Biopsy Score enhancement of our islet isolation technique and our agarose-agarose macroencapsulation methodology bring us significantly closer to realizing clinical porcine islet xenotransplantation for the treatment of insulin-dependent diabetic patients.

Three-dimensional culture of mouse renal carcinoma cells in agarose macrobeads selects for a subpopulation of cells with cancer stem cell or cancer progenitor properties.

The culture of tumor cell lines in three-dimensional scaffolds is considered to more closely replicate the in vivo tumor microenvironment than the standard method of two-dimensional cell culture. We hypothesized that our method of encapsulating and maintaining viable and functional pancreatic islets in agarose-agarose macrobeads (diameter 6-8 mm) might provide a novel method for the culture of tumor cell lines. In this report we describe and characterize tumor colonies that form within macrobeads seeded with mouse renal adenocarcinoma cells. Approximately 1% of seeded tumor cells survive in the macrobead and over several months form discrete elliptical colonies appearing as tumor cell niches with increasing metabolic activity in parallel to colony size. The tumor colonies demonstrate ongoing cell turnover as shown by BrdU incorporation and activated caspase-3 and TUNEL staining. Genes upregulated in the tumor colonies of the macrobead are likely adaptations to this novel environment, as well as an amplification of G(1)/S cell-cycle checkpoints. The data presented, including SCA-1 and Oct4 positivity and the upregulation of stem cell-like genes such as those associated with the Wnt pathway, support the notion that the macrobead selects for a subpopulation of cells with cancer stem cell or cancer progenitor properties.

Hydrophilic agarose macrobead cultures select for outgrowth of carcinoma cell populations that can restrict tumor growth.

Cancer cells and their associated tumors have long been considered to exhibit unregulated proliferation or growth. However, a substantial body of evidence indicates that tumor growth is subject to both positive and negative regulatory controls. Here, we describe a novel property of tumor growth regulation that is neither species nor tumor-type specific. This property, functionally a type of feedback control, is triggered by the encapsulation of neoplastic cells in a growth-restricting hydrogel composed of an agarose matrix with a second coating of agarose to form 6- to 8-mm diameter macrobeads. In a mouse cell model of renal adenocarcinoma (RENCA cells), this process resulted in selection for a stem cell-like subpopulation which together with at least one other cell subpopulation drove colony formation in the macrobeads. Cells in these colonies produced diffusible substances that markedly inhibited in vitro and in vivo proliferation of epithelial-derived tumor cells outside the macrobeads. RENCA cells in monolayer culture that were exposed to RENCA macrobead-conditioned media exhibited cell-cycle accumulation in S phase due to activation of a G(2)/M checkpoint. At least 10 proteins with known tumor suppression functions were identified by analysis of RENCA macrobead-conditioned media, the properties of which offer opportunities to further dissect the molecular basis for tumor growth control. More generally, macrobead culture may permit the isolation of cancer stem cells and other cells of the stem cell niche, perhaps providing strategies to define more effective biologically based clinical approaches to treat neoplastic disease.

Neutralization of endotoxin by a phospholipid emulsion in healthy volunteers.

BACKGROUND: An approach to endotoxin (lipopolysaccharide [LPS]) blockade makes use of the ability of lipoproteins, via surface phospholipids, to bind and neutralize LPS. The aim of the present study was to determine whether the intravenous administration of a protein-free, phospholipid-rich emulsion is an effective method for neutralizing the effects of LPS in healthy persons. METHODS: This was a double-blind, placebo-controlled study in 20 volunteers. Volunteers received Escherichia coli endotoxin (2 ng/kg) intravenously 2 h into a 6-h infusion of either emulsion (210 mg/kg) or placebo (Intralipid diluted 1 : 64). RESULTS: The volunteers who received emulsion had a lower mean clinical score (P<.01), temperature (P<.05), pulse rate (P<.05), neutrophil count (P<.05), tumor necrosis factor- alpha level (P<.05), and interleukin-6 level (P<.05) than did the volunteers who received placebo. Response was related to serum phospholipid level. The greatest effects were observed in the volunteers achieving phospholipid levels of approximately 500 mg/dL or higher. CONCLUSION: Phospholipid emulsion attenuates the clinical and laboratory effects associated with the administration of LPS in humans, suggesting a novel approach to the treatment of endotoxemia.

LDL Apheresis: an effective and safe treatment for refractory hypercholesterolemia.

Through the efforts of Edward H. Ahrens, LDL apheresis became available for the treatment of patients, often with familial hypercholesterolemia, who have no alternative therapy for severely elevated LDL cholesterol levels. In the U.S., the FDA has approved this treatment for individuals on maximum diet and drugs with an LDL cholesterol greater than 300 mg/dL or greater than 200 mg/dL with coronary artery disease. Unlike plasmapheresis, apolipoprotein B-containing lipoproteins (LDL, Lp(a), and VLDL) are selectively removed by heparin precipitation or columns containing dextran sulfate cellulose or antibodies to apolipoprotein B. The acute lowering of LDL-cholesterol by a typical 2 - 3 h treatment is up to 80%, and the time-averaged lowering in the 1 to 2 week interval between treatments is up to 50%, with very few side effects. The lowering of LDL-cholesterol and other cardioprotective effects of LDL apheresis have reduced chest pain, prevented new disability and prolonged life. Whole blood compatible columns in development offer the possibility of simpler and less expensive treatments.

A single intravenous dose of endotoxin rapidly alters serum lipoproteins and lipid transfer proteins in normal volunteers.

Endotoxemia is associated with rapid and marked declines in serum levels of LDL and HDL by unknown mechanisms. Six normal volunteers received a single, small intravenous (iv) dose of endotoxin (Escherichia coli 0113, 2 ng/kg) or saline in a random order, cross-over design. After endotoxin treatment, volunteers had mild, transient flu-like symptoms and markedly increased serum levels of tumor necrosis factor and its soluble receptors, interleukin-6, cortisol, serum amyloid A, and C-reactive protein. Triglyceride (TG), VLDL-TG, and nonesterified fatty acid increased (peak at 3-4 h), then TG declined (nadir at 9 h), and then cholesterol, LDL cholesterol, apolipoprotein B (apoB), and phospholipid declined (nadirs at 12-24 h). HDL cholesterol and apoA-I levels were not affected, but half of the decrease in phospholipid was HDL phospholipid. Lipopolysaccharide binding protein (LBP) rose 3-fold (peak at 12 h), with smaller and later decreases in the activities of phospholipid transfer protein and cholesteryl ester transfer protein. In conclusion, a decline in LDL was rapidly induced in normal volunteers with a single iv dose of endotoxin. The selective loss of phospholipid from HDL may have been mediated by LBP and, after more intense or prolonged inflammation, could result in increased HDL clearance and reduced HDL levels.

Safety and pharmacokinetics of an endotoxin-binding phospholipid emulsion.

BACKGROUND: Lipids and lipoproteins have been shown to bind and neutralize endotoxin and to improve outcomes in animal models of sepsis. OBJECTIVE: To provide safety and pharmacokinetic data for a protein-free, phospholipid-rich emulsion developed as an agent to neutralize endotoxin, and to study the changes in lipids and lipoproteins following emulsion administration. METHODS: Thirty healthy male volunteers (aged 18-45 y) were given an emulsion containing 92.5% soy phospholipid, 7.5% soy triglyceride, and 18 mM sodium cholate using a double-blind, placebo-controlled crossover protocol. Emulsion at 3 escalating doses (75, 150, 300 mg/kg) based on phospholipid content was administered by intravenous infusion over 2 hours in the low- and mid-dose groups and 6 hours in the high-dose group. RESULTS: All subjects completed the protocol without significant toxicities. A slight dose-dependent increase in indirect bilirubin at the 24-hour time point was observed in the emulsion treatment period, with a maximum difference between placebo and emulsion of 0.9 mg/dL. Mean +/- SD peak phospholipid levels were 316 +/- 30, 533 +/- 53, and 709 +/- 86 mg/dL, and phospholipid half-lives were 5.4 +/- 0.6, 5.4 +/- 0.5, and 8.0 +/- 0.8 hours for the low, mid, and high doses, respectively. Increases in total cholesterol, low-density lipoprotein cholesterol and apolipoprotein A-I and B levels were observed. High-density lipoprotein cholesterol decreased immediately following emulsion infusion, but rebounded to above placebo levels by 24 hours. CONCLUSIONS: A unique phospholipid-rich emulsion was shown to have a favorable safety profile and to expand the blood lipid and lipoprotein pool without the use of human-derived blood products. Lipid levels expected to protect against the physiologic effects of bacterial endotoxin were achieved.

Protein-free phospholipid emulsion treatment improved cardiopulmonary function and survival in porcine sepsis.

Lipoprotein phospholipid (PL) plays a major role in neutralization of endotoxin. This study tested the hypothesis that prophylactic administration of a PL-enriched emulsion (PRE), which augments PL content of serum lipoproteins and neutralizes endotoxin in vitro, would preserve cardiovascular function and improve survival in porcine septic peritonitis. A control group was compared with low-, mid-, and high-dose treatment groups that received PRE by primed continuous infusion for 48 h. A fibrin clot containing live Escherichia coli 0111.B4 was implanted intraperitoneally 30 min after the priming dose. Survival increased in a dose-dependent manner and was correlated with serum PL. Infused PL was associated with high-density lipoprotein in the low-dose group and all serum lipoproteins at higher doses. Treatment significantly lowered serum endotoxin and tumor necrosis factor (TNF)-alpha, preserved cardiac output and ejection fraction, and attenuated increases in systemic and pulmonary vascular resistances. This study demonstrated that augmentation of lipoprotein PL via administration of PRE improved survival and offered a novel therapeutic approach to sepsis.