Therapeutic cell encapsulation techniques and applications in diabetes.

The encapsulation of therapeutic cells permits the implantation of allogeneic and xenogeneic cells for the regulation of certain physiological processes damaged by the death or senescence of host tissues. The encapsulation of pancreatic cells for the treatment of diabetes is emphasized; however, many of the techniques are applicable to a wide array of mammalian cell applications. The summary of both established and novel encapsulation techniques, clinical trials, and commercial product developments highlights the metered but steady pace of therapeutic cell encapsulation towards implementation.

Co-encapsulation of bioengineered IGF-II-producing cells and pancreatic islets: effect on beta-cell survival.

Insulin-like growth factor-II (IGF-II) has been shown to promote pancreatic ß-cell survival. We evaluated the effect of co-encapsulating islets and bioengineered IGF-II-producing cells on islet cell survival. IGF-II or green fast protein (GFP) genes were transferred into TM4 cells, and purified using a neomycin resistance gene, leading to pure cell cultures (TM4-IGF-II or TM4-GFP) with a stable overexpression of the transferred gene. Islets were co-encapsulated with TM4-IGF-II or TM4-GFP, or encapsulated alone in alginate microcapsules. Rat and mouse islet cell survival was studied in vitro and in vivo, respectively. After 12 days in culture, islet viability (dual staining, acridine orange/propidium iodide) was 83% with TM4-IGF-II, compared with 51% (P<0.05) and 41% (P<0.001) with TM4-GFP and islets alone, respectively. The study of islet necrotic centers and the evaluation of islet function, using the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) assay, yielded similar results. From 125 days after transplantation, more diabetic mice maintained normoglycemia when they were transplanted with islets co-encapsulated with TM4-IGF-II (4/7). A significant difference for the maintenance of normoglycemia was observed between recipients of islets co-encapsulated with TM4-IGF-II versus islets alone (P=0.023), or with TM4-GFP (P=0.048). In conclusion, the co-encapsulation of islets with bioengineered IGF-II-producing cells promotes islet cell survival.

Biocompatibility and physicochemical characteristics of alginate-polycation microcapsules.

There is a need for better understanding of the biocompatibility of alginate-polycation microcapsules based on their physicochemical characteristics. Microcapsules composed of alginate with 44% (IntG) or 71% (HiG) guluronate, gelled with calcium (Ca) or barium (Ba) and coated with poly-L-lysine (PLL) or poly-l-ornithine (PLO), followed by IntG alginate were compared. For microcapsules with an IntG(Ca) gel core, using PLO instead of PLL resulted in less immune cell adhesion after 2 days in C57BL/6J mice. The PLO microcapsules were also characterized by greater hydrophilicity and superior resistance to swelling and damage under osmotic stress. For microcapsules with a PLL membrane, replacing the IntG(Ca) gel core with IntG(Ba) or HiG(Ca) gel resulted in stronger immune responses (p<0.05). This was explained by poor penetration of PLL into the gel, as demonstrated by Fourier transform infrared spectroscopy analyses and membrane rupturing during osmotic swelling. X-ray photoelectron spectroscopy analyses show that all microcapsules had the same amount of polycation at their surface. Moreover, alginate coatings had non-significant effects on the biocompatibility and physicochemical properties of the microcapsules. Thus, alginate-polycation interactions for membrane formation are more important for biocompatibility than either the quantity of polycation at the surface or the alginate coating.

Similar progression of diabetic retinopathy with insulin glargine and neutral protamine Hagedorn (NPH) insulin in patients with type 2 diabetes: a long-term, randomised, open-label study.

AIMS/HYPOTHESIS: This long-term study was designed to further characterise the retinal safety profile of insulin glargine and human neutral protamine Hagedorn (NPH) insulin in patients with type 2 diabetes mellitus. METHODS: An open-label, 5 year, randomised (1:1), multicentre, stratified, parallel-group study conducted in the USA and Canada enrolled individuals with type 2 diabetes and either no or non-proliferative retinopathy (less than severe; Early Treatment Diabetic Retinopathy Study [ETDRS] level less than 53 in both eyes) who were treated with oral hypoglycaemic agents (OHAs) alone, insulin alone or OHAs with insulin for >/=3 months prior to study entry and a baseline HbA(1c) level of 6.0-12.0%. Patients were randomised by the investigator according to the centralised interactive voice response system to receive twice-daily NPH insulin (n = 509) or once-daily basal insulin glargine (n = 515). The investigator was not blinded to the treatment group to which each participant had been assigned. The main objective of this study was to compare the progression of diabetic retinopathy between treatment groups by analysing the percentage of patients with three or more step progression in the ETDRS retinopathy patient-level severity scale after treatment with either basal insulin. Masked, centralised grading of seven-field stereoscopic fundus photographs was used. RESULTS: Similarly sustained glycaemic control was observed in both the insulin glargine and NPH insulin treatment groups. Despite a slightly greater severity of diabetic retinopathy for the insulin glargine group at baseline, three or more step progression in ETDRS score from baseline to end-of-study was similar between treatment groups (14.2% [53/374] of insulin glargine-treated patients vs 15.7% [57/363] of NPH-treated patients); the difference in the incidence of progression was -1.98% (95% CI -7.02, 3.06%). Other measures of retinopathy-the development of proliferative diabetic retinopathy and progression to clinically significant macular oedema-occurred to a similar degree in both treatment groups. No other safety issues, such as unexpected adverse events for either insulin emerged during the 5 year study. However, NPH insulin treatment was associated with a higher incidence of severe hypoglycaemia compared with insulin glargine. CONCLUSIONS/INTERPRETATION: This study shows no evidence of a greater risk of the development or progression of diabetic retinopathy with insulin glargine vs NPH insulin treatment in patients with type 2 diabetes mellitus. TRIAL REGISTRATION: ClinicalTrials.gov NCT00174824 FUNDING: This study was sponsored by sanofi-aventis.

Hyperglycemia induces insulin resistance on angiotensinogen gene expression in diabetic rat kidney proximal tubular cells.

Clinical and animal studies have shown that treatment with angiotensin-converting enzyme (ACE) inhibitors or angiotensin II (Ang II) receptor antagonists slows the progression of nephropathy in diabetes, indicating that Ang II plays an important role in its development. We have reported previously that insulin inhibits the stimulatory effect of high glucose levels on angiotensinogen (ANG) gene expression in rat immortalized renal proximal tubular cells (IRPTCs) via the mitogen-activated protein kinase (p44/42 MAPK) signal transduction pathway. We hypothesize that the suppressive action of insulin on ANG gene expression might be attenuated in renal proximal tubular cells (RPTCs) of rats with established diabetes. Two groups of male adult Wistar rats were studied: controls and streptozotocin (STZ)-induced diabetic rats at 2, 4, 8 and 12 weeks post-STZ administration. Kidney proximal tubules were isolated and cultured in either normal glucose (i.e. 5 mM) or high glucose (i.e. 25 mM) medium to determine the inhibitory effect of insulin on ANG gene expression. Immunoreactive rat ANG (IR-rANG) in culture media and cellular ANG mRNA were measured by a specific radioimmunoassay and reverse transcription-polymerase chain reaction assay respectively. Activation of the p44/42 MAPK signal transduction pathway in rat RPTCs was evaluated by p44/42 MAPK phosphorylation employing a PhosphoPlus p44/42 MAPK antibody kit. Insulin (10(-7) M) inhibited the stimulatory effect of high glucose levels on IR-rANG secretion and ANG gene expression and increased p44/42 MAPK phosphorylation in normal rat RPTCs. In contrast, it failed to affect these parameters in diabetic rat RPTCs. In conclusion, our studies demonstrate that hyperglycaemia induces insulin resistance on ANG gene expression in diabetic rat RPTCs by altering the MAPK signal transduction pathway.

The Ca2+-binding proteins S100A8 and S100A9 are encoded by novel injury-regulated genes.

To gain insight into the molecular mechanisms underlying cutaneous wound repair, we performed a large scale screen to identify novel injury-regulated genes. Here we show a strong up-regulation of the RNA and protein levels of the two Ca(2+)-binding proteins S100A8 and S100A9 in the hyperthickened epidermis of acute murine and human wounds and of human ulcers. Furthermore, both genes were expressed by inflammatory cells in the wound. The increased expression of S100A8 and S100A9 in wound keratinocytes is most likely related to the activated state of the keratinocytes and not secondary to the inflammation of the skin, since we also found up-regulation of S100A8 and S100A9 in the epidermis of activin-overexpressing mice, which develop a hyperproliferative and abnormally differentiated epidermis in the absence of inflammation. Furthermore, S100A8 and S100A9 expression was found to be associated with partially differentiated keratinocytes in vitro. Using confocal microscopy, both proteins were shown to be at least partially associated with the keratin cytoskeleton. In addition, cultured keratinocytes efficiently secreted the S100A8/A9 dimer. These results together with previously published data suggest that S100A8 and S100A9 are novel players in wound repair, where they might be involved in the reorganization of the keratin cytoskeleton in the wounded epidermis, in the chemoattraction of inflammatory cells, and/or in the defense against microorganisms.

Albuminuria and risk of cardiovascular events, death, and heart failure in diabetic and nondiabetic individuals.

CONTEXT: Microalbuminuria is a risk factor for cardiovascular (CV) events. The relationship between the degree of albuminuria and CV risk is unclear. OBJECTIVES: To estimate the risk of CV events in high-risk individuals with diabetes mellitus (DM) and without DM who have microalbuminuria and to determine whether levels of albuminuria below the microalbuminuria threshold increase CV risk. DESIGN: The Heart Outcomes Prevention Evaluation study, a cohort study conducted between 1994 and 1999 with a median 4.5 years of follow-up. SETTING: Community and academic practices in North and South America and Europe. PARTICIPANTS: Individuals aged 55 years or more with a history of CV disease (n = 5545) or DM and at least 1 CV risk factor (n = 3498) and a baseline urine albumin/creatinine ratio (ACR) measurement. MAIN OUTCOME MEASURES: Cardiovascular events (myocardial infarction, stroke, or CV death); all-cause death; and hospitalization for congestive heart failure. RESULTS: Microalbuminuria was detected in 1140 (32.6%) of those with DM and 823 (14.8%) of those without DM at baseline. Microalbuminuria increased the adjusted relative risk (RR) of major CV events (RR, 1.83; 95% confidence interval [CI], 1.64-2.05), all-cause death (RR, 2.09; 95% CI, 1.84-2.38), and hospitalization for congestive heart failure (RR, 3.23; 95% CI, 2.54-4.10). Similar RRs were seen for participants with or without DM, even after adjusting for other CV risk factors (eg, the adjusted RR of the primary aggregate end point was 1.97 [95% CI, 1.68-2.31] in those with DM and 1.61 [95% CI, 1.36-1.90] in those without DM). Compared with the lowest quartile of ACR (<0.22 mg/mmol), the RRs of the primary aggregate end point in the second quartile (ie, ACR range, 0.22-0.57 mg/mmol) was 1.11 (95% CI, 0.95-1.30); third quartile, 1.38 (95% CI, 1.19-1.60; ACR range, 0.58-1.62 mg/mmol); and fourth quartile, 1.97 (95% CI, 1.73-2.25; ACR range, >1.62 mg/mmol) (P for trend <.001, even after excluding those with microalbuminuria). For every 0.4-mg/mmol increase in ACR level, the adjusted hazard of major CV events increased by 5.9% (95% CI, 4.9%-7.0%). CONCLUSIONS: Our results indicate that any degree of albuminuria is a risk factor for CV events in individuals with or without DM; the risk increases with the ACR, starting well below the microalbuminuria cutoff. Screening for albuminuria identifies people at high risk for CV events.

A 16-week comparison of the novel insulin analog insulin glargine (HOE 901) and NPH human insulin used with insulin lispro in patients with type 1 diabetes.

OBJECTIVE: To determine the safety and efficacy of the long-acting insulin analog, insulin glargine, as a component of basal bolus therapy in patients with type 1 diabetes. RESEARCH DESIGN AND METHODS: Patients with type 1 diabetes receiving basal-bolus insulin treatment with NPH human insulin and insulin lispro were randomized to receive insulin glargine (HOE 901), a long-acting basal insulin analog, once a day (n = 310) or NPH human insulin (n = 309) as basal treatment with continued bolus insulin lispro for 16 weeks in an open-label study NPH insulin patients maintained their prior schedule of administration once or twice a day, whereas insulin glargine patients received basal insulin once a day at bedtime. RESULTS: Compared with all NPH insulin patients, insulin glargine patients had significant decreases in fasting blood glucose measured at home (means +/- SEM, -42.0 +/- 4.7 vs. -12.4 +/- 4.7 mg/dl [-2.33 +/- 0.26 vs. -0.69 +/- 0.26 mmol/l]; P = 0.0001). These differences were evident early and persisted throughout the study More patients in the insulin glargine group (29.6%) than in the NPH group (16.8%) reached a target fasting blood glucose of 119 mg/dl (< 6.6 mmol/l). However, there were no differences between the groups with respect to change in GHb. Insulin glargine treatment was also associated with a significant decrease in the variability of fasting blood glucose values (P = 0.0124). No differences in the occurrence of symptomatic hypoglycemia, including nocturnal hypoglycemia, were observed. Overall, adverse events were similar in the two treatment groups with the exception of injection site pain, which was more common in the insulin glargine group (6.1%) than in the NPH group (0.3%). Weight gain was 0.12 kg in insulin glargine patients and 0.54 kg in NPH insulin patients (P = 0.034). CONCLUSIONS: Basal insulin therapy with insulin glargine once a day appears to be as safe and at least as effective as using NPH insulin once or twice a day in maintaining glycemic control in patients with type 1 diabetes receiving basal-bolus insulin treatment with insulin lispro.

Long-term comparison of losartan and enalapril on kidney function in hypertensive type 2 diabetics with early nephropathy.

BACKGROUND: The objectives of this study were to compare the effects of the angiotensin II receptor blocker, losartan, to those of the angiotensin-converting enzyme inhibitor, enalapril, on albuminuria and renal function in relationship to clinic and ambulatory blood pressure (ABP) in hypertensive type 2 diabetic subjects with early nephropathy. The tolerability of these agents and their effect on the metabolic profile were also evaluated. METHODS: The study was a one-year prospective, double-blind trial with losartan and enalapril administered alone or in combination with hydrochlorothiazide and other antihypertensive agents. ABP and renal and biochemical parameters were measured at baseline and after 12, 28, and 52 weeks of active treatment. Ninety-two hypertensive type 2 diabetics with early nephropathy completed the study. RESULTS: Both losartan and enalapril administered alone or in combination with other agents induced significant reductions in sitting clinic (P < 0.05) and ABP (P < 0.002) without a statistical difference between groups. Geometric means for urinary albumin excretion (UAE) decreased significantly (P < 0.001) in patients treated with losartan from 64. 1 to 41.5 microg/min and in those treated with enalapril from 73.9 to 33.5 microg/min after 52 weeks of therapy. A significant relationship (P < 0.05) between changes in systolic and diastolic ABP and the decrease in UAE at 52 weeks was seen in both groups. The decline in glomerular filtration rate (GFR) was stabilized at the end of therapy and was identical in both treatment groups. Treatment with enalapril was associated with a significantly higher incidence of cough (P = 0.006) and a rise in serum uric acid (P = 0.002) compared with losartan. CONCLUSIONS: Our results indicate that a one-year course of antihypertensive therapy with either losartan or enalapril significantly reduces UAE in hypertensive type 2 diabetic patients with early nephropathy. The reduction in UAE with each treatment is similarly related to decrements in ABP. In addition, the rate of decline in GFR is similar in both treatment groups.

Time course of transforming growth factor-beta(1) (TGF-beta(1)) mRNA expression in the host reaction to alginate-poly-L-lysine microcapsules following implantations into rat epididymal fat pads.

Microencapsulation of islets of Langerhans within semipermeable membranes has been proposed to prevent their immune destruction after transplantation. However, the successful application of this method is impaired by a pericapsular reaction, which eventually induces graft failure. Our goal is to study the role of cytokines in the pathogenesis of this reaction, using the model of alginate-poly-L-lysine microcapsule implantation into Wistar rat epididymal fat pads (EFP). The specific objective of this study was to determine the time course of transforming growth factor (TGF)-beta(1) mRNA expression by semi-quantitative reverse transcriptase-polymerase chain reaction. Microcapsules induced an increase of TGF-beta(1) mRNA expression that reached a maximum 14 days after implantation. Seven, 14, 30, and 60 days after microcapsule implantation, the expression of TGF-beta(1) mRNA was significantly higher in pericapsular infiltrate cells than in nonimplanted EFP cells (p<0.05, p<0.0001, p<0.005, and p<0.01, respectively). Injection of physiological saline induced a small and gradual augmentation of TGF-beta(1) mRNA expression with a maximum 30 days after injection (p<0.01 vs. nonimplanted EFP cells). These results demonstrated that microcapsule implantation, in comparison with saline injection, induce an early, extended, and amplified TGF-beta(1) mRNA expression. This suggests that TGF-beta(1) plays a role in the pathogenesis of the pericapsular host reaction.

Prevalence and determinants of microalbuminuria in high-risk diabetic and nondiabetic patients in the Heart Outcomes Prevention Evaluation Study. The HOPE Study Investigators.

OBJECTIVE: To describe the characteristics of diabetic and nondiabetic participants in the Heart Outcomes Prevention Evaluation (HOPE) Study who are at high risk of developing cardiovascular (CV) disease and who have microalbuminuria (MA), and to identify the key determinants of MA in these two groups. RESEARCH DESIGN AND METHODS: Albuminuria was measured in 97% of patients enrolled in the HOPE Study as part of the MICRO-HOPE (MA, CV, and Renal Outcomes in HOPE) substudy. Baseline clinical characteristics of diabetic and nondiabetic participants with MA were recorded, and the univariate and multivariate relationship between these characteristics and the presence of MA was estimated for both groups. RESULTS: Baseline urinary albumin determinations were available in 3,574 (97.8%) diabetic participants and 5,708 (97.0%) nondiabetic participants. MA was detected in 1,151 (32.2%) diabetic participants and 837 (14.7%) nondiabetic participants. Age, waist-to-hip ratio, diabetes, smoking, hypertension, vascular disease, and left ventricular hypertrophy were independent determinants of MA in all participants. In diabetic participants, the odds of MA increased 16% for every 10.4 years of diabetes duration, and increased 8% for every 0.9% increase in glycated hemoglobin (assuming a GHb assay with an upper limit of 6% in the nondiabetic range). CONCLUSIONS: MA is independently associated with several risk factors for CV and renal disease in both diabetic and nondiabetic individuals at high risk for CV disease.

Improved postprandial glycemic control with Humalog Mix75/25 after a standard test meal in patients with type 2 diabetes mellitus.

OBJECTIVE: This double-blind study was designed to compare the postprandial glucodynamic profile of Humalog Mix75/25, a new premixed insulin analogue containing 75% neutral protamine lispro and 25% insulin lispro with that of human insulin 70/30 (70% neutral protamine Hagedorn insulin and 30% regular human insulin) in patients with type 2 diabetes mellitus. BACKGROUND: Insulin lispro Mix75/25 (Mix75/25) is the first available insulin formulation in which both the rapid-acting and basal components are insulin analogues. METHODS: This randomized, multicenter, double-blind, crossover study monitored patients' postprandial glucodynamic response to Mix75/25 and human insulin 70/30 (70/30) after a standard test meal. Eighty-four patients with type 2 diabetes participated in this study and were randomly assigned to 1 of 2 treatment sequence groups. Patients received an identical test meal on 4 occasions, completing 2 test meals for each treatment. Equal doses of Mix75/25 or 70/30 were administered 5 minutes before each of the 2 test meals, with doses individualized for each patient. Blood samples were collected for 4 hours after the meal for measurement of plasma glucose. From these plasma glucose measurements, fasting plasma glucose, 2-hour postprandial glucose (2pp), 2-hour postprandial glucose excursion (2pp(ex)), maximum glucose excursion (Gex(max)), the area under the glucose concentration versus time curve from 0 to 4 hours (AUC4), and the area under the glucose excursion versus time curve from 0 to 4 hours (AUCex4) were calculated. RESULTS: Because of significant differences in the baseline fasting plasma glucose levels between Mix75/25 and 70/30 (Mix75/25: 8.9+/-2.2 mmol/L [160.2+/-39.6 mg/dL]; 70/30: 8.6+/-1.9 mmol/L [154+/-34 mg/dL), analyses of the excursion parameters provide a truer comparison of the glucodynamic response between insulin formulations. Mix75/25 resulted in significantly lower values for 2pp(ex) (3.35+/-2.28 vs 4.13+/-2.26 mmol/L), Gex(max) (4.51+/-1.88 vs 5.19+/-1.98 mmol/L), and AUCex4 (8.01+/-7.02 vs 10.6+/-6.47 mmol x h/L) compared with 70/30. CONCLUSIONS: In patients with type 2 diabetes mellitus, premeal injection of Mix75/25 resulted in better postprandial glycemic control than did premeal injection of 70/30 in the 4 hours after a standard meal. Mix75/25 is a valuable option for managing postprandial blood glucose in patients with type 2 diabetes mellitus who require insulin.

Studies on small (<350 microm) alginate-poly-L-lysine microcapsules. V. Determination of carbohydrate and protein permeation through microcapsules by reverse-size exclusion chromatography.

Membrane molecular weight (MW) cut-off is a critical factor for immunoprotection of transplanted microencapsulated cells as well as for graft survival. Our goal was to study dextran and protein permeation through small (<350 microm in diameter) alginate-poly-L-lysine microcapsules made with an electrostatic system. Microcapsules were packed into a column, and gel-sieving chromatography was performed with proteins and dextrans of known MW. The objectives of this study were (1) to validate this approach for the assessment of the MW cut-off of <350 microm-in-diameter microcapsules and (2) to evaluate the effect on MW cut-off of changes in experimental conditions. Elution profiles of proteins suggest that the MW cut-off of our small microcapsules lies between 14,500 and 44,000 Da whereas dextrans > or =19,000 Da were excluded. The increase in poly-L-lysine (PLL) concentration from 0.02 to 0.08% reduced the MW cut-off. Increasing the PLL MW from 11.6 to 69.6 kDa induced no change in the MW cut-off. The results also show that the method can be used to discriminate between adsorption and absorption and that insulin diffuses freely across the microcapsule membrane. This method will be useful in establishing the ideal MW cut-off, in optimizing microcapsule characteristics, and in performing routine quality controls.

Humalog Mix25 offers better mealtime glycemic control in patients with type 1 or type 2 diabetes.

To compare the postprandial glucodynamics of Humalog Mix25, (Humalog Mix75/25 in the US; Mix25), to human insulin 30/70 (Humulin 70/30 in the US; 30/70) in patients with type 1 or type 2 diabetes. Ninety-three patients with type 1 diabetes and 84 patients with type 2 diabetes were evaluated in two separate but identical protocols using a randomized, multicenter, double-blind, crossover design. Patients consumed test meals 5 minutes after equal doses of Mix25 or 30/70. Plasma glucose was measured at baseline and 15 minute intervals for 4 hours after the meal. Two-hour postprandial glucose (2pp), 2-hour glucose excursion (2pp(ex) ), glucose versus time area under the curve 0 to 4 hours (AUC(0-4) ) and glucose excursion area under the curve 0 to 2 and 0 to 4 hours (AUCex(0-2), AUCex(0-4) ) were calculated. For the combined patient population, Mix25 resulted in significantly lower 2pp (12.45 +/- 3.59 vs. 13.47 +/- 3.62 mmol/L; p <0.001), AUC(0-4) (44.45 +/- 12.20 vs. 47.25 +/- 11.97 mmol x h/L; p <0.001), and glucose excursion parameters: 2pp(ex) (3.20 +/- 2.72 vs. 4.40 +/- 2.81 mmol/L; p <0.001), AUCex(0-2) (5.45 +/- 3.15 vs 6.60 +/- 3.13 mmol x h/L; p <0.001), and AUCex(0-4) (7.57 +/- 8.37 vs. 11.02 +/- 8.47 mmol x h/L; p <0.001) compared to 30/70. Further analysis of the treatment by type of diabetes indicated that Mix25 provided nearly identical glucose excursion responses in type 1 and type 2 diabetes up to 2 hours after the test meal, in contrast to 30/70. Pre-meal injection of Mix25 resulted in lower postprandial blood glucose levels compared to 30/70. The postprandial blood glucose response following Mix25 was similar in patients with either type 1 or type 2 diabetes.

Studies on smaller (approximately 315 microM) microcapsules: IV. Feasibility and safety of intrahepatic implantations of small alginate poly-L-lysine microcapsules.

UNLABELLED: The most successful transplantation site of nonencapsulated islets of Langerhans is the liver. Because usual alginate poly-L-lysine microcapsules were too large (700-1200 microm diameter) for intravascular implantations and were almost exclusively implanted intraperitoneally, the question of the preferred implantation site of microencapsulated islets has received little attention. The feasibility of implanting smaller (approximately 315 microm) alginate poly-L-lysine microcapsules into the liver and the effect of such implantations on portal pressure and liver histology was evaluated in Wistar rats. A bolus of 10,000 microcapsules of 315 microm diameter was injected intraportally (group 1; n = 22). The portal pressure increased from 6.4 +/- 1.8 mmHg to a maximum of 19 mmHg, returned to basal levels within 2 h, and remained normal after 2 months. In group 2 (n = 3), following the injection of 10,000 larger microcapsules (420 microm), the portal pressure increased to > 60 mmHg and two out of the three rats died within 3 h. When 5,000 microcapsules of 420-microm diameter were injected (group 3; n = 5), the portal pressure peaked to 30 +/- 8 mmHg and remained elevated after 4 h (12 +/- 3 mmHg), but returned to normal (8 +/- 1 mmHg) after 2 weeks. Histological studies showed normal hepatic architecture without collagen deposition into portal tracts occupied by microcapsules. CONCLUSION: intrahepatic implantations of approximately 315-microm alginate poly-L-lysine microcapsules are feasible and safe. These results justify further investigation of this potential implantation site for microencapsulated islets.

Conserved cAMP responsive element and core promoter complex are critical for specificity of the distal T-cell receptor beta chain enhancer for its native promoter.

The Vbeta 8.1 promoter is regulated by T-cell-specific and ubiquitous transcription factors, which bind immediately upstream of and inside the core promoter region. The various Vbeta promoters contain two conserved elements, a cAMP responsive element (CRE) located upstream of the core promoter and a basal initiator flanked by two regulatory motifs. Here we have studied the interplay between the distal enhancer and its native promoter. We show that the remote enhancer acts specifically through its native promoter. Specific enhancer-promoter interplay is mediated through the conserved regions of the Vbeta promoters. Importantly, the conserved CRE serves as a functional recognition element for the enhancer whereas it barely contributes to promoter activity. The other conserved regions surrounding the initiation site are critical for activators that bind at and function through the core promoter region and thereby regulate both promoter and enhancer activity. The enhancer is highly sensitive to E1A-12S, which represses both general and specific enhancer activities. Enhancer activity and promoter-enhancer specificity is, at least in part, mediated by the coactivators CBP/p300.

Studies on small (<350 microm) alginate-poly-L-lysine microcapsules. III. Biocompatibility Of smaller versus standard microcapsules.

Microencapsulation of islets of Langerhans has been proposed as a means of preventing their immune destruction following transplantation. Microcapsules of diameters <350 microm made with an electrostatic pulse system present many advantages relative to standard microcapsules (700-1500 microm), including smaller total implant volume, better insulin kinetics, better cell oxygenation, and accessibility to new implantation sites. To evaluate their biocompatibility, 200, 1000, 1120, 1340, or 3000 of these smaller microcapsules (<350 microm) or 20 standard microcapsules (1247+/-120 microm) were implanted into rat epididymal fat pads, retrieved after 2 weeks, and evaluated histologically. The average pericapsular reaction increased with the number of small microcapsules implanted (p<0.05; 3000 vs. 200, 3000 vs. 1000, and 1000 vs. 200 microcapsules). At equal volume and alginate content, standard microcapsules caused a more intense fibrosis reaction than smaller microcapsules (p<0.05). In addition, 20 standard microcapsules elicited a stronger pericapsular reaction than 200 and 1000 smaller microcapsules (p<0.05) although the latter represented a 3.4-fold larger total implant surface exposed. We conclude that microcapsules of diameters <350 microm made with an electrostatic pulse system are more biocompatible than standard microcapsules.

Accelerated proliferative senescence of rat embryo fibroblasts after stable transfection of multiple copies of the c-Myc DNA-binding sequence.

The protooncogene c-myc positively regulates cellular proliferation whereas it exhibits negative effects on both cellular senescence and differentiation. Ectopic overexpression of c-myc in transfection experiments or titration of the c-myc mRNA by antisense oligonucleotides has demonstrated that small changes of the concentration of cellular c-myc mRNA or protein levels can be crucial for these processes. In view of the role of c-Myc as a transcription factor, most of these effects may be mediated via its binding to specific DNA sequences. Here we studied the cellular reactions after manipulating the cellular concentration of c-Myc DNA-binding sites. Multiple copies of the c-Myc-binding sequence GACCACGTGGTC or, alternatively, the control sequence GACCAGCTGGTC that displays only a poor affinity for c-Myc were stably introduced into the genome of rat embryo fibroblasts. Transfection with the c-Myc-binding sequence yielded much lower clone numbers and sizes than transfection with the control sequence. After polyclonal selection and further subcultivation cells transfected with c-Myc-binding sequence exhibited a reduced growth rate and achieved less than two-thirds of the cumulative population doublings before becoming senescent and irreversibly growth arrested compared to the controls. Southern blot analysis demonstrated that 30 binding sequences on average were integrated into the cellular genome. Our results can be interpreted as competition of the ectopically introduced c-Myc-binding sequences with the functional genomic ones and assume that a fairly low number of the latter exist in the normal cellular genome. Hence, only a low copy number of introduced c-Myc-binding sequences is sufficient to cause signs of accelerated proliferative senescence.

Involvement of negative cofactor NC2 in active repression by zinc finger-homeodomain transcription factor AREB6.

The transcription factor AREB6 contains a homeodomain flanked by two clusters of Krüppel type C2H2 zinc fingers. AREB6 binds to the E-box consensus sequence, CACCTGT, through either the N- or the C-terminal zinc finger cluster. To gain insights into the molecular mechanism by which AREB6 activates and represses gene expression, we analyzed the domain structure of AREB6 in the context of a heterologous DNA-binding domain by transient-transfection assays. The C-terminal region spanning amino acids 1011 to 1124 was identified as a conventional acidic activation domain. The region containing amino acids 754 to 901, which was identified as a repression domain, consists of 40% hydrophobic amino acids displaying no sequence similarities to other known repression domains. This region repressed transcription in vitro in a HeLa nuclear extract but not in reconstituted transcription systems consisting of transcription factor IID (TFIID), TFIIB, TFIIE, TFIIH/F, and RNA polymerase II. The addition of recombinant negative cofactor NC2 (NC2alpha/DRAP1 and NC2beta/Dr1) to the reconstituted transcription system restored the activity of the AREB6 repression domain. We further demonstrated interactions between the AREB6 repression domain and NC2alpha in yeast two-hybrid assay. Our findings suggest a mechanism of transcriptional repression that is mediated by the general cofactor NC2.