SEARCH

SEARCH BY CITATION

Keywords:

  • Apoptosis;
  • islet;
  • isolation;
  • nicotinamide;
  • transplantation;
  • two-layer method

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

We investigated the effects of nicotinamide (NA) supplementation of the processing medium during islet isolation. One hundred and two human pancreata were processed for clinical transplantation after preservation either in the University of Wisconsin (UW) or using the two-layer method (TLM). Pancreata were then divided into four groups and retrospectively analyzed. Group I: UW preservation followed by processing without NA, Group II: UW preservation and processing with NA, Group III: TLM preservation without NA, Group IV: TLM preservation with NA. We observed a significant increase in islet yield in Group II (4343 ± 348 IEQ/g) [mean ± SEM], compared to Group I (2789 ± 348 IEQ/g) (p = 0.005). Similarly, a significant increase in islet yield was observed when NA was used in the processing of organs preserved with TLM (Group IV: 5538 ± 413 vs. Group III: 3500 ± 629; p = 0.02). Furthermore islet yield was higher in Group IV than in Group II (p < 0.05). The percentages of preparations that qualified for transplantation were 25, 47, 45, 69% in Groups I, II, III, IV, respectively. Addition of NA to the processing medium significantly improved islet yields in both the UW and TLM preservation protocols, allowing for a higher percentage of islet preparations to qualify for clinical transplantation.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

The results of clinical islet transplantation have greatly improved due to the introduction of more efficient methods for the separation of islets and more effective immunosuppressive strategies (1–4). Improved islet isolation has been key to the evolution of this procedure (5–8). However, it is still difficult to consistently produce adequate islet numbers for ongoing clinical trials. As a result, sequential or single infusions of islet preparations obtained from multiple donors (generally 2–3) are usually needed to achieve insulin independence (2–4,9).

The isolation procedure consists of a mechanically enhanced enzymatic digestion of the pancreas, which results in dissociation of the islets from the surrounding acinar and ductal tissues. During this process, islets are exposed to a number of insults that may result in cellular damage and functional impairment, which ultimately leads to a reduction of the viable islet mass recovered. We hypothesized that selected reagents may provide desirable and effective cyto-protection for islet cells during processing, therefore increasing islet yield and/or viability.

NA has a demonstrated cyto-protective ability, which could potentially be beneficial for clinical islet transplantation (10–14). Furthermore, it has been recently reported that islets express and synthesize tissue factor (TF), which triggers a detrimental thrombotic reaction after hepatic portal islet infusion (15,16). Other reports have shown that macrophage chemoattractant protein (MCP-1) is also expressed and secreted by islets (17–19). MCP-1 has a potent chemotactic activity for monocytes and MCP-1 levels inversely correlate with the outcome of clinical islet transplantation. NA supplementation of culture medium has been shown to reduce both TF and MCP-1 production (20).

The two-layer method (TLM) of preservation has been suggested to increase oxygen and adenosine triphosphate content in pancreata during preservation, and to maintain cell viability and integrity by supporting active metabolism and sodium/potassium pumping (21–24). Several groups have reported that TLM preservation can lead to increased islet yields (2–4,6,25–27).

In this study, we therefore investigated two strategies for improving the results of clinical islet isolation. Before human islet isolation, we used TLM preservation, and during isolation we added NA to the islet-processing medium. In addition to islet yield, we evaluated the cyto-protective effect of NA against oxidative stress and on TF and MCP-1 production. Our results showed that TLM is a superior method in terms of islet yield, and that addition of NA to the processing medium, regardless of the preservation method, improved islet yields and the efficiency of purification. Moreover, the addition of NA to the processing medium decreased TF and MCP-1 production in a highly significant manner, an observation that may have significant bearing on clinical islet transplantation outcomes.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Human islet isolation

Between March 2001 and July 2004, 102 standardized islet isolations were performed at the Human Islet Cell Processing Facility of the University of Miami DRI Cell Transplant Center. The isolation data were retrospectively analyzed only from donors aged 25–55 with cold ischemic time of less than 12 h. All human islet isolations were performed from pancreata preserved with UW alone or with pre-oxygenated (30 min) two-layer perfluorocarbon/UW solution (PFC/UW, TLM) (27). Following cold preservation, islets were isolated using a modification of the automated method (28) with processing medium without nicotinamide (NA) from March 2001 to February 2002 and with NA (10 mM) from March 2003 to July 2004. Human pancreata were divided into two groups based on the presence or absence of NA in the processing medium (Tables 1 and 2). These two groups were further subdivided based on organ preservation methodology (Tables 3 and 4). In all isolation, Liberase HI (Roche, IN, USA) was used for digestion. Twelve different lots of enzyme were used in Group I, seven in Group II, six in Group III and nine in Group IV. The three same lots of enzyme were used in Group II and IV.

Table 1.  Donor-related variables of human pancreas
GroupGender (F/M)Age (25–55)BMI (kg/m2)Pancreas weight (g)Cold ischemia (h)
Nicotinamide (−)23/2441.0 ± 1.327.8 ± 1.6108 ± 3.78.4 ± 0.3
Nicotinamide (+)19/3641.9 ± 1.029.0 ± 1.1110 ± 3.08.1 ± 0.3
Table 2.  Isolation-related variables
GroupUndigested (g)Tissue volume (mL)Pre-purification yields (IEQ)Post-purification yields (IEQ)
Nicotinamide (−)21.7 ± 1.743.1 ± 2.7424 021 ± 35 643245 214 ± 25 197
Nicotinamide (+)28.3 ± 1.945.0 ± 2.1532 102 ± 27 840405 098 ± 22 806
Table 3.  Donor-related variables of human pancreas
GroupGender (F/M)Age (26–55)BMI (kg/m2)Pancreas weight (g)Cold ischemia (h)
TotalTLM
  1. UW = University of Wisconsin; TLM = two-layer method; NA = nicotinamide.

Group I (n = 36)
 UW + NA(−)20/1639.4 ± 1.528.6 ± 1.7108 ± 4.38.3 ± 0.4NA
Group II (n = 19)
 UW + NA(+)6/1340.7 ± 1.729.8 ± 1.3108 ± 4.48.6 ± 0.6NA
Group III (n = 11)
 TLM + NA(−)3/846.2 ± 2.525.0 ± 1.0108 ± 7.68.5 ± 0.75.6 ± 0.8
Group IV (n = 36)
 TLM + NA(+)12/2442.6 ± 1.128.6 ± 1.2111 ± 4.07.9 ± 0.46.6 ± 0.4
Table 4.  Isolation-related variables
GroupUndigested tissue (g)Tissue volume (mL)Pre-purification yields (IEQ)Post-purification yields (IEQ)
  1. UW = University of Wisconsin; TLM = two-layer method; NA = nicotinamide.

Group I (n = 36)
 UW + NA(−)22.0 ± 2.242.9 ± 2.8389 129 ± 26 939226 718 ± 37 804
Group II (n = 19)
 UW + NA(+)23.6 ± 2.440.7 ± 4.0506 190 ± 49 817364 293 ± 31 672
Group III (n = 11)
 TLM + NA(−)20.6 ± 2.046.2 ± 2.5538 214 ± 83 047305 744 ± 60 728
Group IV (n = 36)
 TLM + NA(+)30.8 ± 2.542.6 ± 1.1545 778 ± 33 734426 634 ± 30 255

Clinical assessment of graft function after islet transplantation

Graft function was monitored using exogenous insulin dose, basal C-peptide and HbA1c at about one month after the first infusion.

Fluorescein diacetate-propidium iodide viability staining

Islet preparations were assessed for islet cell viability using cell membrane exclusion dyes as described previously (8,29). Briefly, a small aliquot of islets was obtained at the end of each isolation procedure, and transferred in phosphate buffered saline (PBS) to a 10 × 35 mm counting Petri dish. Fluorescein diacetate (FDA) and propidium iodide (PI) stock solutions were added to the sample at a final concentration of 0.67 μM and 75 μM, respectively. Using a fluorescent microscope, 50 islets were then assessed for cell viability by estimating the percentage of viable cells (green) versus the percentage of nonviable cells (red) within each islet. The mean and standard deviation of viable cells were then calculated for each preparation. The criterion utilized for the final product release for transplantation was minimal viability >70%.

Static glucose challenge

To determine the in vitro potency of isolated islets, a static glucose challenge was performed as described previously (8). Briefly, after overnight culture, islets (50-100 IE) were incubated with either 2.8 mM or 20 mM glucose in culture medium for 2 h at 37°C. The supernatant was collected and stored at −80°C for insulin assessment by ELISA (Alpco, Salem, NH, USA). The glucose-stimulated insulin release was expressed as stimulation index, calculated as the ratio of insulin released during exposure to high glucose (20 mM) over the insulin concentration released during low-glucose incubation (2.8 mM).

In vitro assessment of the cyto-protective effect of NA against oxidative stress

Human islets were cultured in Miami-defined medium 1 (MM1) without or with 10 mM of NA. To induce cellular damage, an 18-h incubation in either 50 or 200 μM hydrogen peroxide (H2O2; Sigma) was performed during islets cultured in MM1 without or with 10 mM of NA. Islets were dissociated into single cells by incubation with 1 mL of accutase solution (Innovative Cell Technologies, Inc, San Diego, CA, USA) at 37°C for 10 min. For the assessment of islet cell viability, single islet cell suspensions were incubated with 1 μM Newport Green PDF (NG, Molecular Probes) to preferentially label beta-cells and 100 ng/mL of tetramethylrhodamine ethyl ester (TMRE, Molecular Probes) to assess mitochondrial membrane potential (apoptosis) for 60 min at 37°C in PBS without Ca2+ and Mg2+. After washing, cells were stained with 7-Aminoactinomycin D (7-AAD, Molecular Probes), which binds to DNA when cell membrane permeability is altered after cell death. Analysis was performed using CellQuest software on an FACS Calibur flow cytometer, as previously described (Becton Dickinson & Co, Mountain View, CA, USA) (30).

In addition to the pancreata used for transplantation, five research pancreata were also processed using medium with or without NA (10 mM). After digestion, the tissue from each individual organ was split into two comparable aliquots and collected separately in processing medium with or without NA. Two continuous gradient purifications were simultaneously performed using two computed cell processors (Cobe 2991; COBE Laboratories, Inc., Lakewood, CO, USA) (8,31) in a refrigerated (4°C) cell processing room. Following purification, islets were washed with processing medium with or without NA. Islets obtained from these research pancreata were used in selected experiments described herein (e.g. TF and MCP-1 measurement, perifusion).

Measurement of MCP-1 and TF production

Aliquots of pancreatic islets from the research pancreata (500 IEQ/mL) that were processed with or without NA in the processing medium, were cultured in MM1 with or without NA in a 24-well plate (Costar) for 24 h. Culture supernatants were collected and stored at −80°C. MCP-1 was detected using a conventional colorimetric ELISA kit (Quantikine Immunoassay, R&D Systems, MN, USA). At the end of culture, recovered islets were homogenized, and TF was measured by a conventional colorimetric ELISA kit (Imubind Tissue Factor, American Diagnostica, Greenwich, CT, USA). The TF and MCP-1 measurements were normalized by total protein of islet aliquots.

Dynamic glucose-stimulated insulin release

Additional aliquots from research pancreata processed with or without NA were analyzed for glucose responsiveness by means of a previously described dynamic stimulation assay (8,29). Islets cultured with or without NA for 48 h after isolation were pre-perifused in a chromatography column (Bio-gel Fine 45-90nm; Bio-Rad) with a buffer containing 125 mM NaCl, 5.9 mM KCl, 1.28 mM CaCl2, 1.2 mM MgCl2, 25 mM HEPES, 0.1% bovine serum albumin and 3 mM glucose for 20 min at 37°C. Then islets were perifused in the same buffer for 10 min and then sequentially exposed to glucose 11 mM, 3 mM and 25 mM KCl. Fractions of the perifusate were collected every 1 min. The collected fractions were then measured for human insulin concentrations using a commercially available ELISA kit (Alpco).

Statistical analysis

Statistical analysis were preformed using methods appropropriate to each specific analysis including Student's t-tests for two sample comparisons of independent groups, two-factor analysis of variance (ANOVA) for analyses of NA while adjusting for preservation methods, and Wilcoxon's signed-rank tests for paired comparisons, and chi-squared analysis for tests of proportions with ordinal classifications. Analyses were performed using SAS software (SAS v9.1 Cary, NC.). Tests of significance were assessed using a type I error rate of 0.05, thus comparisons were reported as statistically significant where associated p-values were <0.05.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Islet recovery from human pancreata using processing medium with or without NA

As a preliminary analysis, we examined the role of NA in addition to the processing medium overall, without taking into account, the preservation method (UW only or TLM).

The donor characteristics and isolation-related variables of the assessed human pancreata are shown in Tables 1 and 2. There were no significant differences in age, body mass index (BMI), pancreas weight, tissue volume after digestion and cold ischemic time between the groups.

Figure 1 shows pre- and post-purification islet recovery from human pancreata processed with or without NA. A significant increase in islet recovery from human pancreata processed with NA was observed in both pre- and post-purification counts (pre-purification; 5151 ± 480 islet equivalents [IEQ]/g [mean ± SEM] vs. 6716 ± 388 IEQ/g, p = 0.01, post-purification; 2956 ± 305 vs. 5125 ± 299 IEQ/g, p < 0.0001) in groups without and with NA, respectively.

image

Figure 1. Pre-purification and post-purification islet yields from human pancreata processed by NA(+) or NA(−) processing medium. One hundred and two human pancteata were procured with 10 mM NA(−) (n = 47) or NA(+) (n = 55) processing medium for clinical islet transplantation. Pre-purification islet yields/pancreatic weight (IEQ/g) and post-purification islet yields/pancreas weight from two experimental groups were calculated.

Download figure to PowerPoint

Islet recovery from human pancreata accounting for preservation method by UW solution or by using TLM

The human pancreata were then divided into four groups according to the preservation method (UW vs. TLM) and the presence or absence of NA in the processing medium. The donor characteristics and isolation-related variables from the four groups are shown in Tables 3 and 4. There were no significant differences in age, BMI, pancreas weight, tissue volume after digestion and cold ischemic time between groups. Despite no significant difference in pre-purification counts between the groups, there was a substantial increase in the post-purification count of Group II, preserved with UW and processed using medium containing 10 mM NA, when compared to that of Group I, preserved with UW alone and processed using medium without NA. (GI vs. GII, 2789 ± 348 vs. 4343 ± 311 IEQ/g, p = 0.005) The efficiency of purification, as judged by recovery rate, was improved by the addition of NA in the processing medium (GI vs. GII, 59.5 ± 3.6% vs. 74.4 ± 3.7%, p < 0.01). Similar results were observed when Groups III and IV (TLM without NA vs. TLM with NA) were compared: (3500 ± 629 vs. 5538 ± 413 IEQ/g in the post-purification counts, p < 0.01) (56.6 ± 7.0% vs. 78.8 ± 3.0% in the efficiency of purification, p = 0.001) (Figure 2 A,B).

image

Figure 2. Pre- and post-purification islet recovery (A) and the efficiency of purification (B) from human pancreata preserved by UW or UW/PFC (TLM). (A) To assess the effect of NA addition to processing medium in pancreata preserved with different solutions, these pancreata were divided into four groups and analyzed in pre-purification islet yields/pancreatic weight (IE/g) and post-purification islet yields/pancreas weight (IEQ/g). Group I: NA(−) in processing medium preserved by UW, Group II: NA(+) in processing medium preserved by UW, Group III: NA(−) in processing medium preserved by TLM, Group IV: NA(+) in processing medium preserved by TLM. (B) The efficiency of purification was calculated by post-purification/pre-purification islet yields × 100(%) in each group.

Download figure to PowerPoint

ANOVA results reveal TLM does significantly increase pre-purification yield by an average of 1459IEQ/g (p = 0.02) over preparations receiving UW in these data regardless of NA status. However, there is no significant effect of NA on pre-purification count when adjusting for preservation method. Post-purification yield is significantly affected in these data by both preservation method and application of NA to the processing medium. ANOVA results reveal that TLM significantly increases post-purification yield by an estimated 999 IEQ/g (p = 0.03) over preparations receiving UW in these data after adjusting for NA status. In addition, preparations where NA is applied to the processing medium have an average increase in post-purification yield of 1749 IEQ/g (p < 0.001) over those without NA while adjusting for preservation method.

These results indicate that while the TLM preservation improves islet yields, NA could further increase post-purification islet yield not only through cyto-protective mechanisms during preservation, but also by improving the efficiency of purification.

Islet function and viability were examined by static glucose challenge and FDA-PI viability staining, respectively. No significant difference between the groups was observed (Table 5). Isolation success rate was evaluated in two ways: first, by the percentage transplanted per group and second, by the post-purification counts. Islet isolations were defined as ‘successful’ when greater than 300 000 IEQ were recovered in the post-purification count. It should be noted that some preparations were transplanted, despite failing to meet the cell number requirement of a ‘successful’ isolation simply because they were pooled with another consecutive preparation. This allowed for achieving the mass requirement of greater than 5000 IEQ/kg of recipient body weight. The percentages of transplanted preparations in Groups I, II, III, IV were 25, 47, 45, 69%, respectively as illustrated in Table 5. Isolation success rates based on cell count in Groups I, II, III, IV were 22, 58, 55, 72%, respectively. Chi-squared analysis for a trend in success rates with the inclusion of NA, TLM or both demonstrate an improvement in both percentage of transplanted preparations (p < 0.001) and isolation success rate (p < 0.001). For both outcomes, the success rate is greatest in the presence of both NA and TLM.

Table 5.  Human islet and isolation variables
GroupViability (%)Stimulation indexPreparation transplanted (%)Successful isolation (%)
  1. UW = University of Wisconsin; TLM = two-layer method; NA = nicotinamide.

  2. Chi-squared analysis for a trend in success rates with the inclusion of NA, TLM or both demonstrates an improvement in both percentage of transplanted preparations (p < 0.001) and isolation success rate (p < 0.001). For both outcomes, the success rate is greatest in the presence of both NA and TLM.

Group I (n = 36)
 UW + NA(−)85.5 ± 1.82.2 ± 0.32522
Group II (n = 19)
 UW + NA(+)91.7 ± 1.42.1 ± 0.34758
Group III (n = 11)
 TLM + NA(−)91.6 ± 1.52.3 ± 0.34555
Group IV (n = 36)
 TLM + NA(+)94.4 ± 0.72.0 ± 0.36972

Islet transplantation into patients with type I diabetes

To evaluate function of transplanted islets isolated with or without NA in the processing medium, we selected the preparations used as first infusion (Table 6). The preparations processed with and without NA in the processing medium were transplanted into eight and seven recipients, respectively. The final infused tissue volume in both groups was always <10 mL and all islet infusions resulted in a measurable function after implant. There was no significant difference in terms of transplanted IEQ/kg BW, percentage reduction of exogenous insulin requirement, and HbA1c between groups. However, C-peptide levels in the NA(+) group were significantly higher (NA(−) vs. NA(+), 1.08 ± 0.109 vs. 2.08 ± 0.15 p = 0.0002). These results showed that the addition of NA improved not only isolation outcome but also positively affected cellular function as benchmarked by transplantation outcome.

Table 6.  Characteristics of Tx processed without or with NA-containing medium
GroupTransplanted islets (IEQ/kg) p = 0.27Insulin reduction (IU/kg) p = 0.60HbA1c (ng/mL) p = 0.19C-peptide (IEQ) p = 0.0002
  1. NA = nicotinamide; Tx = transplantation.

 Pre-Tx    Post-TxPre-Tx   Post-TxPre-Tx Post-Tx
NA(−) (n = 7)5933 ± 5600.52 ± 0.02 [RIGHTWARDS ARROW] 0.21 ± 0.027.5 ± 0.4 [RIGHTWARDS ARROW] 6.8 ± 0.3<0.11 [RIGHTWARDS ARROW] 1.08 ± 0.11
Reduction (%) 60.2 ± 4.211.2 ± 4.7 
NA(+) (n = 8)7134 ± 8930.48 ± 0.02 [RIGHTWARDS ARROW] 0.22 ± 0.038.2 ± 0.1 [RIGHTWARDS ARROW] 6.7 ± 0.2<0.11 [RIGHTWARDS ARROW] 2.08 ± 0.15
Reduction (%) 53.9 ± 5.518.5 ± 1.8 

Analysis of beta-cell fractional viability/apoptosis reveals the cyto-protective effect of NA against oxidative stress

NA supplementation of the processing medium increased islet yields independent of the preservation method. In order to try to elucidate the mechanisms by which NA exerts its protective effects, we looked at its role in preventing damage induced by oxidative stress, a mechanism that may indeed be operational during in vitro processing in the isolation procedure (32–34). To mimic oxidative stress, hydrogen peroxide (H2O2) was used at 50 or 200 μM for 18 h. We evaluated necrotic cell (%) by 7-AAD and beta-cell fractional apoptosis by TMRE staining, with a novel islet cell assessment method that we have recently described (30). Figure 3 A,B shows that 10 mM NA resulted in decreasing the percentage of 7-AAD positive islet cell (dead cells) as well as the percentage of apoptotic beta-cells. These results showed that NA protected beta-cells from cell death induced by H2O2.

image

Figure 3. The in vitro analysis in cyto-protective effect of NA against oxidative stress. Islets were incubated with/without 10 mM NA in the presence of H2O2 at 0, 50 or 200 μM for 18 h as oxidative stress. After dispersion of human islets into single cell suspensions, cells were stained with 7-AAD, NG and TMRE. The dead cells were evaluated by 7-AAD+ (A), and further analysis was performed after exclusion. Beta-cells identified by NGbright were then analyzed for relative percentage of apoptotic or nonapoptotic cells by TMRE (B). Data are representative of at least five independent experiments using different human islet preparations.

Download figure to PowerPoint

Changes in TF and MCP-1 production in islets processed using the medium with/without NA

Although the presence of NA in culture medium has been reported to reduce TF and MCP-1 production (15,17), the effect of NA in the processing medium had not yet been examined. Thus as assessed the effect of NA on TF and MCP-1 using the five research pancreata that were also processed using medium with or without NA (10 mM). After digestion, the tissue from each individual organ was split in two comparable aliquots and collected separately in processing medium with or without NA. Paired comparisons were then conducted to assess differences in TF and MCP-1 when NA is applied to the processing medium in comparison to preparations without NA within groups where NA was or was not applied to the culture medium. Since the distributional properties of these measures are not clearly understood, and given the small sample size of n = 5, a Wilcoxon's signed-rank test for paired samples was used to assess differences between groups. By comparing islets isolated using processing medium in the presence of NA, we observed notable differences in both TF and MCP-1 production (Figure 4), although the Wilcoxon's signed-rank test did not reach statistical significance in these data (p = 0.0625). We hypothesize the lack of significance in this analysis is probably due to the small sample size. We additionally illustrate a possible additive effect of the use of NA in both processing and culture medium as shown in Figure 4.

image

Figure 4. Change in TF and MCP-1 production in islets processed using the medium with/without NA. The islets were separately isolated using processing medium without or with NA. Each of those islets (500 IEQ/mL) was cultured with NA(+) or NA(−) medium in 24-well plate for 24 h. Culture supernatants were collected and stored at –80°C. MCP-1 was detected using an ELISA kit (A). The recovered islets were homogenized and tissue factor was measured with an ELISA kit (B). The data shown are representative of at least five similar experiments using human islet preparations from independent donors.

Download figure to PowerPoint

In vitro assessment of islet function

The effect of NA in the processing medium in terms of the dynamics of insulin release under glucose stimulation was assessed by perifusion (Figure 5). There was no significant difference observed between the two experimental groups when comparing the dynamics of insulin release after stimulation with 11-mM glucose and 25-mM KCl. The data showed that addition of NA (10 mM) to the processing medium did not affect islet function in terms of insulin release on a per islet basis.

image

Figure 5. In vitro assessment of islet function. The effect of NA in processing medium in terms of the dynamics of insulin release under stimulation was assessed by perifusion. The islets were separately isolated using processing medium without or with NA. There was no significant difference observed between the two experimental groups when comparing the dynamics of insulin release after stimulation with 11-mM glucose and 25 mM KCl. Data are representative of five experiments using human islet preparations from independent donors.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Islet transplantation has been demonstrated as a potential therapeutic alternative for selected patients with type I diabetes (1–4,35). Under the current human islet transplantation protocols, successful islet transplantation is achieved with islets generally obtained from multiple donors (2–4,8,9). While there have been significant recent improvements in the procurement and preservation of human pancreata and in the isolation technology (1–9,36), it is still difficult to obtain islet yields consistently that meet the minimal requirements of 5000 IEQ/kg of recipient weight for transplantation. In this study, we have shown that addition of NA to the processing medium improves human islet isolation outcomes, particularly yield and efficiency of purification independent of the preservation method. Moreover, the addition of NA to the processing and/or culture medium improved cellular viability during culture and reduced the production of TF and MCP-1.

The process of human islet isolation generates sizable stress to the islets, including the induction of apoptosis and necrosis and the production of pro-inflammatory cytokines and chemokines. Recently, Bottino et al. demonstrated that oxidative stress plays a major role in triggering the death of islets during isolation. The activation of nuclear factor-κB (NF-κB) and poly (ADP-ribose) polymerase (PARP), two of the major pathways responsible for cellular responses to stress, are shown to be up-regulated significantly in pancreatic cells during the isolation procedure (37).

This literature clearly indicates that the use of effective cyto-protective reagents during isolation could potentially increase islet yields and improve islet quality.

NA is well established as a cyto-protective compound that ameliorates injury caused by noxious stimuli such as hydrogen peroxide (11) and combinations of cytokines (12,13). The PARP inhibition induced by NA prevents nicotinamide adenine dinucleotide (NAD) consumption and decrease in cellular adenosine triphosphate (ATP) content, thereby preventing necrosis by inducing a shift from necrosis to apoptosis (38). As a result, cell viability is preserved (11). As shown in Figure 3, our results clearly confirm previously reported observation. When the absolute number of apoptotic cells in 200 μl H2O2 treated islets cultured with or without NA (Figure 3 A,B, bottom figure) based on the percentage of 7-AAD(−) and TMRE(−), 0.51 (living cells) × 0.71 (apoptotic cells) in islets cultured without NA and 0.81 × 0.58 with NA are 0.36 and 0.47. Our recently developed method confirmed that NA reduced necrosis but increases apoptosis against H2O2 stimulation.

Our data demonstrate that the addition of NA to the processing medium significantly increased islet yields and led to increased donor pancreata utilization. There is indeed a statistically significant difference in the gender distribution of islet donors between Group I and all others. There are no data in the literature that links gender distribution to islet yield, and we feel confident that this variable is by no means responsible for the observed differences.

One cause of inefficient human islet purification is the cell aggregation exacerbated by proteolytic enzymes released by damaged acinar cells (39). NA could potentially protect acinar and other cell subsets of the islet preparation (unpublished data). Therefore, the cyto-protective effect of NA might lead to the reduction of proteolytic enzymes released into the processing environment by the damaged acinar cells. The reduced proteolytic enzymes may further improve the efficiency of purification through decreased cell aggregation and superior islet cell quality. The augmentation of final islet yields was caused not only by rescuing islet cells from cell death but also improving the efficiency of purification.

It is now obvious that the TLM for pancreas preservation can significantly improve the success rate of islet isolations utilized for clinical transplantation. Perfluorochemicals have an increased solubility for oxygen and act as an oxygen reservoir. The oxygenating characteristics can resuscitate ischemically damaged tissue. Though substantial investigation has been done in this area, the detailed mechanisms of the preservative ability of perfluorochemicals remain undefined.

Several groups (including us) have reported that the maintenance of tissue ATP levels (22), the attenuation of free-radical-mediated effects and the up-regulation of cyto-protective genes might be beneficial for improving isolation outcome (27,40–42). Our data showed that the addition of NA to the processing medium could improve human islet isolation outcome from pancreata preserved by either UW or TLM and furthermore provide additional benefits through different cyto-protective mechanisms. The encouraging data suggest that addition of cyto-protective reagents such as NA into preservation solution may be another target for improving islet isolation outcome, leading the development of pancreas preservation solution specific for islet isolation.

One of the limitations in this study was the difficulty in evaluating the quality of transplanted islets in patients with type I diabetes. One cause of this is the variation in the numbers of islet transplanted into recipients. Another is that some recipients required a second infusion in order to achieve insulin independence. To assess the quality of the post-transplantation islet, we selected the preparations transplanted into patients with type I diabetes as the first infusion, as mentioned above, and compared C-peptide levels, post-transplant insulin requirements and HbA1c levels pre- and post-transplantation between the NA(−) and NA(+) groups. While C-peptide level in NA(+) group was significantly improved, other parameters did not show significant difference. In fact, the transplanted IEQ/kg in NA(+) group were slightly higher (not significantly, p = 0.27) when compared to NA(−) group. We could not exclude that the difference of transplanted islet mass between groups might somehow skew the clinical outcome shown in this study.

While it is apparent that islet quality and yield are critical factors in clinical islet transplantation, other factors such as MCP-1 and TF production are also recent areas of focus and procedural modifications.

Piemonti et al. reported that MCP-1 secreted by islet preparations plays a relevant role in the clinical outcome of islet transplantation in patients with type I diabetes. They showed that low MCP-1 secretion from islet preparation resulted in long-lasting insulin independence (17,18). Moberg et al. (15,20) reported that islet-produced TF triggers detrimental thrombotic reaction at islet infusion. She also demonstrated that the addition of NA to the culture medium could reduce TF and MCP-1 production from islet preparation.

NA is thought to interfere with nuclear factor-κB activation, a trigger for the production of pro-inflammatory cytokines and for the secretion of TF and MCP-1 (43,44). NF-κB activation is a key cause of apoptosis and necrosis initiation during islet isolation, and persists throughout the culture. This, in turn, leads to the production and release of TF and MCP-1 (37). The data shown in this study indicate that the use of NA in the processing medium is at least as efficient at decreasing the production of MCP-1 and TF as its use in the culture medium. Also, there seems to be at least additive effects, when NA is present both in processing and culture media, suggesting the use of prolonged exposure to NA as the ideal strategy.

The effect of NA shown in this study suggests a potential use in clinical islet transplantation not only for improving islet yields but also for the reduction of pro-coagulative activity induced by TF and the relief of the inflammation response to islets caused by MCP-1 although we could not observe significant differences in terms of liver enzyme and coagulation parameters between the groups in this study. The addition of NA in the processing and culture medium could maximize the number of islet preparations utilized for transplantation and furthermore and could improve the clinical outcome of islet transplant in patients with type I diabetes.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

This work was supported in part by NIH-NCRR, GCRC MO1RR16587, NIDDK RO1-DK55347-IU42RR016603, 5R01 DK25802, ICR 5U42RR016603, JDRFI 4-200-946 and 4-2004-361, and the Diabetes Research Institute Foundation. The Authors are grateful to the members of the Human Cell Processing Facility, the Preclinical Cell Processing Laboratory of the Cell Transplant Center, the Clinical Islet Transplant Program, the General Clinical Research Center, the Imaging Core at the Diabetes Research Institute, and the administrative offices at the Diabetes Research Institute, and the Organ Procurement Organizations for invaluable contribution to our research.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  • 1
    Ricordi C, Strom TB. Clinical islet transplantation: advances and immunological challenges. Nat Rev Immunol 2004; 4: 259268.
  • 2
    Shapiro AM, Lakey JR, Ryan EA et al. Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N Engl J Med 2000; 343: 230238.
  • 3
    Hering BJ, Kandaswamy R, Ansite JD et al. Single-donor, marginal-dose islet transplantation in patients with type 1 diabetes. JAMA 2005; 293: 830835.
  • 4
    Froud T, Ricordi C, Baidal DA et al. Islet transplantation in type 1 diabetes mellitus using cultured islets and steroid-free immunosuppression: Miami experience. Am J Transplant 2005; 5: 20372046.
  • 5
    Rose NL, Palcic MM, Helms LM, Lakey JR. Evaluation of Pefabloc as a serine protease inhibitor during human-islet isolation. Transplantation 2003; 75: 462466.
  • 6
    Matsumoto S, Rigley TH, Reems JA, Kuroda Y, Stevens RB. Improved islet yields from Macaca nemestrina and marginal human pancreata after two-layer method preservation and endogenous trypsin inhibition. Am J Transplant 2003; 3: 5363.
  • 7
    Sawada T, Matsumoto I, Nakano M, Kirchhof N, Sutherland DE, Hering BJ. Improved islet yield and function with ductal injection of University of Wisconsin solution before pancreas preservation. Transplantation 2003; 75: 19651969.
  • 8
    Ichii H, Pileggi A, Molano RD et al. Rescue purification maximizes the use of human islet preparations for transplantation. Am J Transplant 2005; 5: 2130.
  • 9
    Markmann JF, Deng S, Huang X et al. Insulin independence following isolated islet transplantation and single islet infusions. Ann Surg 2003; 237: 741750.
  • 10
    Mandrup-Poulsen T, Reimers JI, Andersen HU et al. Nicotinamide treatment in the prevention of insulin-dependent diabetes mellitus. Diabetes Metab Rev 1993; 9: 295309.
  • 11
    Hoorens A, Pipeleers D. Nicotinamide protects human beta cells against chemically-induced necrosis, but not against cytokine-induced apoptosis. Diabetologia 1999; 42: 5559.
  • 12
    Eizirik DL, Sandler S, Welsh N, Bendtzen K, Hellerstrom C. Nicotinamide decreases nitric oxide production and partially protects human pancreatic islets against the suppressive effects of combinations of cytokines. Autoimmunity 1994; 19: 193198.
  • 13
    Andersen HU, Jorgensen KH, Egeberg J, Mandrup-Poulsen T, Nerup J. Nicotinamide prevents interleukin-1 effects on accumulated insulin release and nitric oxide production in rat islets of Langerhans. Diabetes 1994; 43: 770777.
  • 14
    Goto M, Eich TM, Felldin M et al. Refinement of the automated method for human islet isolation and presentation of a closed system for in vitro islet culture. Transplantation 2004; 15; 78: 13671375.
  • 15
    Moberg L, Johansson H, Lukinius A et al. Production of tissue factor by pancreatic islet cells as a trigger of detrimental thrombotic reactions in clinical islet transplantation. Lancet 2002; 360: 20392045.
  • 16
    Johansson H, Lukinius A, Moberg L et al. Tissue factor produced by the endocrine cells of the islets of Langerhans is associated with a negative outcome of clinical islet transplantation. Diabetes 2005; 54: 17551762.
  • 17
    Piemonti L, Leone BE, Nano R et al. Human pancreatic islets produce and secrete MCP-1/CCL2: relevance in human islet transplantation. Diabetes 2002; 51: 55.
  • 18
    Bertuzzi F, Marzorati S, Maffi P et al. Tissue factor and CCL2/monocyte chemoattractant protein-1 released by human islets affect islet engraftment in type 1 diabetic recipients. J Clin Endocrinol Metab 2004; 89: 57245728.
  • 19
    Chen MC, Proost P, Gysemans C, Mathieu C, Eizirik DL. Monocyte chemoattractant protein-1 is expressed in pancreatic islets from prediabetic NOD mice and interleukin-1 beta-exposed human and rat islet cells. Diabetologia 2001; 44: 325332.
  • 20
    Moberg L, Olsson A, Berne C et al. Nicotinamide inhibits tissue factor expression in isolated human pancreatic islets: implications for clinical islet transplantation. Transplantation 2003 Nov 15; 76: 12851288.
  • 21
    Kuroda Y, Kawamura T, Suzuki Y, Fujiwara H, Yamamoto K, Saitoh Y. A new, simple method for cold storage of the pancreas using perfluorochemical. Transplantation 1988; 46: 45760.
  • 22
    Kuroda Y, Fujino Y, Morita A, Tanioka Y, Ku Y, Saitoh Y. The mechanism of action of the two-layer (Euro-Collins' solution/perfluorochemical) cold-storage method in canine pancreas preservation—the effect of 2,4 dinitrophenol on graft viability and adenosine triphosphate tissue concentration. Transplantation 1992; 53: 992994.
  • 23
    Matsumoto S, Kuroda Y, Hamano M et al. Direct evidence of pancreatic tissue oxygenation during preservation by the two-layer method. Transplantation 1996; 62: 16671670.
  • 24
    Tanioka Y, Kuroda Y, Kim Y et al. The effect of ouabain (inhibitor of an ATP-dependent Na+/K+ pump) on the pancreas graft during preservation by the two-layer method. Transplantation 1996; 62: 17301734.
  • 25
    Matsumoto S, Qualley SA, Goel S et al. Effect of the two-layer (University of Wisconsin solution-perfluorochemical plus O2) method of pancreas preservation on human islet isolation, as assessed by the Edmonton Isolation Protocol. Transplantation 2002; 74: 14141419.
  • 26
    Tsujimura T, Kuroda Y, Avila JG et al. Influence of pancreas preservation on human islet isolation outcomes: impact of the two-layer method. Transplantation 2004; 78: 96100.
  • 27
    Ricordi C, Fraker C, Szust J et al. Improved human islet isolation outcome from marginal donors following addition of oxygenated perfluorocarbon to the cold-storage solution. Transplantation 2003; 75: 15241527.
  • 28
    Ricordi C, Lacy PE, Finke EH, Olack BJ, Scharp DW. Automated method for isolation of human pancreatic islets. Diabetes 1988; 37: 413420.
  • 29
    Ranuncoli A, Cautero N, Ricordi C et al. Islet cell transplantation: in vivo and in vitro functional assessment of nonhuman primate pancreatic islets. Cell Transplant 2000; 9: 409414.
  • 30
    Ichii H, Inverardi L, Pileggi A et al. A novel method for the assessment of cellular composition and beta-cell viability in human islet preparations. Am J Transplant 2005; 5: 16351645.
  • 31
    Alejandro R, Strasser S, Zucker PF, Mintz DH. Isolation of pancreatic islets from dogs. Semiautomated purification on albumin gradients. Transplantation 1990; 50: 207210.
  • 32
    LeDoux SP, Hall CR, Forbes PM, Patton NJ, Wilson GL. Mechanisms of nicotinamide and thymidine protection from alloxan and streptozocin toxicity. Diabetes 1988; 37: 10151019.
  • 33
    Hauschildt S, Scheipers P, Bessler WG. Inhibitors of poly (ADP-ribose) polymerase suppress lipopolysaccharide-induced nitrite formation in macrophages. Biochem Biophys Res Commun 1991; 179: 865871.
  • 34
    Mandrup-Poulsen T, Reimers JI, Andersen HU et al. Nicotinamide treatment in the prevention of insulin-dependent diabetes mellitus. Diabetes Metab Rev 1993; 9: 295309.
  • 35
    Ricordi C. Islet transplantation: a brave new world. Diabetes 2003; 52: 15951603.
  • 36
    Linetsky E, Bottino R, Lehmann R, Alejandro R, Inverardi L, Ricordi C. Improved human islet isolation using a new enzyme blend, liberase. Diabetes 1997; 46: 11201123.
  • 37
    Bottino R, Balamurugan AN, Tse H et al. Response of human islets to isolation stress and the effect of antioxidant treatment. Diabetes 2004; 53: 25592568.
  • 38
    Aikin R, Rosenberg L, Paraskevas S, Maysinger D. Inhibition of caspase-mediated PARP-1 cleavage results in increased necrosis in isolated islets of Langerhans. J Mol Med 2004; 82: 389397.
  • 39
    London NJM, James RFL, Bell PRF. Islet purification. In: RicordiC, ed. Pancreatic Islet Cell Transplantation. Austin , TX : Landes, 1992: 113123.
  • 40
    Fujino Y, Suzuki Y, Tsujimura T et al. Possible role of heat shock protein 60 in reducing ischemic-reperfusion injury in canine pancreas grafts after preservation by the two-layer method. Pancreas 2001; 23: 393398.
  • 41
    Heard SO, Puyana JC. The anti-inflammatory effects of perfluorocarbons: let's get physical. Crit Care Med 2000; 28: 12411242.
  • 42
    Bekyarova G, Yankova T, Galunska B. Increased antioxidant capacity, suppression of free radical damage and erythrocyte aggrerability after combined application of alpha-tocopherol and FC-43 perfluorocarbon emulsion in early postburn period in rats. Artif Cells Blood Substit Immobil Biotechnol 1996; 24: 629641.
  • 43
    Mackman N, Brand K, Edgington TS. Lipopolysaccharide-mediated transcriptional activation of the human tissue factor gene in THP-1 monocytic cells requires both activator protein 1 and nuclear factor kappa B binding sites. J Exp Med 1991; 174: 15171526.
  • 44
    Ueda A, Okuda K, Ohno S et al. NF-kappa B and Sp1 regulate transcription of the human monocyte chemoattractant protein-1 gene. J Immunol 1994; 153: 20522063.