Recently, we reported the generation of single-chain antibodies (SCAs) highly specific for rodent and human β-cells. Our current report describes the generation of a fusion protein of one of these SCAs (SCA B1) with a NF-κB essential modifier (NEMO)-binding domain (NBD) peptide, thereby creating a selective inhibitor of NF-κB activation in β-cells. The SCA B1-NBD fusion protein was cloned in the pIRES-EGFP, expressed in bacteria, and purified by metal affinity chromatography; the newly generated complex was then administered intravenously to rodents and evaluated for its ability to protect β-cells against cytokines in vitro and diabetogenic agents in vivo. First, it was shown clearly that our SCA B1-NBD fusion protein binds highly selective to CD rat β-cells in vivo. Second, we observed that SCA B1-mediated in vivo delivery of the NBD peptide completely blocked IL-1β + IFNγ- and TNFα + IFNγ-mediated induction of NF-κB as well as islet dysfunction in culture. Finally, repeated intravenous injection of SCA B1-NBD prior to multiple low-dose administration of streptozotocin in CD mice not only induced a striking resistance to diabetes development but also preserved β-cell mass. In conclusion, our data show for the first time that a SCA B1-NBD fusion peptide reliably protects β-cells against cytokines in vitro and allows protection from diabetes development in CD mice in vivo.
- fusion protein
- β-cell mass
- nuclear factor-κB essential modifier
pancreatic β-cell mass is markedly reduced in patients with type 1 or type 2 diabetes, most likely because of an increased rate of β-cell apoptosis (1, 17). Therefore, strategies for preserving human β-cell mass are critically needed. Proinflammatory cytokines such as IL-1β and TNFα (both in combination with IFNγ) play an important role in the initial destruction of β-cells, leading to the development of diabetes (4, 11). The cytokine-induced activation of the transcription factor NF-κB is an important cellular signal for initiating the cascade of events culminating in β-cell death, and blocking NF-κB activation protects β-cells against IL-1β + IFNγ- or TNFα + IFNγ-induced apoptosis (11). This observation has been confirmed by clinical trials showing that blockade of IL-1 receptor with anakinra improved islet function in patients with type 2 diabetes (2).
In resting cells, most of the NF-κB complex is bound to two kinases (IκBα and IκBβ), and the cytoplasmatic regulatory protein NF-κB essential modifier (NEMO) prevents the NF-κB complex from its translocation to the nucleus as well as its DNA association. Signals from various stimuli (e.g., cytokines, nitric oxide) induce phosphorylation, ubiquitination, and rapid degradation of IκBs, thereby freeing NF-κB, which in turn enters the nucleus, binds to DNA, and activates transcription. In this context, it has been described that intracellular expression of an IκB mutant (which is nonphosphorylatable and thus unable to be degraded) stops the nuclear translocation of the NF-κB proteins even in the presence of cytokines (7). Moreover, it has been shown that the interaction between NEMO and IκBs (which is required for activation of NF-κB) could be blocked by the NEMO-binding domain (NBD) peptide (10). This observation is supported by the finding that intraperitoneal injection of NBD ameliorates inflammatory responses in two mouse models of acute inflammation (8). In a recently published study, a transduction-fusion peptide (PTD-5-NBD) was infused in situ through the bile duct; after subsequent islet isolation, the authors discovered an improved function and viability of islet cells in vitro (12). However, caveats of this method include the lack of β-cell selectivity as well as its invasiveness, creating considerable uncertainty with respect to the application of this approach to studies on islet transplantation or other emerging fields of research. Therefore, novel transport agents with high specificity for β-cells are needed, allowing for a specific and noninvasive delivery of selected therapeutic agents to islet β-cells in vivo.
We have reported recently the isolation of five single-chain antibodies (SCAs) highly specific for pancreatic β-cells in vivo (14, 15). These SCAs (termed SCA B1 to SCA B5) have features indicating that they allow highly selective in vivo delivery to β-cells, i.e., rapid, specific, and high-volume uptake into the cytoplasm of β-cells coupled with rapid elimination of unbound particles from the circulation and lack of cytotoxicity. The present study describes the generation of a SCA B1-NBD fusion protein and its ability to protect islets against the detrimental effects of cytokines in vitro and diabetogenic agents in vivo.
MATERIALS AND METHODS
Generation of SCA B1-NBD fusion protein.
The NBD peptide (TALDWSWLQTE) was COOH-terminal fused to the SCA B1 with a flexible glycine-serine linker. The SCA B1-NBD construct was cloned in the multiple cloning site of pIRES-EGFP, and the SCA B1-NBD-IRES-EGFP fusion protein was then expressed in BL21 bacteria. With respect to the latter, the fusion protein was transformed into BL21 E. coli cells and plated on LB plates (50 μg/ml kanamycin) at 37°C overnight. A single colony was grown further in LB medium (50 μg/ml kanamycin) at 37°C until the OD600 reached 0.9. Isopropyl β-d-thiogalactoside (1 mM final concentration) was added to induce expression of SCA B1-NBD peptide, and finally the SCA B1-NBD peptide was purified from the supernatant of this culture by metal affinity chromatography (Nunc, Langenselbold, Germany). The purity of the sample was checked by SDS gel electrophoresis and Western blotting.
In vivo biodstribution of SCA B1-NBD fusion protein.
All animal studies were approved by the Landesamt für Naturschutz (No. 50.10.32.08.037; Recklinghausen, Germany). Female nondiabetic CD rats were purchased from Charles River Laboratories (Sulzfeld, Germany). The CD rats received intravenous injections of either 100 μl of phosphate-buffered saline (PBS; n = 5) or SCA B1-NBD (100 μg of protein in 100 μl of PBS; n = 5). CD rats were euthanized 2 h after injection, and organs were harvested and fixed with formalin. Staining of paraffin-embedded rat tissue sections (5 μm) was performed as follows; sections were deparaffinized and subsequently permeabilized by heating in the microwave oven for 10 min in an antigen-unmasking solution (pH 6), followed by a cooling interval of 45 min. Blocking was done at 24°C for 1 h with PBS containing 2% (wt/vol) bovine serum albumin (BSA; Sigma Aldrich, Steinheim, Germany). Primary and secondary antibodies were diluted in PBS with 2% BSA. Primary antibodies were incubated at 4°C overnight, except for anti-insulin and anti-glucagon antibodies, with an incubation period of 1 h (at 37°C). Secondary antibodies were incubated at 24°C for 30 min, using Cy2- and Cy3-conjugated streptavidin reagents. The following primary antibodies and dilutions were used: sheep anti-His-tag, 1:200 (Antikörper-online.de); mouse anti-c Myc, 1:200 (Cell Signaling, Technology, Frankfurt am Main, Germany); guinea pig anti-swine insulin, 1:400 (Dako, Carpinteria, CA); and mouse anti-glucagon, 1:200 (Thermo Scientific, Rockford, IL). Secondary antibodies were biotinylated anti-sheep IgG, 1:100 (Linaris, Wertheim-Bettingen, Germany); Cy3-conjugated goat anti-mouse IgG, 1:200 (Jackson Immuno Research Europe, Suffolk, UK); and Cy3-conjugated goat anti-guinea pig IgG, 1:800 (Jackson Immuno Research Europe). The tertiary reagent was Cy2-conjugated streptavidin, 1:200 (Jackson ImmunoResearch Europe). Tissue slides were analyzed using a Zeiss Axioplan microscope.
Female nondiabetic CD rats were injected intravenously either with 100 μl of PBS (n = 5), SCA B1, or SCA B1-NBD (100 μg of protein in 100 μl of PBS; n = 5). Two hours after injection, islets were isolated as described previously (14). Briefly, CD rats were anesthetized by intraperitoneal pentobarbital administration (60 mg/kg). Afterward, a midline abdominal incision was performed. The pancreas was exposed and injected via the pancreatic duct with Hanks' balanced salt solution (HBSS; Biochrom, Berlin, Germany) containing 0.5 mg/ml collagenase (Serva PanPlus, Heidelberg, Germany). After the animal was euthanized, the pancreatic tissue was surgically removed and incubated at 37°C for 7 min in the collagenase solution. Mechanical disruption of the digested pancreatic tissue was achieved by further incubation in collagenase solution at 37°C for 7 min, interrupted every 80 s by shaking for 15 s. Digestion was stopped by addition of 4°C cold HBSS plus 10% BSA. Islet purification was performed using a discontinuous three-phase Ficoll density gradient (densities: 1.090, 1.077, and 1.040). Subsequently, the islets were cultured in 5% CO2 at 37°C overnight in RPMI 1640 medium (Biochrom) supplemented with 100 mg/dl glucose, 10% BSA, and various antibiotics (100 U/ml penicillin, 100 μg/ml streptomycin), with the latter purchased from Gibco BRL (Paisley, UK).
Assessment of viability, glucose-induced insulin secretion, and frequency of apoptosis of CD rat islets in vitro.
To determine the protective effects of NF-κB inhibition in the presence or absence of cytokines, islet viability, frequency of apoptosis, and glucose-stimulated insulin release were analyzed. For this, in vivo-delivered CD rat islets with SCA B1-NBD, SCA B1, or PBS (with the latter serving as control islets) were incubated in the presence or absence of either IL-1β + IFNγ or TNFα + IFNγ for a period of 24 h. The following cytokine concentrations were used: 2 ng/ml recombinant rat IL-1β (R & D Systems, Minneapolis, MN), 1,000 U/ml recombinant murine TNFα (Innogenetics, Gent, Belgium), and 0.036 μg/ml recombinant rat IFNγ (R & D systems, Abingdon, UK). Subsequently, the cytokine-containing medium was removed, and the CD rat islets were washed twice in RPMI 1640 medium without BSA. For viability assessment, the islets were incubated at 37°C for 30 min in the presence of 16.3 μg/ml Calcein AM (Invitrogen, Karlsruhe, Germany) and 10 μg/ml propidium iodide (Invitrogen, Karlsruhe, Germany), as described previously (9). Subsequently, pictures were taken using a two-photon confocal microscope, and the percentage of viable cells was analyzed by the MetaMorph software package version 4.6r9 (Universal Imaging, Downingtown, PA), scoring green vs. red fluorescence in at least 25–30 CD rat islet cell aggregates.
For insulin secretion studies, 12 CD rat islets were hand-picked and transferred in triplicate into a Millicell PCF culture insert with a membrane pore diameter of 12 μm (Millipore, Schwalbach, Germany). The insert was then transferred in a well of a 24-well culture plate (Peske, Aindling-Arnhofen, Germany), and CD rat islets were incubated in 5% CO2 at 37°C for 1.5 h in RPMI medium with a low glucose concentration (100 mg/dl). Afterward, the RPMI medium was removed and stored before being replaced by RPMI medium with high glucose levels (350 mg/dl). The latter was incubated for another 1.5 h in 5% CO2 at 37°C. Finally, insulin was determined (see below). To normalize insulin secretion, the islet protein content of each culture insert was determined.
Apoptotic cells were detected using the TUNEL (Tdt-mediated dUTP nick-end labeling) technique. Following cytokine treatment under high glucose concentrations (350 mg/dl), CD rat islets were incubated for 15 min with trypsin-EDTA (0.25% trypsin, 1 mM EDTA-4Na in HBSS without Ca2+, and Mg2+ at 37°C), and islet cells were gently dispersed. After washing with PBS, cells were cytospun on poly-12-lysine-coated slides, fixed in 4% methanol-free formaldehyde solution in PBS for 25 min at 4°C, and stored in 70% ethanol at −20°C until detection of apoptotic cells by TUNEL assay. The TUNEL assay was performed according to the manufacturer's instructions (Apoptosis Detection System, Fluorescein; Promega). The fluorescein-12-dUTP-labeled DNA was directly visualized by fluorescence microscopy, with excitation at 520 ± 20 nm, to allow counting of the percentage of apoptotic cells (nuclei with green fluorescence). Cell nuclei were stained with propidium iodide (red fluorescence). Apoptotic and total nuclei were counted, in a blinded fashion, >1,000 cells with two slides per condition and per experiment.
NF-κB p65 transcription factor assay following in vivo delivery of SCA B1-NBD and exposure of CD rat islets to cytokines in vitro. The protective effect of the SCA B1-NBD peptide on NF-κB activity in vitro (after prior cytokine exposure) was examined by comparing isolated islets of CD rats treated with SCA B1-NBD, SCA B1, or PBS. Briefly, CD rat islets derived from each of the three study groups were cultured in the presence or absence of either IL-1β + IFNγ or TNFα + IFNγ for 24 h (see above for the respective concentrations). Afterward, the CD rat islets were washed twice with cold PBS and stored at −20°C until whole cell extracts were prepared. In total, 10 μg of cellular protein was taken from each group and analyzed for p65 binding activity using the Trans-AM NF-κB p65 transcription factor enzyme-linked immunosorbent assay (ELISA) from ActiveMotif (Carlsbad, CA). NF-κB binding activity was measured at 450 nm, and the OD reading was normalized to its protein content.
Multiple low-dose streptozotocin diabetes model and quantification of β-cell mass.
CD mice (age: 6 wk; body weight: 20–25 g) were purchased from Charles River Laboratories. CD mice received intraperitoneal injections (40 mg/kg body wt) of streptozotocin (STZ; Sigma Aldrich, Steinheim, Germany) on 5 consecutive days, with the latter being dissolved in fresh citrate buffer (100 mM citrate, pH 4,5). Ten CD mice were treated on a daily basis with SCA B1-NBD (50 μg of protein in 50 μl of PBS) via tail vein injection 2 h before administration of STZ, whereas control CD mice received either SCA B1 (n = 10) or PBS (n = 10). Blood samples for measurement of blood glucose were always taken from the tail vein (on day 0 before any injection and as stated). Hyperglycemia was defined as a nonfasting blood glucose level >200 mg/dl in three sequential measurements.
On day 28, the mice were euthanized for determination of β-cell mass, as described previously (9). Briefly, the pancreas was removed, weighed, fixed, and embedded, and then three 50-μm longitudinal sections of the pancreas were taken and stained for insulin, and images of each section were captured at a magnification of ×100 (×10 objective) with a Zeiss Axioplan microscope. The insulin-positive and total pancreas area were quantified with Zeiss Axiovision version 4.5 software, and the relative ratio of insulin-positive areas to the entire pancreas area was determined. The β-cell mass was calculated by multiplying the relative ratio by the total weight of the pancreas.
For insulin staining, the sections were deparaffinized and permeabilized (see above) and incubated with the primary guinea pig antibody against insulin (1:400, diluted in PBS with 2% BSA; Dako) at 4°C overnight. Insulin was quantified with the streptavidin-alkaline/phosphatase method (Dako) according to the manufacturers' protocol.
Analysis of pancreatic tissue sections for immune cell infiltrates and β-cell apoptosis.
In CD mice (age: 6 wk; body weight: 20–25 g; Charles River), diabetes was induced by multiple low-dose STZ (MLDS) treatment (see above). The CD mice were treated on a daily basis with either SCA B1-NBD (n = 6), SCA B1 (n = 6), or PBS (n = 6) prior to the STZ injection according to the protocol described above. In this experiment, the CD mice were euthanized 5 days after the last STZ injection, and the pancreas was removed, fixed, embedded, and hematoxylin and eosin stained. Moreover, six control CD mice were euthanized before any injection of STZ (day 0). Pancreatic islet mononuclear cell infiltration was quantified as described previously (7a). Briefly, mononuclear cell infiltration of the pancreatic islets was ranked according to an arbitrary scale (0–3) as follows: rank 0, no lymphocytic infiltration; rank 1, <10% of the islets infiltrated; rank 2, 10–50% of the islets infiltrated; rank 3, >50% of the islets infiltrated. Finally, a mean score for each animal was calculated as follows: total score for all islets examined/total number of islets examined. To blind the analysis, the examiner was unaware of the origin of the pancreatic sections.
To determine the frequencies of apoptosis in β-cells, the total number of β-cells positive for TUNEL were determined in one pancreatic section from the head, body, and tail region and expressed in relation to the total number of β-cells. All islets present in the section were included in this analysis. Apoptosis was determined using the TUNEL method (In Situ Cell Death Detection Kit, Fluorescein; Roche, Mannheim, Germany) according to the manufacturer's protocol. All tissue sections stained for TUNEL were simultaneously stained for insulin.
Glucose concentrations were measured with a glucose oxidase method, using a clinical analyzer from Nova Biomedical (Rödermark, Germany). Rat insulin was quantified in duplicate (with a sample volume of each 10 μl), applying a rat insulin ELISA (Mercodia, Uppsala, Sweden). The detection limit of this assay was ≤1.5 μg/l.
Data are expressed as means ± SE, and parametric comparisons of continuous data were calculated with one-way ANOVA, followed by Tukey's honest significance test to correct for α-error accumulation in the setting of multiple testing. Differences in the appearance of diabetes, comparing the percentage of diabetic mice in each group, were calculated by means of Fisher's exact test. Calculations have been perfomed with custom scripts written in the language S in the statistical environment R for Mac OS X. A P value of <0.05 was considered statistically significant.
To protect β-cells against the detrimental effects of cytokines in vivo, the SCA B1-NBD fusion protein was cloned in the pIRES-EGFP, expressed in BL21 bacteria, and purified by metal affinity chromatography (the amino acid sequence is shown in Fig. 1). Subsequently, the SCA B1-NBD was injected intravenously in nondiabetic CD rats before pancreata were harvested and prepared for immunohistochemical analyses. By these means, we confirmed β-cell-specific accumulation of the SCA B1-NBD peptide in the pancreas (Fig. 2), whereas it was not found in any of the control tissues (liver, spleen, kidney, brain; data not shown). These results are comparable to the in vivo biodistribution of the β-cell-specific SCAs (SCA B1 to SCA B5), which had been reported previously (15, 16).
To determine whether the SCA B1-NBD peptide protects islet cells against the detrimental effects of cytokines in vitro, we administered the newly generated fusion peptide to normal CD rats and isolated islets thereafter. After an overnight culture, the isolated islets were exposed to either IL-1β + IFNγ or TNFα + IFNγ for 24 h before the islet viability was evaluated. We thereby demonstrated that treatment with SCA B1-NBD completely blocked the detrimental effects of cytokines on islet viability. Furthermore, SCA B1-NBD treatment resulted in a significantly increased percentage of viable cells compared with cytokine-exposed islets derived from CD rats treated with either PBS or SCA B1 (Fig. 3A). In this line, the apoptotic cell rates in β-cells of SCA B1-NBD-treated animals cultured in high glucose (350 mg/dl) plus the aforementioned cytokine combinations were comparable with that in β-cells without cytokine exposure. Moreover, the frequency of apoptosis in cytokine-exposed β-cells of SCA B1-NBD-treated animals was significantly lower than in PBS- or SCA B1-treated CD rats (Fig. 3B). To confirm that improved cell survival due to prior treatment with the SCA B1-NBD peptide resulted in preserved islet function, we examined the islet response to a glucose challenge and detected that prior treatment with SCA B1-NBD was able to maintain insulin secretion in islets exposed to cytokines, whereas insulin secretion was completely absent in the control groups (Fig. 3C).
To evaluate whether in vivo delivery of the SCA B1-NBD inhibits NF-κB activation, we analyzed NF-κB binding activity in CD rat islets. For this, a NF-κB p65 transcription factor assay was used. As shown in Fig. 4, NF-κB activity was significantly increased in islets of CD rats treated with either SCA B1 or PBS (after application of IL-1β + IFNγ or TNFα + IFNγ). Of note, exposure to IL-1β + IFNγ resulted in a higher degree of NF-κB activation than incubation with TNFα + IFNγ. However, in CD rat islets treated with the SCA B1-NBD peptide, exposure to either IL-1β + IFNγ or TNFα + IFNγ led to a NF-κB level similar to that of control islets. These results demonstrate that selective in vivo delivery of the NBD peptide into β-cells sufficiently blocked cytokine-mediated induction of NF-κB binding activity.
The level of β-cell protection against in vivo administration of diabetogenic agents was analyzed by treating CD mice with intravenous injections of SCA B1-NBD. For this, the newly generated peptide was administered prior to multiple low-dose STZ-treatment, which was repeated over several days to monitor for diabetes development. When comparing the outcome of SCA B1-NBD-treated CD mice with that of SCA B1- or PBS-treated animals, we observed a striking resistance to diabetes development in the CD mice that received our newly generated fusion protein. In detail, 10 of 10 (100%) of the SCA B1- and eight of 10 (80%) of the PBS-treated CD mice gradually developed diabetes 5 to 10 days after the last injection of STZ, whereas only three of 10 (30%) of the SCA B1-NBD-treated CD mice did so (Fig. 5).
Twenty-eight days after multiple low-dose STZ treatment in CD mice, we analyzed plasma concentrations of glucose and insulin during an intraperitoneal glucose tolerance test. When comparing SCA B1-NBD-treated CD mice to SCA B1- or PBS-treated CD mice, our experiments revealed that the control groups had significantly higher glucose concentrations (both after fasting and postchallenge), whereas their postchallenge insulin levels were significantly lower (data not shown). Moreover, pancreatic β-cell area and pancreatic weight were determined in SCA B1-NBD-treated CD mice and control animals 28 days after the last injection of STZ. The fractional β-cell area was 0.90 ± 0.13% in SCA B1-NBD-treated CD mice (n = 10), whereas the fractional β-cell areas amounted to 0.12 ± 0.03 (n = 10) and 0.53 ± 0.12% (n = 10) in SCA B1- and PBS-treated CD mice, respectively. Thus, β-cell mass was significantly higher in SCA B1-NBD-treated animals with 0.71 ± 0.11 mg compared with 0.11 ± 0.02 mg in SCA B1-treated animals (P < 0.01) and 0.38 ± 0.16 mg in PBS-treated animals (P < 0.05) (Fig. 6B). Of interest, an immunohistochemical analyses of pancreata from CD mice 28 days after the last STZ injection revealed that the SCA B1-NBD fusion protein was still detectable in insulin-producing β-cells (Fig. 6C).
To evaluate the mechanisms of how SCA B1-NBD treatment protect β-cells from STZ-induced β-cell death in CD mice in vivo, we euthanized SCA B1-NBD-, SCA B1-, and PBS-treated CD mice 5 days after the last injection of STZ and analyzed pancreatic tissue sections for immune cell infiltrates and β-cell apoptosis. The results were compared with control CD mice without any STZ injection. By these means, a mononuclear cell infiltrate was detectable in ∼10% of the islets in SCA B1-NBD-treated CD mice, and the level was similar to that of CD mice treated with either PBS or SCA B1 (Fig. 7, A and B). In contrast, in control animals without STZ injection no signs of mononuclear cell infiltration were observed. Interestingly, the apoptosis rate in the β-cells of SCA B1-NBD-treated CD mice was very low and comparable with that of control CD mice, whereas the apoptosis rate in the β-cells of SCA B1- and PBS-treated CD mice was significantly increased (Fig. 7C). Taken together, these results suggest that SCA B1-NBD treatment makes β-cells in vivo resistant to STZ-induced immune mediated β-cell apoptosis.
Here we report the successful generation of a SCA B1-NBD fusion protein that accumulates highly selectively in β-cells in vivo. Importantly, we observed that treatment of rodents with intravenously injected SCA B1-NBD prevents CD rat islet cells from cytokine-induced reduction of viability. Furthermore, treatment with SCA B1-NBD maintains glucose-stimulated insulin secretion of CD rat islet cells in vitro and sufficiently protects CD mice against STZ-induced diabetes in vivo.
The main pathophysiological mechanism that underlies both type 1 and type 2 diabetes is a reduction in pancreatic β-cell mass, which is due mainly to apoptosis or programmed cell death (1, 17). Several studies showed that β-cell-specific activation of the transcription factor NF-κB is critical for this process (6, 11, 12). Thus, one might speculate that β-cell-specific inhibition of NF-κB may be a powerful strategy to prevent β-cell death not only in the early stages of diabetes development but also after islet transplantation. In recent years, several strategies have been used to generate NF-κB inhibitors, which were then applied to islet cells in culture or to rodents in situ (e.g., adenoviral-mediated transfer or in situ transduction). Although some of these approaches were promising with respect to viability and function of pancreatic islets (3, 12), noninvasive techniques for β-cell-specific delivery in vivo have not been described so far. However, it is obvious that invasive techniques are poorly suited as therapeutics, whereas noninvasive strategies would allow for a widespread use.
Antibodies have been used frequently for cell-specific delivery of various agents (e.g., chemotherapeutics) in vivo (3). Therefore, it is reasonable to speculate that antibodies directed against a highly specific β-cell molecule are of clinical value because they represent an appropriate method for in vivo delivery of therapeutic compounds. However, so far, such an approach has not been reported, most likely because of a lack of antibodies suitable for β-cell-specific in vivo transport. Recently, we reported the generation of such antibodies: SCAs with rapid (∼5 min), specific, and high-volume (∼650,000 antibodies/β-cell) intracellular accumulation not only in β-cells of mice and rats in vivo but also in human β-cells in situ (15, 16). Other important characteristics of these SCAs that may facilitate their future therapeutic application are the rapid elimination of unbound particles from the circulation (∼20 min) and the lack of cytotoxicity. Finally, it has been shown that the biodistribution of one of these SCAs (SCA B1) strongly predicts β-cell mass. Taken together, these data indicate that our SCAs are suitable for β-cell-specific transport of therapeutic compounds in vivo.
Here, we report the successful generation of a fusion protein, connecting the SCA B1 mentioned above with a selective inhibitor of NF-κB activation termed NBD peptide. After intravenous injection, our newly generated SCA B1-NBD fusion protein was found to be highly specific for β-cells of rodents in vivo, whereas binding to other cell types was negligible. We also observed that SCA B1-mediated in vivo delivery of the NBD peptide (prior to islet isolation) was able to completely block cytokine-mediated induction of NF-κB in CD rats as well as islet dysfunction in culture. The impressive in vitro data were strengthened by the finding that β-cell-specific SCA B1-mediated in vivo delivery of the NBD peptide induced a remarkable resistance to diabetes development in CD mice receiving multiple low-dose STZ treatment. Twenty-eight days after the last injection of STZ, the SCA B1-NBD-treated CD mice also had a significantly higher β-cell mass than the control groups. In accord with this finding after intraperitoneal glucose injection, the postchallenge plasma concentrations of glucose were significantly lower, with a well-preserved insulin response (data not shown).
As shown previously (7a), MLDS treatment induces insulin deficiency and hyperglycemia in mice, typical of type 1 diabetes. In particular, MLDS lead to β-cell destruction through a direct toxicity of the drug to β-cells and a STZ-induced insulitis. Of note, STZ-induced mononuclear cell infiltration is seen early after STZ treatment is started (7–14 days) and declines rapidly thereafter. However, although the methodology applied in this study of using SCA B1-NBD for in vivo protection of β-cells is novel and preserves β-cell mass, a theoretical concern could be that the protective effect may be related simply to protection of β-cells from direct STZ-induced death since SCA B1-NBD is given prior to STZ. Against this, by analysis of pancreatic tissue sections of CD mice euthanized 5 days after the last STZ administration, we were able to show that SCA B1-NBD treatment did not alter the extent of immune cells infiltrating the islets; however, the rate of β-cell apoptosis was significantly lower compared with PBS- or SCA B1-treated CD mice. Therefore, the data provide strong evidence that SCA B1-mediated in vivo delivery of NBD to pancreatic β-cells protected β-cells from direct STZ-induced death and immune-mediated effects.
In conclusion, we have generated a SCA B1-NBD fusion protein that binds specifically to β-cells in vivo. After in vivo administration, the SCA B1-NBD reliably protects rodent β-cells against a diabetes-inducing immune reaction. Therefore, our novel, noninvasive strategy of SCA-mediated β-cell-specific delivery may represent a future therapeutic option, preventing β-cell death both in the early stages of diabetes development and after islet transplantation.
This work was supported by research grants from the Beta Cell-Biology Consortium (DK-072473 to S. Schneider), the Deutsche Forschungsgemeinschaft (SCHN 702/2-1 to S. Schneider), the European Foundation for the Study of Diabetes (EFSD/MSD to S. Schneider), and the Menarini Projektförderung (to S. Schneider) of the German Diabetes Association.
S. Schneider and R. Schirrmacher are patentees of the SCAs and the SCA B1-NBD fusion protein described. The remaining authors declare that there is no duality of interest associated with this article.
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