Pimitespib in patients with advanced gastrointestinal stromal tumor (CHAPTER-GIST-301): a randomized, double-blind, placebo-controlled phase III trial.

BACKGROUND: Prognosis of advanced gastrointestinal stromal tumors (GIST) refractory to tyrosine kinase inhibitors (TKIs) is poor. This randomized, placebo-controlled, phase III trial evaluated the efficacy and safety of pimitespib, a novel heat shock protein 90 inhibitor, in advanced GIST refractory to standard TKIs. PATIENTS AND METHODS: Patients with histologically confirmed GIST refractory to imatinib, sunitinib, and regorafenib were randomized 2 : 1 to oral pimitespib 160 mg/day or placebo for 5 consecutive days per week in 21-day cycles. Following disease progression by blinded central radiological review (BCRR), cross-over to open-label pimitespib was permitted. The primary endpoint was progression-free survival (PFS) by BCRR in the full analysis set. Secondary endpoints included overall survival (OS) adjusted using the rank-preserving structural failure time (RPSFT) method to reduce the expected confounding impact of cross-over. RESULTS: From 31 October 2018 to 30 April 2020, 86 patients were randomized to pimitespib (n = 58) or placebo (n = 28). Median PFS was 2.8 months [95% confidence interval (CI) 1.6-2.9 months] with pimitespib versus 1.4 months (0.9-1.8 months) with placebo [hazard ratio (HR) 0.51 (95% CI 0.30-0.87); one-sided P = 0.006]. Pimitespib showed an improvement in cross-over-adjusted OS compared with placebo [HR 0.42 (0.21-0.85), one-sided P = 0.007]. Seventeen (60.7%) patients receiving placebo crossed-over to pimitespib; median PFS after cross-over was 2.7 months (95% CI 0.7-4.1 months). The most common (≥30%) treatment-related adverse events (AEs) with pimitespib were diarrhea (74.1%) and decreased appetite (31.0%); the most common (≥10%) grade ≥3 treatment-related AE was diarrhea (13.8%). Treatment-related AEs leading to pimitespib discontinuation occurred in three (5.2%) patients. CONCLUSIONS: Pimitespib significantly improved PFS and cross-over-adjusted OS compared with placebo and had an acceptable safety profile in patients with advanced GIST refractory to standard TKIs.

Application of ultra-high voltage electron microscope tomography to 3D imaging of microtubules in neurites of cultured PC12 cells.

Electron tomography methods using the conventional transmission electron microscope have been widely used to investigate the three-dimensional distribution patterns of various cellular structures including microtubules in neurites. Because the penetrating power of electrons depends on the section thickness and accelerating voltage, conventional TEM, having acceleration voltages up to 200 kV, is limited to sample thicknesses of 0.2 µm or less. In this paper, we show that the ultra-high voltage electron microscope (UHVEM), employing acceleration voltages of higher than 1000 kV (1 MV), allowed distinct reconstruction of the three-dimensional array of microtubules in a 0.7-µm-thick neurite section. The detailed structure of microtubules was more clearly reconstructed from a 0.7-µm-thick section at an accelerating voltage of 1 MV compared with a 1.0 µm section at 2 MV. Furthermore, the entire distribution of each microtubule in a neurite could be reconstructed from serial-section UHVEM tomography. Application of optimised UHVEM tomography will provide new insights, bridging the gap between the structure and function of widely-distributed cellular organelles such as microtubules for neurite outgrowth. LAY DESCRIPTION: An optimal 3D visualisation of microtubule cytoskeleton using ultra-high voltage electron microscopy tomography The ultra-high voltage electron microscope (UHVEM) is able to visualise a micrometre-thick specimen at nanoscale spatial resolution because of the high-energy electron beam penetrating such a specimen. In this study, we determined the optimal conditions necessary for microtubule cytoskeleton imaging within 0.7-µm-thick section using a combination with UHVEM and electron tomography method. Our approach provides excellent 3D information about the complex arrangement of the individual microtubule filaments that make up the microtubule network.

Adjunctive perampanel in partial-onset seizures: Asia-Pacific, randomized phase III study.

OBJECTIVES: To evaluate the efficacy, safety, and tolerability of perampanel, a selective, non-competitive, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist, as an adjunctive treatment for patients with refractory partial-onset seizures (POS) from Asia-Pacific. MATERIALS & METHODS: This multicenter, randomized, double-blind, placebo-controlled trial (ClinicalTrials.gov identifier: NCT01618695) involved patients aged ≥12 years with refractory POS (receiving 1-3 antiepileptic drugs). Patients were randomized (1:1:1:1) to receive once-daily placebo or perampanel 4, 8, or 12 mg over a 6-week titration and 13-week maintenance double-blind period. Enzyme-inducing antiepileptic drugs were equally stratified between groups. The primary efficacy endpoint was percent change in POS frequency per 28 days (double-blind phase vs baseline). Other efficacy endpoints included ≥50% responder rate and seizure freedom. Treatment-emergent adverse events (TEAEs) were also monitored. RESULTS: Of 710 randomized patients, seizure frequency data were available for 704 patients. Median percent changes in POS frequency per 28 days indicated dose-proportional reductions in seizure frequency: -10.8% with placebo and -17.3% (P = .2330), -29.0% (P = .0003), and -38.0% (P < .0001) with perampanel 4, 8, and 12 mg, respectively. In total, 108 (15.3%) patients discontinued treatment; 44 (6.2%) due to TEAEs. TEAEs occurring in ≥5% of patients, and reported at least twice as frequently with perampanel vs placebo, included dizziness and irritability. CONCLUSIONS: Adjunctive perampanel (8 and 12 mg/d) significantly improved seizure control in patients with refractory POS. Safety and tolerability were acceptable at daily doses of perampanel 4-12 mg.

Low-intensity pulsed ultrasound (LIPUS) treatment of cultured chondrocytes stimulates production of CCN family protein 2 (CCN2), a protein involved in the regeneration of articular cartilage: mechanism underlying this stimulation.

OBJECTIVE: CCN family protein 2/connective tissue growth factor (CCN2/CTGF) promotes cartilage regeneration in experimental osteoarthritis (OA) models. However, CCN2 production is very low in articular cartilage. The aim of this study was to investigate whether or not CCN2 was promoted by cultured chondrocytes treated with low-intensity pulsed ultrasound (LIPUS) and to clarify its mechanism. METHODS: Human chondrocytic cell line (HCS)-2/8, rat primary epiphyseal and articular cartilage cells, and Ccn2-deficient chondrocytes that impaired chondrocyte differentiation, were treated with LIPUS for 20 min at 3.0 MHz frequency and 60 mW/cm2 power. Expressions of chondrocyte differentiation marker mRNAs were examined by real-time PCR (RT-PCR) analysis from HCS-2/8 cells and Ccn2-deficient chondrocytes at 30 min and 1 h after LIPUS treatment, respectively. CCN2 production was examined by Western blotting after 5 h of LIPUS treatment. Moreover, Ca2+ influx was measured by using a Fluo-4 probe. RESULTS: The gene expression of chondrocyte differentiation markers and CCN2 production were increased in cultured chondrocytes treated with LIPUS. In addition, Ca2+ influx and phosphorylation of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK)1/2 were increased by LIPUS treatment, and the stability of TRPV4 and BKca channel mRNAs was decreased by siRNA against CCN2. Consistent with those findings, the LIPUS-induced the gene expressions of type II collagen (COL2a1) and Aggrecan (ACAN) observed in wild-type cells were not observed in the Ccn2-deficient chondrocytes. CONCLUSION: These data indicate that chondrocyte differentiation represented by CCN2 production was mediated via MAPK pathways activated by LIPUS-stimulated Ca2+ influx, which in turn was supported by the induced CCN2 molecules in articular chondrocytes.

Comparison of insulin degludec with insulin detemir in type 1 diabetes: a 1-year treat-to-target trial.

The long-term safety and tolerability of insulin degludec (IDeg) was compared with that of insulin detemir (IDet), as basal treatment in participants with type 1 diabetes mellitus (T1DM). In the present multinational, 26-week core + 26-week extension, controlled, open-label, parallel-group trial, adults with T1DM were randomized to IDeg or IDet as basal insulin treatment combined with meal-time bolus insulin aspart. IDeg was administered once daily, whilst IDet was administered once or twice daily depending on patients' glycaemic control. After 1 year, IDeg provided a 33% lower rate of nocturnal hypoglycaemia compared with IDet: estimated rate ratio (IDeg : IDet) 0.67 [95% confidence interval (CI) 0.51; 0.88]; p < 0.05. IDeg improved glycated haemoglobin after 1 year of treatment, similarly to IDet, but IDeg also provided a significantly greater reduction in fasting plasma glucose compared with IDet: estimated difference (IDeg - IDet) -1.11 (95% CI -1.83; -0.40) mmol/l; p < 0.05. The present study confirmed the long-term safety and tolerability profile of IDeg in patients with T1DM. IDeg provided a lower risk of nocturnal confirmed hypoglycaemia than IDet.

Laparoscopic intraoperative navigation surgery for gastric cancer using real-time rendered 3D CT images.

PURPOSE: Recent advances in laparoscopic surgical technology have made it possible to perform advanced high-level surgery, such as lymph node dissection for malignancy. Grasping the anatomy during such procedures is important for a safe operation. We have developed a new image information system that provides three-dimensional (3D) reconstructed CT images synchronized with the motion of the laparoscope. This study assesses this new navigation system. METHODS: Enhanced CT using a custom-made software program can provide 3D angiography images reconstructed as a laparoscopic view. A motion sensor mounted on the laparoscope can detect the direction angle of the laparoscope. The real-time rendered 3D CT images are synchronized with the laparoscopic video images according to the motion of the scope. These 3D CT images are projected on another monitor close to the laparoscopic video monitor. Lymph node dissection can be performed with the help of the real-time navigation system that provides a detailed 3D view of the vasculature. RESULTS: Ten laparoscopic gastrectomies were performed using this navigation system. Real-time intraoperative navigation of the vasculature was available, allowing for an excellent surgical outcome. No complications occurred in this series. CONCLUSION: Our intraoperative navigation system allows for safe laparoscopic gastric lymph node dissection.

Genetic Diversity of mtDNA D-loop Polymorphisms in Laotian Native Fowl Populations.

Here, we studied the genetic diversity of native fowls in Laos by analyzing a mitochondrial DNA (mtDNA) sequence polymorphism. A 546-bp fragment of the mtDNA D-loop region was sequenced in 129 chickens from the areas of Vientiane, Luang Prabang and Pakse. In total, 29 haplotypes were identified and formed five clades. Haplotype diversity and nucleotide diversity of the native fowls in Laos were 0.85536±0.0172 and 0.010158±0.005555, respectively. Although the Laotian native fowls were distributed across five clades, most of them were clustered in two main clades (A and B), which were originated in China. The other haplotypes were contained in clades D, F, and I, which originated from continental southeast Asia. These results suggest that multiple maternal lineages were involved in the origin of domestic chicken in Laos. Moreover, there appear to be at least two maternal lineages, one from China and the other from the southeast Asian continent.

Impaired glycolytic metabolism causes chondrocyte hypertrophy-like changes via promotion of phospho-Smad1/5/8 translocation into nucleus.

OBJECTIVE: Hypertrophy-like changes are often observed in chondrocytes during the development of osteoarthritis (OA). These changes play a crucial part in the OA-associated cartilage degradation and osteophyte formation. However, the pathogenesis leading to such changes is still unknown. In this study, we investigated the mechanism by which these hypertrophy-like changes are induced from the viewpoint of impaired glycolytic metabolism. METHODS: The effect of sodium fluoride (NaF) on glycolytic metabolism of cultured chondrocytes was confirmed by measurement of intracellular adenosine triphosphate (ATP) production. Translocation of phosphorylated Smad1/5/8 to the nucleus was evaluated by subcellular fractionation and Western blotting. Chondrocyte hypertrophy-like changes were investigated by real-time RT-PCR and Western blot analysis of differentiation markers. RESULTS: ATP production was dose-dependently decreased by NaF in the human chondrocytic cell line HCS-2/8. In addition, both chondrocyte proliferation and differentiation were inhibited, whereas cell death was promoted by treatment with NaF. Interestingly, combinational treatment with NaF and lactate enhanced translocation of phospho-Smad1/5/8 to the nucleus, as well as gene expression of ALP, VEGF, COL10a1, and matrix metalloproteinase13 (MMP13), which were the markers of late mature and hypertrophic chondrocytes. Furthermore, the production of type X collagen and activation of MMP9 were also promoted under the same conditions. CONCLUSIONS: These findings suggest that decreased ATP production by NaF promotes hypertrophy-like changes via activation of phospho-Smad1/5/8 in the presence of lactate. Novel metabolic aspects of OA pathogenesis are indicated herein.

The impact of Lactobacillus plantarum TUA1490L supernatant on in vitro rumen methanogenesis and fermentation.

This study investigated the suppression of in vitro rumen methane (CH4) output by the supernatant of Lactobacillus plantarum TUA1490L. Hydrogen peroxide (H2O2) was detected in the supernatant. Although CH4 output was reduced by 72%, the supernatant had an adverse effect on total volatile fatty acid (VFA) concentration.

Esophageal submucosal dissection under steady pressure automatically controlled endoscopy (SPACE): a randomized preclinical trial.

BACKGROUND AND STUDY AIMS: A new overtube system has been developed for steady pressure automatically controlled endoscopy (SPACE) in the gastrointestinal tract. The objectives of this study were to validate the feasibility and safety of SPACE in the esophagus, and to evaluate its potential advantages over conventional (manually insufflating) endoscopy in endoscopic submucosal dissection (ESD). METHODS: This was a multicenter preclinical trial using acute porcine models (n = 20). In Experiment 1 (feasibility/safety study), SPACE was attempted in the esophagus with continuous monitoring of cardiopulmonary parameters and intraluminal pressures in the downstream bowel. Different insufflation pressures were tested to optimize the insufflation condition. Each session was video-recorded and scored by blinded reviewers. In Experiment 2 (randomized trial), esophageal ESD was attempted using either SPACE or conventional endoscopy, and results were compared. RESULTS: In Experiment 1, SPACE was performed safely without intraluminal pressure elevation in the downstream bowel. According to video review, SPACE provided more stable, reproducible, and rapid visualization than conventional endoscopy. The insufflation pressure was optimized at 14 mmHg for esophageal SPACE. In Experiment 2, ESD was completed in all animals. The ESD time was significantly shorter with SPACE compared with conventional endoscopy (1326 vs. 1616 seconds; P = 0.009). Responses to questionnaires showed that 94 % - 100 % of participants considered SPACE to provide improved exposure and more uniform tissue tension than conventional endoscopy. Other data were comparable. CONCLUSIONS: SPACE is feasible, safe, and potentially effective for complicated endoscopic procedures, such as ESD. SPACE improves and standardizes endoscopic exposure and tissue tension. A clinical study is required to further confirm its safety and clinical effectiveness.

Grain size dependent photoresponsivity in GaAs films formed on glass with Ge seed layers.

The strong correlation between grain size and photoresponsivity in polycrystalline GaAs films on glass was experimentally demonstrated using Ge seed layers with a wide range of grain sizes (1‒330 µm). The crystal evaluations using Raman spectroscopy, scanning electron microscopy, electron backscatter diffraction, and transmission electron microscopy revealed that 500-nm-thick GaAs films epitaxially grown from the Ge seed layers at 550 °C inherited the grain boundaries and crystal orientations in Ge. With increasing grain size, the photoresponsivity corresponding to GaAs increased from 0.01 to 3 A W-1 under a bias voltage of 0.3 V. The maximum value approached that of the GaAs film formed simultaneously on a single-crystal Ge wafer, indicating the high potential of the large-grained GaAs film. Knowledge gained from this study will be essential for designing advanced solar cells based on polycrystalline III-V compound semiconductors using inexpensive substrates.

Insulin-like growth factor-binding protein-2 and -3 in gingival crevicular fluid.

BACKGROUND AND OBJECTIVE: Insulin-like growth factor-binding proteins (IGFBPs) are crucial regulators of insulin-like growth factor (IGF). They enhance or inhibit IGF functions, but also exhibit IGF-independent effects. In a previous study, we detected, qualitatively, IGFBP-2 and -3 in gingival crevicular fluid using a cytokine antibody array. Here we extended these results using an ELISA to determine the concentrations of IGFBP-2 and -3 in gingival crevicular fluid. In addition, we explored whether the expression of IGFBP-2 and IGFBP-3 correlates with periodontal disease severity. MATERIAL AND METHODS: Gingival crevicular fluid samples from 92 sites of 12 patients affected with periodontal disease and from 100 sites of 19 healthy volunteers, were collected, divided into two groups and analyzed by ELISA for IGFBP-2 and -3 expression. The potential correlation among probing depth, gingival index and the concentrations of IGFBP-2 and -3 was analyzed. RESULTS: Positive correlations were observed between the concentration of IGFBP-2 and probing depth and gingival index, but not for IGFBP-3. The IGFBP-2 concentrations at bleeding on probing-positive sites and at sites with a probing depth of ≥ 4 mm were higher than at bleeding on probing-negative sites and at sites with a probing depth of ≤ 3 mm. CONCLUSION: These results indicate that IGFBP-2 is a potential novel marker for periodontal disease progression. As IGFBP-2 modulates bone metabolism and cell migration, IGFBP-2 in the gingival crevicular fluid may reflect periodontal disease activity.

Utility of the Eating Assessment Tool-10 (EAT-10) in Evaluating Self-Reported Dysphagia Associated with Oral Frailty in Japanese Community-Dwelling Older People.

OBJECTIVES: The aim of the present study was to verify the associations between dysphagia as screened by the Eating Assessment Tool-10 (EAT-10) and indicators in the 100-mL water swallowing test (WST) or medical history among community-dwelling older people. STUDY DESIGN: A cross-sectional study. SETTING AND PARTICIPANTS: The study participants were 202 community-dwelling older Japanese adults aged ≥65 years. MEASUREMENTS: We investigated the participants' basic attributes, including age, sex, body mass index, medical history (cerebrovascular disease, respiratory disease: chronic obstructive pulmonary disease [COPD], and history of pneumonia within the previous year), and number of prescribed medications. Dysphagia assessment was performed using the EAT-10 and the 100-mL WST as subjective and objective examinations, respectively. The 100-mL WST used four indicators (SC: swallowing capacity, VS: volume per swallow, TS: time per swallow, and choking signs). Patients with and without dysphagia according to the EAT-10 were divided into two groups according to a cutoff score of 3, and the two groups were then compared in terms of their characteristics including medical history and 100-mL WST indicators. A multiple logistic regression model was used to determine whether the indicators of the 100-mL WST or medical history were independently associated with dysphagia in the EAT-10. RESULTS: The multiple logistic regression analysis revealed that dysphagia in the EAT-10 was independently associated with male sex (odds ratio [OR] = 2.78; 95% confidence interval [CI] = 0.98-7.90), COPD (OR = 14.68; 95% CI = 3.14-68.85), and VS and TS in the 100-mL WST (OR = 0.85; 95% CI = 0.80-0.90 and OR = 3.03; 95% CI = 1.78-5.16, respectively). CONCLUSIONS: Our results revealed that the EAT-10 was independently associated with the 100-mL WST and respiratory disease. We propose that swallowing rehabilitation incorporating respiratory training could be effective for older people screened using the EAT-10.

Cytokeratins 18 and 8 are poor prognostic markers in patients with squamous cell carcinoma of the oesophagus.

BACKGROUND: Cytokeratins (CKs) are structural marker proteins specific for epithelial cells. However, recent studies indicate their involvement in cancer progression. METHODS: We evaluated CK18 and its filament partner, CK8 expression, by immunohistochemistry in 210 resected specimens from patients with oesophageal squamous cell carcinoma (OSCC). We also analysed the relationship between their expression and various clinicopathological parameters including prognosis. RESULTS: Neither CK18 nor CK8 was expressed in non-cancerous squamous epithelium whereas proper oesophageal glands expressed both CKs. Ninety (42.9%) tumours were CK18 positive and 85 (40.5%) CK8 positive, and the concordance rate for immunohistochemical classification for CK18 and CK8 was 82.4%. CK18 expression correlated with poorly differentiated tumours, use of neo-adjuvant chemotherapy, and advanced stage. Prognosis of patients with CK18-positive tumours was poorer than that of patients with negative OSCC (P<0.001). A similar trend was noted for CK8 expression. Multivariate analysis identified pT (P=0.020), pN number (P=0.001), and CK18 expression (P=0.004) as independent prognostic factors. CK18 expression in 83 pretreatment biopsy specimens was detected in 47 cases (56.6%) and also correlated with prognosis (P=0.045). CONCLUSION: CK18/CK8 expression correlated with progression of OSCC. The significant correlation with prognosis and stable expression in biopsy specimen suggest usefulness of CK18 in selection of treatment strategies for OSCC.

Fibronectin promotes epithelial migration of cultured rabbit cornea in situ.

We investigated the effect of fibronectin on epithelial migration onto the stroma in cultured rabbit cornea. Rabbit plasma fibronectin was purified by affinity chromatography using gelatin-Sepharose 4B, and its purity was confirmed by SDS polyacrylamide slab gel electrophoresis. Antibody against rabbit plasma fibronectin raised in guinea pigs formed a single precipitin line against rabbit plasma and purified rabbit plasma fibronectin by Ouchterlony double diffusion test. When rabbit cornea was cut into small blocks and cultured in TCM-199 medium alone, corneal epithelial cells began to migrate on the cut edge of the corneal stroma. The addition of purified rabbit plasma fibronectin to the culture medium significantly enhanced epithelial migration. The degree of enhancement depended on the amount of fibronectin added. When guinea pig IgG anti-rabbit plasma fibronectin was added, epithelial migration was significantly inhibited when compared with that in control cultured corneal blocks. The results demonstrate that fibronectin promotes epithelial migration in the cornea and thus plays an important role in corneal wound healing.

Retinal tumor induced in the baboon by human adenovirus 12.

Three of 21 newborn baboons injected intraocularly with human adenovirus type 12 developed an intravitreal mass 12 to 36 months later. Two of the masses were indistinguishable from human retinoblastoma, a retinal tumor that afflicts children. To our knowledge this is the first time a retinoblastoma-like tumor has been induced experimentally by adenovirus type 12 in a nonhuman primate.

Gain-of-function mutations of c-kit in human gastrointestinal stromal tumors.

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors in the human digestive tract, but their molecular etiology and cellular origin are unknown. Sequencing of c-kit complementary DNA, which encodes a proto-oncogenic receptor tyrosine kinase (KIT), from five GISTs revealed mutations in the region between the transmembrane and tyrosine kinase domains. All of the corresponding mutant KIT proteins were constitutively activated without the KIT ligand, stem cell factor (SCF). Stable transfection of the mutant c-kit complementary DNAs induced malignant transformation of Ba/F3 murine lymphoid cells, suggesting that the mutations contribute to tumor development. GISTs may originate from the interstitial cells of Cajal (ICCs) because the development of ICCs is dependent on the SCF-KIT interaction and because, like GISTs, these cells express both KIT and CD34.

Adenovirus-mediated gene expression of the human c-FLIP(L) gene protects pig islets against human CD8(+) cytotoxic T lymphocyte-mediated cytotoxicity.

Cell-mediated immunity, especially of human CD8+ cytotoxic T lymphocytes (CTLs) is believed to have an important role in the long-term survival of pig islet xenografts. Protection against human CD8+ CTL cytotoxicity may reduce the direct damage to pig islets and enable long-term xenograft survival in pig-to-human islet xenotransplantation. We have previously reported that c-FLIP(S/L) genes, which are potent inhibitors of death receptor-mediated proapoptotic signals through binding competition with caspase-8 for recruitment to the Fas-associated via death domain (FADD), markedly suppress human CD8+ CTL-mediated xenocytotoxicity. In addition, the cytoprotective effects of c-FLIP(L) seem to be significantly stronger than those of c-FLIP(S). Accordingly, in the present study, expression of c-FLIP(L) was induced in intact pig islets by adenoviral transduction. Consequently, the cytoprotective capacity of the transgene in pig islets was examined in in vitro and in vivo exposure to human CD8+ CTLs. Cells from untransduced islets or mock islets were sensitive to CD8+ CTL-mediated lysis (59.3% +/- 15.9% and 64.0% +/- 8.9% cytotoxicity, respectively). In contrast, cells from pig islets transduced with the c-FLIP(L) gene were markedly protected from lysis (30.5% +/- 3.5%). Furthermore, prolonged xenograft survival was elicited from pig islets transduced with this molecule as assessed using an islet transplant model using the rat kidney capsule. Thus, these data indicate that intact pig islets can be transduced to express c-FLIP(L) with adenovirus. Pig islets expressing c-FLIP(L) are significantly resistant to human CTL killing and further exhibit beneficial effects to prolong xenograft survival.

In vivo controlling of cellular response to pig islet xenografts by adenovirus-mediated expression of either membrane-bound human FasL or human decoy Fas.

The critical problem with clinical islet transplantation for patients with type 1 diabetes is the severe shortage of human donors. Pig islet xenotransplantation has the potential to provide a virtually unlimited source of donor pancreata. However, our previous studies demonstrated that cell-mediated rejection, especially human CD8(+) cytotoxic T lymphocyte (CTL)-mediated cytotoxicity, remains a major obstacle for long-term islet xenograft survival. Moreover, we have demonstrated that the overexpression of either membrane-bound human FasL (mFasL) or human decoy Fas antigen (decoy Fas) in pig islets not only prevented CTL xenocytotoxicity in vitro, but also prolonged histological survival of pig islet xenografts in vivo. Therefore, the aim of the present study was to determine whether adenoviral transfer of these genes into pig islets ex vivo prior to transplantation had a beneficial effect on posttransplantation glycemic control of diabetic recipients. Isolated pig islets were transfected with adenovirus vector carrying complementary DNA (cDNA) of either mFasL or decoy Fas. The transfected islets were transplanted under the kidney capsule of diabetic recipient rats. Rats transplanted with either mFasL- or decoy Fas-transfected pig islet grafts showed significantly suppressed blood glucose levels from 12 hours to 18 hours posttransplantation compared with control groups transplanted with empty vector-transfected pig islets. Unfortunately, blood glucose levels of these groups were increased, with no significant difference observed at 24 hours posttransplantation. However, transgenic expression of these molecules with clinically tolerable amount of immunosuppressants may be more effective to achieve islet xenograft survival in the future.