Comparative efficacy of treatments for a first-time traumatic anterior shoulder dislocation: a systematic review and network meta-analysis.

BACKGROUND: First-time traumatic anterior shoulder dislocation (FASD) is a common trauma associated with shoulder dysfunction. Although several randomized controlled trials have compared conservative and surgical treatments for FASD, the comparative efficacy of these treatments is poorly understood. In this network meta-analysis (NMA), we compared the available evidence on the efficacy of various interventions in patients with FASD. METHODS: We searched the Cochrane Central Register of Controlled Trials, MEDLINE, and EMBASE databases in March 2023. This NMA included randomized controlled trials comparing conservative and surgical treatments for FASD, including arthroscopic Bankart repair, arthroscopic lavage, external rotation (ER) immobilization, and internal rotation (IR) immobilization. The primary outcomes were redislocation rates, Western Ontario Shoulder Instability Index (WOSI) scores, and adverse events (AEs). We conducted random-effects NMA within the frequentist framework. To rank the treatments, the Surface Under the Cumulative Ranking curve was calculated using a Bayesian framework. We evaluated confidence in each outcome using the CINeMA tool. RESULTS: Of the 2999 reviewed studies, 15 were included and analyzed. Regarding the primary outcomes, arthroscopic Bankart repair likely results in a large reduction in redislocation rates compared to IR immobilization (risk ratio [RR], 0.15; 95% confidence interval [CI], 0.07-0.33). Both arthroscopic lavage (RR, 0.47; 95% CI, 0.20-1.11) and ER immobilization (RR 0.70; 95% CI, 0.50-1.00) may reduce the redislocation rates slightly compared with IR immobilization. According to these results, arthroscopic Bankart repair ranked first in terms of reducing the redislocation rate, followed by arthroscopic lavage, ER and IR immobilization. Regarding the WOSI score, no substantial differences were observed in the WOSI scores among the four treatments. AEs showed that ER immobilization tended to cause greater shoulder stiffness than IR immobilization, and postoperative erythema, swelling, and adhesive capsulitis were observed after arthroscopic Bankart repair and lavage. However, a meta-analysis was not performed because the definitions of AEs differed between the studies. CONCLUSION: Arthroscopic Bankart repair showed a significant effect in reducing the redislocation rate compared to IR immobilization. Although both arthroscopic lavage and ER immobilization seemed to be effective in reducing the redislocation rates, it was not statistically significant. Moreover, these four treatments may result in little to no difference in disease-specific quality of life and there is no clear evidence of AEs.

Incidence of Re-Dislocation/Instability After Arthroscopic Bankart Repair: Analysis via Telephone Interviews.

INTRODUCTION: Current advances in arthroscopic surgery have led to good outcomes for arthroscopic Bankart repair (ABR) for recurrent anterior shoulder dislocation. However, recent studies have reported recurrence rates of 4%-19% after ABR. In our survey conducted from February 2002 to December 2010, the post-ABR re-dislocation rate was 8.8%. In 2011, we began performing the ABR with open Bristow (B) procedure or Remplissage (R) procedure in patients with large glenoid or humeral head bone defects and in patients who play collision sports. Therefore, the present study is the second series evaluating the incidence of re-dislocation and instability after recurrent anterior shoulder dislocation. METHOD: Surgery was performed for 84 cases of shoulder instability from January 2011 to August 2017. After excluding 7 open surgeries, 6 reoperations, and 2 patients with multidirectional instability, telephone interviews were conducted with 69 patients. The average follow-up duration was 46.9 months (range, 13-92 months). RESULT: ABR alone was performed 61 patients; the B procedure was added for 3 patients, and the R procedure was added for 5 patients. Telephone interviews were conducted with 61 patients. There were no cases of re-dislocation or reoperation. Four patients who underwent only ABR experienced postoperative instability, but not to the extent that their daily lives were affected. CONCLUSION: This study showed that the addition of R or B technique to ABR for recurrent anterior shoulder dislocation resulted in a 0% re-dislocation rate.

Midterm Functional and Structural Outcomes of Large/Massive Cuff Tears Treated by Arthroscopic Partial Repair.

BACKGROUND: Previous studies have shown good clinical outcomes in patients with irreparable large or massive rotator cuff tears treated using arthroscopic partial repair (APR); however, few studies have evaluated both functional and structural outcomes in these patients. PURPOSE: To evaluate both functional and structural outcomes in patients with large or massive rotator cuff tears treated using APR. STUDY DESIGN: Case series; Level of evidence, 4. METHODS: Between March 2009 and November 2016, a total of 30 patients underwent APR because of the irreparability of their large or massive rotator cuff tears during surgery. Of these patients, 24 completed the minimum 24-month follow-up (mean, 61.8 ± 27.1 months; range, 24-112 months) and were included in this study. Functional outcome measures included the Japanese Orthopaedic Association (JOA) and University of California Los Angeles (UCLA) scores and the visual analog scale for pain. Structural outcome measures comprised preoperative fatty degeneration, mediolateral tear size, residual tendon attachment area, and glenohumeral (GH) arthritic changes evaluated on magnetic resonance imaging scans or plain radiographs before and after surgery. Functional and structural outcomes were evaluated preoperatively, at 3 months postoperatively, and at the final follow-up. RESULTS: The JOA scores for all patients significantly improved from 67.9 ± 11.3 preoperatively to 85.4 ± 15.6 postoperatively (P < .0001). Similarly, the UCLA scores significantly improved from 15.8 ± 4.20 preoperatively to 29 ± 6.69 at final follow-up postoperatively (P < .0001). The mediolateral tear size were significantly decreased at 3 months postoperatively (P < .001) and at the final follow-up (P < .001). Compared with preoperative scores, the novel score evaluating the residual tendon attachment area improved from 3.08 ± 0.46 to 3.54 ± 0.41 (P < .001) after surgery overall, although it significantly deteriorated from 3 months postoperatively to the final follow-up. GH osteoarthritis progressed in 6 patients (25%). Patients who developed osteoarthritis had lower JOA and UCLA scores than did those who did not (JOA, P = .010; UCLA, P = .037). CONCLUSION: In irreparable large or massive rotator cuff tears treated using APR, functional outcome improved after surgery. Although the residual tendon attachment area improved, functional outcome after APR corresponded to the GH alterations at the midterm follow-up. Longer-term follow-up is needed to further elucidate the effect of APR on clinical outcomes in these patients.

Clinical outcomes of the Cadenat procedure in the treatment of acromioclavicular joint dislocations.

We report our clinical experience using the modified Cadenat method to treat acromioclavicular joint dislocation, and discuss the usefulness of this method. This study examined 6 shoulders in 6 patients (5 males, 1 female) who were diagnosed with acromioclavicular joint dislocation and treated with the modified Cadenat method at our hospital. Average age at onset was 49.3 years (26-78 years), average time interval from injury until surgery was 263.8 days (10 to 1100 days), and the average follow-up period was 21.7 months (12 to 42 months). Post-operative assessment was performed using plain radiographs to determine shoulder joint dislocation rate and Japanese Orthopaedic Association (JOA) score. The average post-operative JOA score was 94.1 points (91 to 100 points). The acromioclavicular joint dislocation rate improved from 148.7% (72 to 236%) before surgery to 28.6% (0 to 60%) after surgery. Conservative treatment has been reported to achieve good outcomes in acromioclavicular joint dislocations. However, many patients also experience chronic pain or a sensation of fatigue upon putting the extremity in an elevated posture, and therefore ensuring the stability of the acromioclavicular joint is crucial for highly active patients. In this study, we treated acromioclavicular joint dislocations by the modified Cadenat method, and were able to achieve favorable outcomes.

Role of the mTOR complex 1 pathway in the in vivo maintenance of the intestinal mucosa by oral intake of amino acids.

AIM: Oral intake of nutrients is often compromised in elderly, multimorbid patients, but parenteral nutrition causes intestinal atrophy and impairs intestinal function. To uncover the molecular mechanisms by which amino acids are involved in intestinal atrophy and recovery, we studied whether the rapamycin-sensitive mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) pathway is involved in this process. METHODS: C57BL/6N mice were fed a glucose solution alone, glucose solution with amino acids or normal chow diet for various lengths of time. Intestinal sections were prepared from these mice and the villus height and villus density were quantified. As a readout for the mTORC1 pathway, the phosphorylation of the ribosomal S6 protein (S6) was analyzed by immunostaining and immunoblotting. To confirm the role of the mTORC1 pathway, the inhibitory effect of a specific mTOR inhibitor, rapamycin, was examined. RESULTS: Inducing fasting in mice fed only glucose caused time-dependent intestinal mucosal atrophy, whereas supplementation with amino acids protected the intestinal mucosa from atrophy. Phosphorylation of S6 decreased in the intestinal mucosa of mice fed only glucose, whereas supplementation with amino acids increased S6 phosphorylation. Importantly, intraperitoneal injection of rapamycin attenuated the protective effect of amino acids on the intestinal mucosa in a pattern consistent with the decrease of S6 phosphorylation. CONCLUSIONS: These results indicate that the mTORC1 pathway plays a crucial role in the in vivo maintenance of the intestinal mucosa by the oral intake of amino acids.

Acceleration of autoimmune diabetes in Rheb-congenic NOD mice with ß-cell-specific mTORC1 activation.

The protein Ras homolog enriched in brain (Rheb) is a Ras-like small GTPase that activates the mechanistic target of rapamycin complex 1 (mTORC1), which promotes cell growth. We previously generated transgenic C57BL/6 mice overexpressing Rheb in ß-cells (B6(Rheb)), which exhibited increased ß-cell size and improved glucose tolerance with higher insulin secretion than wild type C57BL/6 mice. The mice also showed resistance to obesity-induced hyperglycemia, a model of type 2 diabetes, and to multiple low-dose-streptozotocin (MLDS)-induced hyperglycemia, a model of type 1 diabetes (T1D). To investigate whether the effects of mTORC1 activation by Rheb in B6(Rheb) mice would also be evident in NOD mice, a spontaneous autoimmune T1D model, we created two NOD mouse lines overexpressing Rheb in their ß-cells (NOD(Rheb); R3 and R20). We verified Rheb overexpression in ß-cells, the relative activation of mTORC1 and ß-cell enlargement. By 35 weeks of age, diabetes incidence was significantly greater in the R3 line and tended to be greater in the R20 line than in NOD mice. Histological analysis demonstrated that insulitis was significantly accelerated in 12-week-old R3 NOD(Rheb) mice compared with NOD mice. Furthermore, serum insulin autoantibody (IAA) expression was significantly higher than that of NOD mice. We also examined whether complete Freund's adjuvant (CFA) treatment alone or with glucagon-like peptide-1 (GLP-1) analog would reverse the hyperglycemia of NOD(Rheb) mice; unexpectedly, almost none achieved normoglycemia. In summary, diabetes progression was significantly accelerated rather than prevented in NOD(Rheb) mice. Our results suggest that the ß-cell enlargement might merely enhance the autoimmunity of pathogenic T-cells against islets, leading to acceleration of autoimmune diabetes. We conclude that not only enlargement but also regeneration of ß-cells in addition to the prevention of ß-cell destruction will be required for the ideal therapy of autoimmune T1D.

Usefulness of 18F-fluorodeoxyglucose positron emission tomography for diagnosis of asymptomatic giant cell arteritis in a patient with Alzheimer's disease.

It is often difficult to diagnose disease in elderly patients, in particular those with dementia, who do not present with typical symptoms. This report describes our experience of an elderly patient (an 83-year-old woman) who presented with a chief complaint of memory loss, showed a marked inflammatory response, and was diagnosed with large-vessel giant cell arteritis (GCA) on the basis of 18F-fluorodeoxyglucose positron emission tomography (FDG-PET) findings. She had no symptoms typical of GCA including jaw claudication, visual field defect and heavy headed feeling. Corticosteroid therapy resulted in a trend toward improvement in the inflammatory response and then she first recognized that she might have experienced slight dull headache before treatment of GCA. This was probably because this patient had large-vessel GCA, which produces a few symptoms in the head and neck, and because she had Alzheimer's disease and could not accurately describe her symptoms. Our experience suggests the usefulness of FDG-PET for the diagnosis of GCA, particularly in elderly patients without typical symptoms.

Administration of a determinant of preproinsulin can induce regulatory T cells and suppress anti-islet autoimmunity in NOD mice.

Antigen-specific immunotherapy is expected to be an ideal strategy for treating type 1 diabetes (T1D). We investigated the therapeutic efficacy of a peptide in the leader sequence of preproinsulin, which was selected because of its binding affinity to the MHC I-A(g7) molecule. Preproinsulin-1 L7-24 peptide (L7-24) emulsified in Freund's incomplete adjuvant was administered subcutaneously to NOD mice. Administration of L7-24 increased the proportion of regulatory T cells in the spleen. Splenocytes of NOD mice immunized with this peptide secreted IL-4 and IL-10 in response to L7-24. This peptide also significantly prevented the development of diabetes and cured some newly diabetic NOD mice without recurrence. L7-24 peptide, which has a high affinity for pockets of I-A(g7), induced regulatory T cells and showed therapeutic effects. This peptide may provide a new approach for developing antigen-specific immunotherapy for autoimmune diabetes.

Regulatory CD8+ T cells induced by exposure to all-trans retinoic acid and TGF-beta suppress autoimmune diabetes.

Antigen-specific regulatory CD4(+) T cells have been described but there are few reports on regulatory CD8(+) T cells. We generated islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)-specific regulatory CD8(+) T cells from 8.3-NOD transgenic mice. CD8(+) T cells from 8.3-NOD splenocytes were cultured with IGRP, splenic dendritic cells (SpDCs), TGF-beta, and all-trans retinoic acid (ATRA) for 5days. CD8(+) T cells cultured with either IGRP alone or IGRP and SpDCs in the absence of TGF-beta and ATRA had low Foxp3(+) expression (1.7+/-0.9% and 3.2+/-4.5%, respectively). In contrast, CD8(+) T cells induced by exposure to IGRP, SpDCs, TGF-beta, and ATRA showed the highest expression of Foxp3(+) in IGRP-reactive CD8(+) T cells (36.1+/-10.6%), which was approximately 40-fold increase compared with that before induction culture. CD25 expression on CD8(+) T cells cultured with IGRP, SpDCs, TGF-beta, and ATRA was only 7.42%, whereas CD103 expression was greater than 90%. These CD8(+) T cells suppressed the proliferation of diabetogenic CD8(+) T cells from 8.3-NOD splenocytes in vitro and completely prevented diabetes onset in NOD-scid mice in cotransfer experiments with diabetogenic splenocytes from NOD mice in vivo. Here we show that exposure to ATRA and TGF-beta induces CD8(+)Foxp3(+) T cells ex vivo, which suppress diabetogenic T cells in vitro and in vivo.

Enhancement of anti-tumor immunity by high levels of Th1 and Th17 with a combination of dendritic cell fusion hybrids and regulatory T cell depletion in pancreatic cancer.

Regulatory T cells (Tregs) play an important role in immunological self-tolerance and protect the host from autoimmune disease. However, in cancer immunity, Tregs might block anti-tumor immune responses. Therefore, the depletion of Tregs using a specific agent that suppresses its function or population, such as an anti-CD25 antibody, could promote anti-tumor immune responses. In the present study, a cytotoxicity assay, enzyme-linked immunosorbent spot (ELISPOT) assay and measuring cytokine secretion, were used to study the efficacy of Treg depletion by anti-CD25 antibody added to a dendritic cell/tumor cell (DC/TC) fusion hybrid vaccine in a murine pancreatic cancer model. All the mice treated with the combined therapy of fusion hybrid vaccine and Treg depletion rejected tumor growth in a challenging test, although the rejection rate was 20% both for mice that received the fusion hybrids alone or Treg depletion alone. In addition, combined therapy showed a significantly improved survival in comparison to other treatment or control groups. The NK cell activity for DC/TC fusion + Treg depletion was significantly higher than that for the other treatment groups. Cytotoxic T lymphocyte (CTL) activity for DC/TC could potentially be enhanced by the addition of Treg depletion therapy. The treatments including DC/TC fusion induced IFN-gamma secreting effector cells in ELISPOT assays. Furthermore, a cytometric beads array assay used to measure cytokine secretion showed that DC/TC fusion + Treg depletion stimulated the highest levels of IFN-gamma Th1/Th2 ratios and Th17. These results demonstrate that Treg depletion combined with DC/TC fusion hybrid vaccine enhanced the efficacy of immunotherapy in pancreatic cancer by activating CTLs and NK cells.

Upregulation of the mammalian target of rapamycin complex 1 pathway by Ras homolog enriched in brain in pancreatic beta-cells leads to increased beta-cell mass and prevention of hyperglycemia.

OBJECTIVE: Components of insulin/IGF-1 receptor-mediated signaling pathways in pancreatic beta-cells have been implicated in the development of diabetes, in part through the regulation of beta-cell mass in vivo. Studies in vitro have shown that the protein Ras homolog enriched in brain (Rheb) plays a key role as a positive upstream regulator of the mammalian target of rapamycin complex 1 (mTORC1) pathway in integrating inputs from nutrients and growth factors for cell growth. Our objective was to investigate the role of the mTORC1 pathway in the regulation of beta-cell mass in vivo. RESEARCH DESIGN AND METHODS: We generated transgenic mice that overexpress Rheb in beta-cells. We examined the activation of the mTORC1 pathway and its effects on beta-cell mass, on glucose metabolism, and on protection against hyperglycemia. RESULTS: Immunoblots of islet extracts revealed that the phosphorylation levels of ribosomal protein S6 and eukaryotic initiation factor 4E binding protein 1, downstream effectors for mTORC1, were upregulated in transgenic beta-cells. Immunostaining of the pancreatic sections with anti-phospho-S6 antibody confirmed upregulation of the mTORC1 pathway in beta-cells in vivo. The mice showed improved glucose tolerance with higher insulin secretion. This arose from increased beta-cell mass accompanied by increased cell size. The mice also exhibited resistance to hyperglycemia induced by streptozotocin and obesity. CONCLUSIONS: Activation of the mTORC1 pathway by Rheb led to increased beta-cell mass in this mouse model without producing obvious unfavorable effects, giving a potential approach for the treatment of beta-cell failure and diabetes.

NO-mediated cytotoxicity contributes to multiple low-dose streptozotocin-induced diabetes but not to NOD diabetes.

Type 1 diabetes (T1D) is caused mostly by autoimmune destruction of pancreatic beta-cells, the precise mechanism of which remains unclear. Two major effector mechanisms have been proposed: direct cell-mediated and indirect cytokine-mediated cytotoxicity. Cytokine-mediated beta-cell destruction is presumed mainly caused by NO production. To evaluate the role of iNOS expression in T1D, this study used a novel iNOS inhibitor ONO-1714. ONO-1714 significantly reduced cytokine-mediated cytotoxicity and NO production in both MIN6N9a cells and C57BL/6 islets in the presence of IL-1beta, TNF-alpha, and IFN-gamma. To evaluate whether NO contributes to diabetes progression in vivo, ONO-1714 was administered to four different mouse models of autoimmune diabetes: multiple low-dose STZ (MLDS)-induced C57BL/6, CY-induced, adoptive transfer and spontaneous NOD diabetes. Exposure to STZ in vitro induced NO production in MIN6N9a cells and C57BL/6 islets, and in vivo injection of ONO-1714 to MLDS-treated mice significantly reduced hyperglycemia and interestingly, led to complete suppression of cellular infiltration of pancreatic islets. In contrast, when ONO-1714 was injected into spontaneous NOD mice and CY-induced and adoptive transfer models of NOD diabetes, overt diabetes could not be inhibited in these models. These findings suggest that NO-mediated cytotoxicity significantly contributes to MLDS-induced diabetes but not to NOD diabetes.

Regulation of hypoxia-inducible factor 1 by glucose availability under hypoxic conditions.

Hypoxia-inducible transcription factor 1 (HIF-1), consisting of HIF-1 alpha and HIF-1 beta subunits, regulates the expression of a variety of genes involved in diverse adaptive processes in response to hypoxia. While oxygen availability regulates HIF-1 alpha by proteolytic degradation, some growth factors regulate HIF-1 alpha by protein synthesis in part through mammalian target of rapamycin complex 1 (TORC1) pathway. We herein report the role of nutrient availability on the regulation of HIF-1. A reduced availability of glucose, not amino acids, results in a decrease of the expression of HIF1-dependent genes and HIF-1 alpha protein in response to hypoxia. HIF-1 alpha mRNA expression was not significantly suppressed and DMOG, an inhibitor for proteasomal degradation of HIF-1 alpha, did not induce HIF-1 alpha protein expression under hypoxia combined with glucose depletion. In comparison to the effect in the presence of glucose, glucose depletion under hypoxia induced a much stronger activation of the AMP-dependent kinase pathway and phosphorylation of eIF2 alpha, and nearly complete inhibition of the TORC1 pathway. These findings imply that the reduced availability of glucose under hypoxia downregulates HIF-1 in part through the inhibition of HIF-1 alpha mRNA translation, which is occasionally observed in pathophysiological situations such as ischemic diseases.

Low-dose warfarin functions as an immunomodulator to prevent cyclophosphamide-induced NOD diabetes.

Warfarin has been used as an anticoagulant for a long time. Recently, the pleiotropic effect of warfarin has been investigated. As low-dose warfarin has been reported to have anti-inflammatory effect through suppression of IL-6 secretion and inhibit the immune-associated signal between Tyro3 and its ligand, Gas6, the effect of low-dose warfarin on autoimmune diabetes in NOD mice was examined. To investigate the anti-inflammatory effect of warfarin, IL-6 secretion by splenocytes was examined in the presence of various concentrations of warfarin. Low concentration of warfarin inhibited IL-6 secretion. mRNA expression of Rse, one of the Tyro3 receptor family members, and Gas6 were analyzed in NOD mice. It was detected in islets, splenocytes and bone-marrow derived dendritic cells. 0.25 mg/l or 0.50 mg/l of warfarin was orally administered to NOD mice as a cyclophosphamide-induced diabetes model. Oral administration of warfarin at much lower doses than those clinically used as an anticoagulant significantly reduced the degree of insulitis and diabetes incidence in this model. We previously demonstrated that anti-FasL Ab-treatment led to complete prevention of autoimmune diabetes in NOD mice. As Fas/FasL signaling is reported to be essential for cyclophosphamide-induced diabetes model, we extracted RNA from lymphocytes of the inguinal lymph nodes of anti-FasL Ab-treated NOD mice and performed real-time PCR to determine expression of Rse gene. Interestingly, the expression of Rse gene related to the blockade of Fas/FasL signaling was reduced to less than half the level of untreated mice. In conclusion, low-dose warfarin is a potential immunomodulator which can prevent autoimmune diabetes.

One amino acid difference is critical for suppression of the development of experimental autoimmune diabetes (EAD) with intravenous injection of insulinB:9-23 peptide.

InsulinB:9-23 peptide (insB:9-23) reactive T cells has been reported as crucial for type 1 diabetes. In this study, experimental autoimmune diabetes (EAD) mice, which subcutaneous immunization of ins1 or 2B:9-23 induced autoimmune diabetes in F1(B7.1B6 x BALB/c), was investigated for antigen specific therapy to delete pathogenic T cells. Intravenous injection of ins1 or 2B:9-23 significantly delayed the development of diabetes on the corresponding peptide-induced EAD (ins1EAD or ins2EAD) concomitant with reduced insulitis and insulin autoantibodies expression. Population of Foxp3(+) CD4(+) T cell was unchanged whereas the level of anti-insB:9-23 specific IgG(2a) but not IgG(1) were specifically decreased, suggesting reduction of pathogenic insB:9-23 reactive T cells. Most interestingly, intravenous administration of ins2B:9-23, whose amino acid sequence had one amino acid difference at position 9 delayed the development of diabetes in both ins1EAD and ins2EAD whereas ins1B:9-23 administration delayed diabetes in the ins1EAD but not ins2EAD, suggesting that one amino acid difference gives critical influence on the effect of intravenous injection of antigenic peptide for type 1 diabetes.

Prevention of recurrent but not spontaneous autoimmune diabetes by transplanted NOD islets adenovirally transduced with immunomodulating molecules.

Pancreatic islet transplantation has the potential to maintain normoglycemia in patients with established type 1 diabetes, thereby obviating the need for frequent insulin injections. Our previous study showed that recombinant IL-12p40-producing islets prevented the recurrence of NOD diabetes. First, to see which immunomodulating molecule-secreting islet grafts can most powerfully prevent diabetes development in NOD mice without immunosuppressant, NOD islets were transfected with one of the following adenoviral vectors: Ad.IL-12p40, Ad.TGF-beta, Ad.CTLA4-Ig, or Ad.TNF-alpha after which they were transplanted under the renal capsule of acutely diabetic NOD mice. The immunomodulating molecules produced by these adenovirus-transfected islets in vitro were 74+/-19ng, 50+/-4ng, 821+/-31ng, and 77+/-18ng/100 islets, respectively. Transplantation of IL-12p40, TNF-alpha, and CTLA4-Ig but not TGF-beta-secreting islets displayed enhanced survival and delayed diabetes recurrence in recent-onset diabetic recipients. IL-12p40-producing islet grafts most powerfully prevented recurrent diabetes in NOD mice. In addition, local production of TNF-alpha and CTLA4-Ig significantly prolonged islet graft survival. In second series of experiment, these manipulated islets were transplanted under the renal capsule of 10-week-old NOD recipients and were also transplanted subcutaneously into 2-week-old NOD recipients. Transplantation of these islets into 2- or 10-week-old pre-diabetic mice failed to protect them from developing diabetes; in fact, transplantation of Ad.TNF-alpha-transfected islets into 2-week-old mice actually accelerated diabetes onset. Taken together, this approach was ineffectual as a prophylactic protocol. In conclusion, this study showed comparisons of the immunomodulating effects of 4 different adenoviral vectors in the same transplantation model and local production of IL-12p40, TNF-alpha and CTLA4-Ig significantly prevented recurrent diabetes in NOD mice.

Intravenous administration of proinsulin 1 or 2-expressing fiber-mutant recombinant adenovirus vector protects against the development of diabetes in NOD mice.

Insulin has been reported as a major autoantigen in both human and murine type 1 diabetes (T1D). Insulin1-knockout NOD mice with only insulin2 are protected against the development of autoimmune diabetes, suggesting that insulin1 has strong immunogenicity and insulin2 has weak immunogenicity or a possible protective role in the pathogenesis of type 1 diabetes. In this study, we have developed fiber-mutant adenovirus vectors that express murine proinsulin1 or proinsulin2 (named Ad.Pins1-RGD/Ad.Pins2-RGD) and administered those virus vectors to the NOD mouse to evaluate modulation of autoimmune responses. The intravenous administration of either Ad.Pins1-RGD or Ad.Pins2-RGD at 3 and 5 weeks of age strongly suppressed the development of overt diabetes, accompanied by a significant reduction of insulin autoantibody (IAA), and suppression of disease was similar between administration of Ad.Pins1-RGD and that of Ad.Pins2-RGD. Our study suggests that systemic administration of fiber-mutant adenovirus vectors, which induce transient expression of proinsulin, may be applicable to a gene therapy inducing tolerance to insulin.

Autoreactive T cell response in CD25-negative fraction of peripheral blood mononuclear cells in established type 1 diabetes.

CD4(+)CD25(+) T cells (Tregs) play a critical role in maintaining dominant peripheral tolerance, and pathogenic autoreactive T cells may be frequent in the CD25-negative fraction of peripheral blood mononuclear cells (PBMCs) from patients with autoimmune disease. We therefore investigated whether T cell autoimmune responses to recombinant GAD65 can be detected by the use of ELISPOT assay in the CD25-negative fraction of PMBCs from Japanese type 1 diabetes (T1D) patients. The frequency of CD4(+)CD25(+) T cells was not different among patients with newly developed T1D, established T1D, and healthy controls. The CD25 positive cell-depleted fraction was obtained by negative selection with antihuman CD25 magnetic beads, reducing the number of CD4(+)CD25(+) T cells from 4-5% to less than 1%. In whole PBMC fraction, there was a significant elevation of IFN-gamma spots in PBMCs from recently diagnosed patients with T1D (P < 0.05), whereas the number of IFN-gamma spots from patients with established T1D was not significant. In the CD25-negative fraction, unlike whole PBMCs, we observed the significant IFN-gamma spots to GAD65 in the fraction from patients with established T1D (P < 0.05), but not in those with recently diagnosed disease. The phenomena were not observed for IL-4 spots. Our data suggest a possible role of Tregs maintaining dominant peripheral tolerance in T1D and application of further improved T cell assay detecting autoimmunity even in established T1D.

Immunological aspects of 'fulminant type 1 diabetes'.

'Fulminant diabetes' has been recognized as a super-acute onset and non-autoimmune type 1 diabetes. To evaluate autoimmunity against pancreatic beta cell in fulminant diabetes, ELISPOT assay was applied to the peripheral blood of these patients. In our ELISPOT system, GAD65-reactive and insulin B9-23-reactive IFN-gamma spots were detected in 46.3 and 26.0% of autoantibody-positive type 1 diabetes. Also, in fulminant type 1 diabetic patients, IFN-gamma spots in response to GAD65 and insulin B9-23 peptide were detected in 69.2 and 25.0%, respectively. These results suggest that anti-beta cell autoimmunity contributes to develop fulminant type 1 diabetes. Fulminant type 1 diabetes is known to have IDDM-resistant HLA DR2 with similar frequency of non-T1D subjects. In a mouse model, when islet-reactive CD8 cells are transferred to young NOD mice, the recipients develop overt diabetes within 1 week with massive insulitis. In (NOD x Balb/c) F1 mice, which hold idd-resistant genes, transfer of islet-reactive CD8 cells induced diabetes to 60% F1 recipients within 1 week with the later disappearance of insulitis. This mouse model shows very similar feathers to fulminant type 1 diabetes; idd-resistant HLA and no insulitis. These results implicated that once anti-islet immunity is optimally activated, subjects with partially resistant alleles could become overt diabetes.