Mid-term results of cyanoacrylate closure for the treatment of incompetent great and small saphenous veins: Findings from a Japanese prospective consecutive multi-center registry: Mid-term results of cyanoacrylate closure.

OBJECTIVES: We evaluated the efficacy and safety of cyanoacrylate closure (CAC) for endovascular treatment of varicose veins with cyanoacrylate adhesive (VenaSeal® closure system) in Japan. METHODS: A multicenter prospective consecutive registry study was conducted at 12 centers in Japan on 125 patients with primary varicose veins who underwent CAC. The patients were evaluated on target vein occlusion, postoperative complications, Visual Analogue Scale (VAS) for pain, revised Venous Clinical Severity Score (rVCSS), Aberdeen Varicose Vein Questionnaire (AVVQ), and EuroQol 5 dimensions 5-level (EQ-5D-5L) for 1-year after the surgery. RESULTS: The closure rate was 92.6% at 1 year postoperatively, and 95.0% and 90.2% for GSV and SSV respectively with little difference (p = .491). The mean VAS in the immediate postoperative period was 18.9 ± 23.4. Postoperative complications were observed in 20 patients (16%). Hypersensitivity-type phlebitis occurred in 7 patients (5.6%). Infection of the treated vein resulted in resection of GSV. The rVCSS and AVVQ improved significantly after 90 days and 1 year postoperatively (p < .001), while the EQ-5D-5L have not changed. CONCLUSION: Cyanoacrylate Closure was considered generally a safe and minimally invasive treatment with good mid-term outcomes including SSV. However further study is required for some CAC specific complications.

Supplement of Clinical Practice Guidelines for Endovenous Thermal Ablation for Varicose Veins: Overuse for the Inappropriate Indication.

While endovenous thermal ablation (ETA) become first choice of treatment for varicose veins, overuse of ETA for the inappropriate indication is growing problem. ETA is performed not only on varicose cases without symptom but also non diseased cases with segmental reflux of saphenous veins or no reflux. Indications of ETA was demonstrated in "the Clinical Practice Guidelines for ETA for Varicose Veins 2019" by Japanese Society of Phlebology. Purpose of this supplement is description of basics of correct indication for ETA. We also demonstrate the typical case of overuse of ETA for wrong indication. (This is a translation of Jpn J Phlebol 2020; 31: 39-43.).

Endovenous Thermal Ablation for a Varicose Vein Patient with Factor XII Deficiency: A Case Report.

Factor XII (FXII) deficiency is a rare coagulation disorder, and its potential relationship with venous thrombosis was reported. Here we present a case of a 67-year-old woman with FXII deficiency who successfully underwent endovenous thermal ablation (ETA) for primary varicose vein due to the incompetent great saphenous vein (GSV). The FXII deficiency was revealed through preoperative examinations, and the patient underwent ETA as a day surgery. For prophylaxis of thrombosis, she received compression therapy alone. Her postoperative course was uneventful, without any kind of thrombosis. In the presence of FXII deficiency, ETA could be safely performed.

Administration of donor splenocytes via the respiratory tract generates CD8α+ regulatory dendritic cells and induces hyporesponsiveness to fully allogeneic cardiac grafts.

BACKGROUND: We previously showed that pretreatment with intratracheal delivery (ITD) of alloantigen induced prolonged cardiac allograft survival and generated regulatory T cells (Tregs) in mice. In this study, we examined the role of splenic dendritic cells (DCs) in the ITD model. METHODS: CBA mice were treated with ITD from C57BL/10 splenocytes and 7 days later received transplantation of C57BL/10 hearts. In adoptive transfer studies, splenic DCs from ITD-treated mice were transferred into naïve CBA recipients that received C57BL/10 hearts immediately after the transfer. In addition, to determine the role of splenic DCs isolated from ITD-treated mice, the cells were incubated under stimulation with lipopolysaccharide (LPS). RESULTS: ITD-treated CBA recipients had markedly prolonged allograft survival (median survival time [MST], 67 days) while naïve recipients rejected allografts acutely (MST, 8 days). In adoptive transfer studies, CBA recipients of the transfer of splenic DCs from ITD-treated mice had prolonged allograft survival (MST, 85 days), while CBA recipients of the transfer of splenic DCs from naïve mice did not have prolonged allograft survival (MST, 8 days). In another transfer study, CBA recipients of the transfer of splenic CD8α+ DCs from ITD-treated mice had prolonged allograft survival (MST, 79 days), while those receiving splenic CD8α- DCs from ITD-treated mice did not have prolonged allograft survival (MST, 8 days). In vitro studies showed that ITD-treated splenic DCs produced more IL-10 and less IL-12 than naïve splenic DCs under stimulation with LPS. CONCLUSIONS: ITD pretreatment induces regulatory DCs, which produce high amounts of IL-10 resulting in the prolongation of graft survival in our model.

Asymptomatic Isolated Calf Deep Vein Thrombosis: Does It Worsen after Varicose Vein Surgery?

In our varicose vein center, on a trial basis, among the patients with asymptomatic calf deep vein thrombosis (CDVT) we carefully selected the patients for varicose vein surgery using the requirements as follows; 1) the patients had varicose veins with incompetent saphenous veins, 2) sequential examination including DUS confirmed stability and clinical insignificance of asymptomatic CDVT, 3) the patients do not have any risk factors for DVT such as a coagulation profile disorder (antithrombin deficiency, protein C deficiency, protein S deficiency, or antiphospholipid syndrome) or malignancies, 4) surgery is possible under local anesthesia alone, and 5) the patients can understand the concept of asymptomatic CDVT and undergo the surgery on their own will and informed consent. The patients who fulfilled these conditions underwent the varicose vein surgery. Twenty-eight patients with 30 limbs with varicose veins had asymptomatic CDVT, found by preoperative duplex ultrasonography (DUS). Among CDVT, 91% of CDVT existed in the soleal veins. After the diagnosis of the asymptomatic CDVT, serial DUS was performed and showed no changes in the status of the thrombus. Then varicose vein surgery (high ligation of the saphenous junctions either with or without stripping of the saphenous veins) was performed. After the surgery, the CDVT was re-evaluated by DUS. In 27 limbs, CDVT did not show any changes in the status of the thrombus, and in 3 limbs the CDVT was partially resolved. These data suggest that, at least, as far as the patients fulfilled these conditions, varicose vein surgery did not worsen the asymptomatic CDVT. (This is a translation of Jpn J Phlebol 2016; 27: 405-412.).

Prevalence of Isolated Asymptomatic Deep Vein Thrombosis in Varicose Vein Patients with Superficial Thrombophlebitis: A Single Center Experience in Japan.

OBJECTIVE: Prevalence of asymptomatic deep vein thrombosis (DVT) in patients with primary varicose veins remains unclear. MATERIALS AND METHODS: Here, we conducted a retrospective study to clarify the incidence of asymptomatic DVT in patients with varicose veins, especially focusing on those with superficial thrombophlebitis (STP). RESULTS: Among 431 patients with primary varicose veins with saphenous vein incompetence, 20 (4.64%) had asymptomatic DVT. The presence of STP was a significant risk factor for asymptomatic DVT as 10 of the 24 (41.7%) patients with STP had asymptomatic DVT, and all cases having calf muscle vein thrombosis. In contrast, of the patients with primary varicose veins without STP only 2.46% had asymptomatic DVT. CONCLUSIONS: In patients with primary varicose veins with STP, significant risk factors for DVT were being over C3 on the clinical, etiological, anatomical, and pathophysiological (CEAP) classification. (This article is a translation of Jpn J Phlebol 2014; 25: 13-19.).

Induction of regulatory T cells and prolongation of survival of fully allogeneic cardiac grafts by administration of Tokishakuyaku-san in mice.

BACKGROUND: The Japanese herbal medicine Tokishakuyaku-san (TJ-23) has been used to treat neurodegenerative, immune, and respiratory tract diseases, as well as many gynecologic disorders, with few adverse effects. This study investigated the effect of TJ-23 on alloimmune responses in a murine model of cardiac allograft transplantation. METHODS: CBA mice underwent transplantation of a C57BL/6 heart and received oral administration of 2 g/kg per day of TJ-23 or 1 of 16 other commonly used Japanese herbal medicines from the day of transplantation until 7 days afterward. An adoptive transfer study was conducted to determine whether regulatory cells were generated. Histologic and cell proliferation studies, cytokine measurements, and flow cytometry analyses were also performed. RESULTS: Of the 17 herbal medicines studied, only TJ-23, given in a dose of 2 g/kg per day, induced significantly prolonged allograft survival (median survival time [MST], >100 days). TJ-23 also suppressed proliferation of splenocytes and production of interleukin-2, interleukin-6, and interferon-γ. Adoptive transfer of either whole splenocytes or CD4(+) or CD4(+) CD25(+) cells from TJ-23-treated allograft recipients resulted in indefinite survival of allografts in naive secondary recipients (MST >100 days). Flow cytometry studies showed that the CD4(+) CD25(+) forkhead/winged-helix (FOXP3)(+) regulatory cell population was increased in transplant recipients given TJ-23. CONCLUSION: TJ-23 induced hyporesponsiveness to fully allogeneic cardiac allografts and generated CD4(+) CD25(+) regulatory cells in our model.

Depletion of alveolar macrophages abrogates prolongation of cardiac allograft survival induced by intratracheal delivery of alloantigen.

BACKGROUND: We previously showed that pretreatment with intratracheal delivery (ITD) of alloantigen induced prolonged cardiac allograft survival and generated regulatory cells in mice. In this study, we examined the role of alveolar macrophages (AM) in our ITD model. METHODS: Some CBA mice were given ITD of C57BL/6 splenocytes and underwent transplantation of C57BL/6 hearts 7 days later. In others, AM were depleted with clodronate-loaded liposomes 3 days before ITD. In adoptive transfer studies, whole splenocytes were obtained from ITD-treated CBA mice and administered to naïve CBA secondary recipients, which were given C57BL/6 hearts immediately afterward. Interleukin-10 concentrations in bronchoalveolar lavage fluid were assessed by enzyme-linked immunosorbent assays. Immunohistologic and flow cytometric studies were performed after ITD. RESULTS: C57BL/6 splenocytes given by ITD were ingested by AM in 2 days and undetectable in paratracheal lymph nodes or spleen tissue. CBA mice given ITD of C57BL/6 splenocytes had markedly prolonged allograft survival (median survival time [MST], 86 days), whereas naïve CBA mice rejected allografts acutely (MST, 8 days). AM-depleted, ITD-treated mice also rejected allografts (MST, 5.5 days). Naïve secondary recipients given adoptive transfer of splenocytes from ITD-treated mice had prolonged allograft survival (MST, >100 days), whereas secondary recipients given adoptive transfer of splenocytes from AM-depleted, ITD-treated mice rejected the grafts (MST, 15.5 days). Interleukin-10 expression in bronchoalveolar lavage fluid was down-regulated in AM-depleted mice compared with naïve mice. CONCLUSIONS: AM have an important role in the induction of regulatory cells in our model of ITD of alloantigen.

Immunomodulatory effects of eicosapentaenoic acid through induction of regulatory T cells.

Dietary intake of omega-3 polyunsaturated fatty acids (PUFAs) has been found to affect inflammation and metabolism, and many researchers have shown that omega-3 PUFAs provide benefits in immunologic and metabolic disorders. These effects were assumed to result mainly from a modification in the production of inflammatory mediators and the suppression of inflammatory leukocytes. Among PUFAs, eicosapentaenoic acid (EPA), a component of fish oil, apparently has the most potent effect. Recently, much research has focused on regulatory T cells (Tregs) as controllers of immune responses not only to self-antigens but also to non-self-antigens, including donor alloantigens. Therefore, induction of antigen-specific Tregs may be an attractive strategy for managing autoimmune diseases and transplant rejection. Peroxisome proliferator-activated receptor γ (PPARγ), a ligand-activated nuclear receptor that regulates lipid and glucose metabolism, can be activated by thiazolidinediones, fatty acids, and eicosanoids, including EPA. PPARγ was recently found to have immunoregulatory effects, and a PPARγ agonist inhibited immune responses in a rat model of autoimmune disease. Furthermore, in a murine model, one high dose of purified EPA given the day of transplantation induced marked prolongation of cardiac allograft survival in a dose-dependent manner. These findings suggest that EPA induced Tregs by means of a PPARγ-dependent mechanism. This review describes the immunomodulatory effects of PUFAs, especially EPA, and summarizes recent research that may have implications for the development of therapies for autoimmune diseases and transplant rejection that are based on induction of Tregs.

Induction of regulatory T cells and indefinite survival of fully allogeneic cardiac grafts by ursodeoxycholic acid in mice.

BACKGROUND: Ursodeoxycholic acid (UDCA) has been used to treat patients with cholestatic and autoimmune liver diseases. Several studies have addressed whether UDCA can inhibit graft rejection in experimental and clinical transplantation, but the results have varied. We investigated the effect of UDCA and the mechanism of its effect on alloimmune responses in a murine model of cardiac transplantation. METHODS: CBA mice underwent transplantation of a C57BL/10 heart and received a single dose of UDCA. Survival times of the allografts were recorded. An adoptive transfer study was conducted to determine whether regulatory cells were generated. The effects on graft survival of adding FK506 or cyclosporine A (CyA) to UDCA treatment were assessed. Histologic, cell proliferation, and cytokine assessments were performed. RESULTS: CBA recipients given UDCA (25 mg/kg) had indefinite allograft survival (median survival time [MST], >100 days). UDCA also suppressed proliferation of splenocytes and production of interleukin (IL)-2, IL-6, and interferon-gamma, and up-regulated IL-10 production. Adoptive transfer of either whole splenocytes or CD4+ cells from UDCA-treated allograft recipients resulted in indefinite survival of allografts in naive secondary recipients (MST, >100 days). Adoptive transfer of CD4+ CD25+ cells from UDCA-treated recipients significantly prolonged allograft survival in naive secondary recipients (MST, >80 days). FK506 (0.1 mg/kg/day) was compatible with the induction of indefinite allograft survival by UDCA, whereas CyA (10 mg/kg/day) abrogated the effect of UDCA. CONCLUSION: UDCA induced unresponsiveness to fully allogeneic cardiac allografts and generated CD4+ CD25+ regulatory cells in our model. FK506, but not CyA, was compatible with UDCA treatment.

Prolonged survival of fully mismatched cardiac allografts and generation of regulatory cells by Sairei-to, a Japanese herbal medicine.

Sairei-to (TJ114), a 12-component Japanese herbal medicine, is used to treat immune-related diseases. We investigated the effects of oral administration of TJ114 in a murine model of cardiac transplantation with fully mismatched allografts. Untreated CBA mice rejected C57BL/6 hearts acutely (median survival time [MST], 7 days), whereas survival of allografts from mice given TJ114 was significantly prolonged (MST >100 days). Secondary CBA recipients of C57BL/6 hearts also had prolonged allograft survival (MST >100 days) after adoptive transfer of whole or CD4 splenocytes from primary CBA allograft recipients given TJ114. None of the individual components of TJ114 prolonged allograft survival, suggesting that its effects require administration of the combination agent. In mixed leukocyte cultures, proliferation of splenocytes from TJ114-treated CBA recipients was markedly suppressed compared with that of splenocytes from untreated mice, and interferon-gamma production was significantly reduced. Thus, in our model, TJ114 treatment induced hyporesponsiveness to cardiac allografts and generated CD4 regulatory cells.

Prolongation of survival of fully allogeneic cardiac grafts and generation of regulatory cells by a histamine receptor 2 antagonist.

BACKGROUND: The effects of histamine on immunologic responses via the histamine receptor 2 (HR2) have been studied, but few investigations explored the immunomodulatory role of histamine in vivo. We examined whether the HR2 antagonist ranitidine affects the alloimmune response in a murine model of cardiac transplantation. METHODS: CBA (H-2k) recipients were given no treatment or one intravenous injection of ranitidine on the day of transplantation of a heart from C57BL/10 (H-2b) donors. Survival of the allografts was recorded. The effect of the ranitidine treatment on cell proliferation and cytokine production was assessed by mixed leukocyte culture and enzyme-linked immunosorbent assays. An adoptive transfer study was conducted to determine whether regulatory cells were generated. The effect on graft survival of adding FK506 to the ranitidine treatment was also examined. RESULTS: CBA recipients given ranitidine (60 mg/kg) had prolonged graft survival (median survival time [MST], 87 days). Ranitidine treatment also suppressed the proliferation of splenocytes and production of interleukin (IL)-2 and up-regulated IL-10 production. Adoptive transfer of splenocytes and CD4 cells from ranitidine-treated allograft recipients induced significant prolongation of allograft survival in naive secondary recipients (MST, 71 and >100 days, respectively). CBA recipients given both ranitidine and FK506 (0.1 mg/kg/day for 14 days) had indefinite survival of cardiac allografts (MST, >100 days). CBA recipients treated with FK506 alone rejected the allografts (MST, 27 days). CONCLUSION: In our model, ranitidine treatment induced significantly prolonged survival of fully allogeneic cardiac grafts, generated CD4 regulatory cells, and indefinite survival when combined with FK506 (0.1 mg/kg/day).

Induction of indefinite survival of fully mismatched cardiac allografts and generation of regulatory cells by sarpogrelate hydrochloride.

BACKGROUND: At initiation of the immunologic response, platelets rapidly release chemical mediators such as serotonin (5-hydroxytryptamine, [5-HT]) and cytokines. Sarpogrelate hydrochloride (SH), a selective 5-HT2-receptor antagonist, is used to treat patients with peripheral arterial disease. We investigated the effect of SH on the alloimmune response in a murine cardiac transplantation model. METHODS: CBA mice underwent transplantation of a C57BL/10 heart and received a short course of SH treatment. Survival of the allograft was recorded. An adoptive transfer study was performed to determine whether regulatory cells were generated. Immunohistochemistry studies of intercellular adhesion molecule 1 (ICAM-1), histological, cell-proliferation, and cytokine assessments were performed. RESULTS: Untreated CBA mice rejected C57BL/10 cardiac grafts acutely (median survival time [MST], 8 days). In mice given 10 mg/kg of SH, all allografts survived indefinitely (MST, >100 days); these mice also had significantly prolonged survival of donor-specific skin grafts but acute rejection of third-party skin grafts. Secondary CBA recipients given not only whole but also CD4 splenocytes from primary SH-treated CBA recipients with C57BL/10 cardiac allograft had indefinite survival of C57BL/10 hearts (MST, >100 days). SH inhibited upregulation of ICAM-1 on endothelial cells in the allografts. Graft acceptance and hyporesponsiveness were confirmed by the histological and cell-proliferation studies, respectively. Production of interleukin-4 and interleukin-10 from splenocytes of SH-treated transplant recipients increased compared to that from splenocytes of untreated recipients. CONCLUSION: SH induced indefinite survival of fully allogeneic cardiac allografts, generated CD4 regulatory cells, inhibited ICAM-1 expression in the allografts, and upregulated IL-4 and IL-10 production.

Selective cyclooxygenase 2 inhibitor induces indefinite survival of fully allogeneic cardiac grafts and generates CD4+ regulatory cells.

BACKGROUND: Selective inhibition of cyclooxygenase 2 has been reported to have not only anti-inflammatory effects but also effects on the immune response. We investigated ability of a cyclooxygenase 2 inhibitor to inhibit alloimmune response in a murine cardiac transplantation model. METHODS: CBA (H2(k)) mice underwent transplantation of C57BL/10 (H2(b)) hearts. On the day of transplantation, the recipients received either no treatment or single administration of aspirin (a cyclooxygenase 1 and 2 inhibitor) or the selective cyclooxygenase 2 inhibitor NS-398. Naive CBA mice (secondary recipients) underwent adoptive transfer of splenocytes from treated mice with long-surviving grafts (primary recipients) to determine whether regulatory cells developed after NS-398 treatment. Histologic, cell-proliferation, and cytokine studies were also performed. RESULTS: Untreated CBA mice rejected C57BL/10 cardiac grafts acutely (median survival time, 8 days). The majority of recipients given aspirin rejected their grafts within 20 days (median survival time, 11 days). In mice given NS-398, the majority of the grafts survived indefinitely (median survival time, >100 days). Secondary CBA recipients given CD4+ splenocytes from primary CBA recipients treated with NS-398 also had indefinite survival of C57BL/10 hearts (median survival time, >60 days). Graft acceptance and proliferative hyporesponsiveness were also confirmed by the histologic and cell-proliferation studies, respectively. Production of interleukin 4 and 10 from splenocytes of the recipients treated with NS-398 were significantly higher than that from untreated recipients. CONCLUSIONS: In our model administration of cyclooxygenase 2 inhibitor induced indefinite survival of fully mismatched cardiac grafts and generated CD4+ regulatory cells. Cyclooxygenase 2 inhibitor could warrant consideration for use as an immunomodulating agent in clinical transplantation.

Effects of immunosuppressants on induction of regulatory cells after intratracheal delivery of alloantigen.

BACKGROUND: We previously reported that intratracheal delivery (ITD) of alloantigen generated regulatory cells in mice. Here, we examined the effect of various doses of conventional immunosuppressants (FK506, cyclosporine A, azathioprine, mycophenolate mofetil, and rapamycin) on inducing regulatory cells in our model. METHODS: CBA mice (primary recipients) were given C57BL/6 splenocytes by ITD and either no additional treatment or various doses of an immunosuppressant. Seven days later, splenocytes from these mice were adoptively transferred into naive secondary CBA recipients that underwent C57BL/6 cardiac grafting the same day. RESULTS: Adoptive transfer from primary recipients given ITD of splenocytes alone induced prolonged allograft survival in secondary recipients (median survival time [MST], 50 days), suggesting that regulatory cells were generated. When ITD of alloantigen was combined with daily administration of 0.1 mg/kg FK506 or 0.2 mg/kg rapamycin, graft survival was similarly prolonged (MST 55 and 50 days, respectively). When combined with 20 or 40 mg/kg MMF or 0.4 mg/kg rapamycin, the majority of recipients demonstrated indefinite survival (MST, >100 days in all groups). When ITD of alloantigen was combined with 0.3, 0.5, or 1.0 mg/kg FK506; 5, 10, or 25 mg/kg cyclosporine A; or 1.0 or 2.0 mg/kg azathioprine, allografts were rejected acutely (MST 7-13 days). CONCLUSION: Generation of regulatory cells by ITD of alloantigen was facilitated by mycophenolate mofetil and high doses of rapamycin but abrogated by cyclosporine A, azathioprine, and high doses of FK506. Low doses of rapamycin and of FK506 did not interfere with generation of regulatory cells.

Interleukin-10 but not transforming growth factor-beta is essential for generation and suppressor function of regulatory cells induced by intratracheal delivery of alloantigen.

BACKGROUND: We previously reported that intratracheal delivery of alloantigen-induced regulatory cells in mouse heart-transplantation model. Here, we investigated roles of interleukin (IL)-10 and transforming growth factor (TGF)-beta in induction and effector phases of the regulatory cells. METHODS: CBA mice were pretreated with intratracheal delivery of C57BL/10 splenocytes and administration of neutralizing anti-IL-10 or anti-TGF-beta monoclonal antibody (mAb). Seven days after the pretreatment, naive CBA mice (secondary recipients) were given adoptive transfer of splenocytes from the pretreated mice and underwent heart grafting from C57BL/10 mice. To determine roles of these cytokines in the effector phase of the regulatory cells, anti-IL-10 or anti-TGF-beta mAb was administered weekly into the secondary recipients after the adoptive transfer. RESULTS: Adoptive transfer of splenocytes from CBA mice that had been pretreated with intratracheal delivery of C57BL/10 splenocytes significantly prolonged the survival of C57BL/10 allograft (median survival time [MST] 68 days) as compared with adoptive transfer from untreated CBA mice (MST 12 days). In the induction phase, anti-IL-10 mAb abrogated development of the regulatory cells that afforded prolonged allograft survival in the secondary recipients (MST 20 days), whereas anti-TGF-beta mAb did not abrogate it (MST 88 days). In the effector phase, anti-IL-10 mAb abrogated prolonged allograft survival afforded by adoptive transfer of the regulatory cells in the secondary recipients (MST 27 days), whereas anti-TGF-beta mAb did not abrogate suppressor function of the regulatory cells (MST 53 days). CONCLUSION: IL-10 but not TGF-beta was required for generation and suppressor function of the regulatory cells induced by intratracheal delivery of alloantigen.

Rational development of LEA29Y (belatacept), a high-affinity variant of CTLA4-Ig with potent immunosuppressive properties.

Current success in organ transplantation is dependent upon the use of calcineurin-inhibitor-based immunosuppressive regimens. Unfortunately, current immunotherapy targets molecules with ubiquitous expression resulting in devastating non-immune side effects. T-cell costimulation has been identified as a new potential immunosuppressive target. The best characterized pathway includes CD28, its homologue CTLA4 and their ligands CD80 and CD86. While an immunoglobulin fusion protein construct of CTLA4 suppressed rejection in rodents, it lacked efficacy in primate transplant models. In an attempt to increase the biologic potency of the parent molecule a novel, modified version of CTLA4-Ig, LEA29Y (belatacept), was constructed. Two amino acid substitutions (L104E and A29Y) gave rise to slower dissociation rates for both CD86 and CD80. The increased avidity resulted in a 10-fold increase in potency in vitro and significant prolongation of renal allograft survival in a pre-clinical primate model. The use of immunoselective biologics may provide effective maintenance immunosuppression while avoiding the collateral toxicities associated with conventional immunsuppressants.

Development of a chimeric anti-CD40 monoclonal antibody that synergizes with LEA29Y to prolong islet allograft survival.

In recent years, reagents have been developed that specifically target signals critical for effective T cell activation and function. Manipulation of the CD28/CD80/86 and CD40/CD154 pathways has exhibited extraordinary efficacy, particularly when the pathways are blocked simultaneously. Despite the reported efficacy of anti-CD154 in rodents and higher models, its future clinical use is uncertain due to reported thromboembolic events in clinical trials. To circumvent this potential complication, we developed and evaluated a chimeric Ab targeting CD40 (Chi220, BMS-224819) as an alternative to CD154. Although Chi220 blocks CD154 binding, it also possesses partial agonist properties and weak stimulatory potential. The anti-CD40 was tested alone and in combination with a rationally designed, high affinity variant of CTLA4-Ig, LEA29Y (belatacept), in a nonhuman primate model of islet transplantation. Although either agent alone only modestly prolonged islet survival (Chi220 alone: 14, 16, and 84 days; LEA29Y alone: 58 and 60 days), their combination (LEA29Y and Chi220) dramatically facilitated long term survival (237, 237, 220, >185, and 172 days). We found that the effects of Chi220 treatment were not mediated solely through deletion of CD20-bearing cells and that the combined therapy did not significantly impair established antiviral immunity.

Programmed death-1-programmed death-L1 interaction is essential for induction of regulatory cells by intratracheal delivery of alloantigen.

BACKGROUND: Programmed death (PD)-1 has been implicated in peripheral tolerance. The authors investigated the roles of PD-1 and its ligands, PD-L1 and PD-L2, in the induction of regulatory cells by intratracheal delivery of alloantigen. METHODS: CBA (H-2k) mice were pretreated with intratracheal delivery of C57BL/10 (H-2b) splenocytes and administration of monoclonal antibody (mAb) specific for PD-1, PD-L1, or PD-L2. Seven days later, C57BL/10 hearts were transplanted into the pretreated CBA mice. Some naive CBA mice underwent adoptive transfer of splenocytes from the pretreated CBA mice and transplantation of C57BL/10 heart. RESULTS: Untreated CBA mice rejected C57BL/10 cardiac grafts acutely (median survival time [MST], 7 days). Pretreatment with intratracheal delivery of C57BL/10 splenocytes prolonged graft survival significantly (MST, 65 days). Administration of control immunoglobulin (Ig) G or anti-PD-L2 mAb did not significantly affect the prolongation (MST, 72 and 68 days, respectively). In contrast, anti-PD-1 or anti-PD-L1 mAb abrogated the prolongation (MST, 18 and 17 days, respectively). Adoptive transfer from mice pretreated with intratracheal delivery of alloantigen plus control IgG or anti-PD-L2 mAb prolonged survival of C57BL/10 grafts in secondary CBA recipients (MST, 72 and 56 days, respectively). However, concurrent administration of anti-PD-1 or anti-PD-L1 mAb abrogated prolonged survival after the adoptive transfer (MST, 14 and 20 days, respectively). CONCLUSIONS: PD-1-PD-L1 interaction was essential for induction of regulatory cells by intratracheal delivery of alloantigen.

Intravital microscopy identifies selectins that regulate T cell traffic into allografts.

T cell homing to sites of injury and inflammation is a critical step for adaptive immune responses. While much has been learned regarding T cell homing to lymphoid tissues, few studies have directly observed trafficking events during an effector response. In this study, we developed a model that uses intravital fluorescence videomicroscopy to determine the molecules critical to T cell rolling within skin allograft microvasculature during the effector phase of the rejection response. Additional studies were performed to quantify T cell infiltrates as rejection progressed. We found that P-selectin and E-selectin expressed on postcapillary venules play overlapping roles in the recruitment of activated T cells in a SCID reconstitution model of skin graft rejection and are important in T cell accumulation at the graft site. Surprisingly, we also found that naive T cells are recruited and accumulate via constitutive T cell L-selectin and upregulated L-selectin ligands on rejecting allograft vasculature. These data indicated that a specific retinue of molecules is upregulated during the rejection response, and they suggest potential future therapeutic targets.