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OBJECTIVE: To investigate why bisphosphonates are less effective at preventing focal bone loss in rheumatoid arthritis (RA) patients than in those with generalized osteoporosis, and the mechanisms involved. METHODS: The response of osteoclasts to alendronate (ALN) in tumor necrosis factor-transgenic (TNF-Tg) mice that develop erosive arthritis and in wild-type littermates was studied. TNF-Tg and wild-type mice were given ALN, and the osteoclast numbers in the inflamed joints and in the long bones were compared. The expression levels of Bcl-xL in the osteoclasts of TNF-Tg and wild-type mice were examined by immunostaining. The effect of overexpression of Bcl-xL and Ets-2 proteins on ALN-induced osteoclast apoptosis was determined using an in vitro osteoclast survival assay and retrovirus transfer approach. RESULTS: ALN reduced osteoclast numbers in the metaphyses by 97%, but by only 46% in the adjacent inflamed joints. Bcl-xL expression was markedly higher in osteoclasts in the joints than in those in the metaphyses of TNF-Tg mice. Bcl-xL or Ets-2 overexpression protected osteoclasts from ALN-induced apoptosis, and TNF stimulated Bcl-xL and Ets-2 expression in osteoclasts. Overexpression of Ets-2 increased Bcl-xL messenger RNA in osteoclasts, while a dominant-negative form of the Ets-2 blocked the protective effect of Bcl-xL or TNF on ALN-induced apoptosis. CONCLUSION: The reduced efficacy of bisphosphonates to stop bone erosion in the inflamed joints of RA patients may result from local high levels of TNF up-regulating Ets-2 expression in osteoclasts, which in turn stimulates Bcl-xL expression in them and reduces their susceptibility to bisphosphonate-induced apoptosis. [ABSTRACT FROM AUTHOR]

Introduction: Assessment of surgical resident technical performance is an integral component of any surgical training program. Timely assessment delivered in a structured format is a critical step to enhance technical skills, but residents often report that the quality and quantity of timely feedback received is lacking. Moreover, the absence of written feedback with specificity can allow residents to seemingly progress in their operative milestones as a junior resident, but struggle as they progress into their postgraduate year 3 and above. We therefore designed and implemented a web-based intraoperative assessment tool and corresponding summary "dashboard" to facilitate real-time assessment and documentation of technical performance., Materials and Methods: A web form was designed leveraging a cloud computing platform and implementing a modified Ottawa Surgical Competency Operating Room Evaluation instrument; this included additional, procedure-specific criteria for select operations. A link to this was provided to residents via email and to all surgical faculty as a Quick Response code. Residents open and complete a portion of the form on a smartphone, then relinquish the device to an attending surgeon who then completes and submits the assessment. The data are then transferred to a secure web-based reporting interface; each resident (together with a faculty advisor) can then access and review all completed assessments., Results: The Assessment form was activated in June 2021 and formally introduced to all residents in July 2021, with residents required to complete at least one assessment per month. Residents with less predictable access to operative procedures (night float or Intensive Care Unit) were exempted from the requirement on those months. To date a total of 559 assessments have been completed for operations performed by 56 trainees, supervised by 122 surgical faculty and senior trainees. The mean number of procedures assessed per resident was 10.0 and the mean number per assessor was 4.6. Resident initiation of Intraoperative Assessments has increased since the tool was introduced and scores for technical and nontechnical performance reliably differentiate residents by seniority., Conclusions: This novel system demonstrates that an online, resident-initiated technical assessment tool is feasible to implement and scale. This model's requirement that the attending enter performance ratings into the trainee's electronic device ensures that feedback is delivered directly to the trainee. Whether this aspect of our assessment ensures more direct and specific (and therefore potentially actionable) feedback is a focus for future study. Our use of commercial cloud computing services should permit cost-effective adoption of similar systems at other training programs., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Human leukocyte antigen antibodies are important immunologic mediators of renal allograft loss and are difficult to control. The inability to permanently eliminate donor-specific antibodies (DSA) is partly due to an incomplete understanding of the cellular mechanisms driving alloantibody formation, recurrence, and maintenance. Memory T follicular helper (mTfh) cells rapidly interact with memory B cells upon antigen re-exposure for anamnestic humoral responses, but little is known about Tfh memory in transplantation. We hypothesized that alloreactive mTfh cells form after transplantation and play a critical role in DSA formation following alloantigen re-encounter. To test this hypothesis, we utilized murine skin allograft models to identify and characterize Tfh memory and interrogate its ability to mediate alloantibody responses. We identified alloreactive Tfh memory as a mediator of accelerated humoral alloresponses independent of memory B cells and primary germinal center, or DSA, formation. Furthermore, we demonstrate that mTfh-driven alloantibody formation is susceptible to CD28 costimulation blockade. These findings provide novel insight into a pathologic role for memory Tfh in alloantibody responses and strongly support shifting therapeutic focus from the singular targeting of B cell lineage cells and alloantibodies themselves to multimodal strategies that include inhibition of mTfh cells to treat DSA., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:I. Raul Badell reports a relationship with Bristol Myers Squibb Co that includes: funding grants. I. Raul Badell reports a relationship with Veloxis Pharmaceuticals Inc that includes: consulting or advisory., (Copyright © 2023 American Society of Transplantation & American Society of Transplant Surgeons. Published by Elsevier Inc. All rights reserved.)

Belatacept is the first costimulatory blockade agent clinically approved for transplant immunosuppression. Although more than 10 years of study have demonstrated that belatacept offers superior long-term renal allograft and patient survival compared to conventional calcineurin inhibitor (CNI)-based immunosuppression regimens, the clinical adoption of belatacept has continued to lag because of concerns of an early risk of acute cellular rejection (ACR) and various logistical barriers to its administration. In this review, the history of the clinical development of belatacept is examined, along with the findings of the seminal BENEFIT and BENEFIT-EXT trials culminating in the clinical approval of belatacept. Recent efforts to incorporate belatacept into novel CNI-free immunosuppression regimens are reviewed, as well as the experience of the Emory Transplant Center in using a tapered course of low-dose tacrolimus in belatacept-treated renal allograft patients to garner the long-term outcome benefits of belatacept without the short-term increased risks of ACR. Potential avenues to increase the clinical adoption of belatacept in the future are explored, including surmounting the logistical barriers of belatacept administration through subcutaneous administration or more infrequent belatacept dosing. In addition, belatacept conversion strategies and potential expanded clinical indications of belatacept are discussed for pediatric transplant recipients, extrarenal transplant recipients, treatment of antibody-mediated rejection (AMR), and in patients with failed renal allografts. Finally, we discuss the novel immunosuppressive drugs currently in the development pipeline that may aid in the expansion of costimulation blockade utilization., (© 2023 International Society of Nephrology. Published by Elsevier Inc.)

Maintenance immunosuppression with belatacept following kidney transplantation results in improved long-term graft function as compared with calcineurin inhibitors. However, broad application of belatacept has been limited, in part related to logistical barriers surrounding a monthly (q1m) infusion requirement., Methods: To determine whether every 2-mo (q2m) belatacept is noninferior to standard q1m maintenance, we conducted a prospective, single-center randomized trial in low-immunologic-risk, stable renal transplant recipients. Here, post hoc analysis of 3-y outcomes, including renal function and adverse events, are reported., Results: One hundred sixty-three patients received treatment in the q1m control group (n = 82) or q2m study group (n = 81). Renal allograft function as measured by baseline-adjusted estimated glomerular filtration rate was not significantly different between groups (time-averaged mean difference of 0.2 mL/min/1.73 m 2 ; 95% confidence interval: -2.5, 2.9). There were no statistically significant differences in time to death or graft loss, freedom from rejection, or freedom from donor-specific antibodies (DSAs). During the extended 12- to 36-mo follow-up, 3 deaths, 1 graft loss occurred in the q1m group, compared with 2 deaths, and 2 graft losses in the q2m group. In the q1m group, 1 patient developed DSAs and acute rejection. In the q2m group, 3 patients developed DSAs and 2 associated with acute rejection., Conclusions: Based on the similar renal function and survival at 36 mo compared with q1m, q2m belatacept is a potentially viable maintenance immunosuppressive strategy in low immunologic risk kidney transplant recipients that may facilitate increased clinical utilization of costimulation blockade-based immunosuppression., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2023 The Author(s). Transplantation Direct. Published by Wolters Kluwer Health, Inc.)

Background: Belatacept improves long-term graft survival, but control of some primary viral infections may be impaired. We evaluated the impact of belatacept and tacrolimus on cytomegalovirus (CMV) viral control, remission and relapse in CMV high-risk and moderate-risk recipients., Methods: Using a multistate Markov model, we evaluated viral load state transitions of 173 kidney transplant recipients with at least one episode of viremia within 1 year after transplant: state 1, undetectable/low viral load; state 2, moderate viremia; and state 3, severe viremia., Results: Among high-risk recipients, belatacept-treated recipients exhibited a significantly higher probability of entering moderate viremia (.36; 95% CI = .31, .41) than tacrolimus-treated recipients (.20; 95% CI = .13, .29). The expected number of days in viremic states differed. High-risk belatacept-treated recipients persisted in moderate viremia for significantly longer (128 days, 95% CI = 110, 146) than did tacrolimus-treated recipients (70.0 days, 95% CI = 45.2, 100) and showed a trend of shorter duration in low/undetectable viral load state (172 days, 95% CI = 148, 195) than did tacrolimus-treated recipients (239 days, 95% CI = 195, 277). Moderate-risk recipients showed better viral load control and with no differences by immunosuppression., Conclusion: High-risk belatacept-treated recipients showed defects in sustaining viral control relative to tacrolimus-treated recipients. Avoidance of initial use belatacept in high-risk recipients or development of modified management protocols should be considered., (© 2022 The Authors. Transplant Infectious Disease published by Wiley Periodicals LLC.)

The adoption of de novo belatacept in kidney transplant (kTx) recipients was hampered by an increased risk of acute cellular rejection (ACR) with variation in adopted belatacept based immunosuppressive therapies across centers. We used data from the Scientific Registry of Transplant Recipients (SRTR) to evaluate the temporal trends in belatacept use and describe the associated induction and maintenance regimens in US adult kTx recipients transplanted between June 2011 and December 2018. The number of patients receiving de novo-belatacept based immunosuppressive therapy increased from .74% in 2011 to 3.11% in 2016. In 2016, 66/207 centers used de novo belatacept-based regimen with 3.03% using it in over 50% of their patients. The use of T-cell depleting agents increased with time. Since 2012, the rate of calcineurin inhibitor (CNI) use in combination with belatacept remained stable around 50% and ∼30% remained under belatacept/CNI combination at 1-year post-transplantation. The adoption of belatacept as de novo immunosuppressive regimen has been slow and its use remains low in the United States. Various regimens have been used to modulate the risk of ACR. Further studies evaluating the long-term outcomes of these regimens and assessing their safety especially with regard to the risk of infection are needed., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Competing Interests: The authors declare no conflict of interest.

Donor-specific antibodies (DSA) are a recognized cause of allograft injury, yet biomarkers that indicate their development posttransplant or guide management are not available. CXCL13 (chemokine [C-X-C motif] ligand 1) is a chemoattractant produced within secondary lymphoid organs necessary for germinal center (GC) and alloantibody formation. Perturbations in serum CXCL13 levels have been associated with humoral immune activity. Therefore, CXCL13 may correlate with the formation of HLA antibodies following transplantation., Methods: A murine skin graft model was utilized to define the production and kinetics of CXCL13 in response to alloantigen. Human Tfh:B-cell in vitro cocultures were performed to evaluate CXCL13 production by human lymphocytes, and serum from healthy controls and human transplant recipients with and without de novo DSA was tested for CXCL13., Results: CXCL13 was detectable in the blood of allografted mice and correlated with Tfh and GC B-cell responses. Greater CXCL13 expression was observed in the draining lymph nodes of allografted mice as compared with naïve or syngeneic graft recipients, and serum levels preceded the detection of DSA posttransplant. Similarly, productive human Tfh:B-cell interactions that led to plasmablast differentiation and IgG formation also exhibited CXCL13 expression. CXCL13 levels in human transplant recipients with de novo DSA were greater than in healthy controls and stable transplant patients and also correlated with the development of alloantibodies in a small cohort of serially monitored recipients., Conclusions: CXCL13 indicates GC alloreactivity and alloantibody formation and correlated with DSA formation in kidney transplant recipients, thereby introducing CXCL13 as a potential biomarker for HLA antibodies., Competing Interests: The authors declare no funding or conflicts of interest., (Copyright © 2021 The Author(s). Transplantation Direct. Published by Wolters Kluwer Health, Inc.)

Background: Costimulatory blockade with belatacept has demonstrated long-term benefits in renal transplantation, but de novo use in liver transplant recipients has resulted in increased rejection, graft loss, and death. However, belatacept conversion as a calcineurin inhibitor (CNI) avoidance strategy has not been studied and may be of benefit in liver transplantation where CNI-induced renal dysfunction and toxicity are barriers to improved outcomes., Methods: Using clinical data extracted from our institutional medical record, we report on 8 patients who underwent kidney after liver transplantation and were treated with belatacept-based immunosuppression and transient CNI therapy., Results: All patients tolerated belatacept therapy without any patient deaths or graft losses. No episodes of rejection, de novo donor-specific antibody formation, or major systemic infections were observed, and all patients demonstrated preserved liver and excellent renal allograft function. Patients received belatacept for a median duration of 13.2 mo, and at a median follow-up of 15.9 mo post-kidney transplant, 6 of 8 patients continued on belatacept with 3 completely off and 3 poised to transition off CNI., Conclusions: These findings are the first evidence that in liver transplant recipients requiring subsequent kidney transplantation, belatacept-based therapy can potentially facilitate CNI-free maintenance immunosuppression. This supports the possibility of belatacept conversion in stand-alone liver transplant recipients as a viable method of CNI avoidance., Competing Interests: The authors declare no funding or conflicts of interest., (Copyright © 2021 The Author(s). Transplantation Direct. Published by Wolters Kluwer Health, Inc.)

Belatacept results in improved kidney transplant outcomes, but utilization has been limited by logistical barriers related to monthly (q1m) intravenous infusions. Every 2-month (q2m) belatacept has potential to increase utilization, therefore we conducted a randomized noninferiority trial in low immunologic risk renal transplant recipients greater than 1-year posttransplant. Patients on belatacept were randomly assigned to q1m or q2m therapy. The primary objective was a noninferiority comparison of renal function (eGFR) at 12 months with a noninferiority margin (NIM) of 6.0 ml/min/1.73 m 2 . One hundred and sixty-six participants were randomized to q1m (n = 82) or q2m (n = 84) belatacept, 163 patients received treatment, and 76 q1m and 77 q2m subjects completed the 12-month study period. Every 2-month belatacept was noninferior to q1m, as the difference in mean eGFR adjusted for baseline renal function did not exceed the NIM. Two-month dosing was safe and well tolerated, with no patient deaths or graft losses. Four rejection episodes and three cases of donor-specific antibodies (DSAs) occurred among q2m subjects; however, only one rejection and one instance of DSA were observed in subjects adherent to the study protocol. Every 2-month belatacept therapy may facilitate long-term utilization of costimulation blockade, but future multicenter studies with long-term follow-up will further elucidate immunologic risk. (ClinicalTrials.gov NCT02560558)., (© 2021 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Introduction: Cytomegalovirus (CMV) remains associated with poor outcomes after kidney transplantation (kTx). The impact of belatacept on CMV infection remains understudied. In this study, we assessed the impact of belatacept on patient and graft survivals., Methods: CMV seronegative kTx recipients were included. Patient and graft survival were studied using Kaplan-Meier method, log-rank test. Cox models were used to compare outcomes by CMV risk and immunosuppressive regimen. Incidence and persistence of CMV viremia under belatacept vs tacrolimus were compared., Results: Among 308 CMV seronegative recipients, 168 CMV high-risk and 203 belatacept-treated patients were included. High-risk CMV status was associated with lower patient survival and graft survival. Among the CMV high-risk group, patients treated with belatacept presented a higher incidence of CMV viremia, a higher rate of first-line treatment failure and a longer time to virus clearance. They had a nonsignificant trend toward a lower graft survival., Conclusion: Belatacept-based maintenance immunosuppression is associated with an increased risk of CMV primary-infection and a prolonged course of viral replication in CMV high-risk patients. Further studies are needed to confirm the nonsignificant trend towards a lower graft survival in CMV high-risk patients treated with belatacept and whether it is explained by the higher risk of CMV reactivation and infection., (© 2020 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Anti-donor antibodies cause immunologic injury in transplantation. CD28 blockade with CTLA-4-Ig has the ability to reduce the incidence of these donor-specific antibodies (DSA), but its mechanism is suboptimal for the inhibition of alloimmunity in that CTLA-4-Ig blocks both CD28 costimulation and CTLA-4 coinhibition. Thus selective CD28 blockade that spares CTLA-4 has potential to result in improved inhibition of humoral alloimmunity. To test this possibility, we utilized a full allogeneic mismatch murine transplant model and T follicular helper (Tfh):B cell co-culture system. We observed that selective blockade with an anti-CD28 domain antibody (dAb) compared to CTLA-4-Ig led to superior inhibition of Tfh cell, germinal center, and DSA responses in vivo and better control of B cell responses in vitro. CTLA-4 blockade enhanced the humoral alloresponse and, in combination with anti-CD28 dAb, abrogated the effects of selective blockade. This CTLA-4-dependent inhibition was Tfh cell specific in that CTLA-4 expression by Tfh cells was necessary and sufficient for the improved humoral inhibition observed with selective CD28 blockade. As CD28 blockade attracts interest for control of alloantibodies in the clinic, these data support selective CD28 blockade as a superior strategy to address DSA via the sparing of CTLA-4 and more potent targeting of Tfh cells., (© 2020 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Recent evidence suggests that belatacept reduces the durability of preexisting antibodies to class I and class II human leukocyte antigens (HLAs). In this case series of 163 highly sensitized kidney transplant candidates whose calculated panel-reactive antibody (cPRA) activity was ≥98% to 100%, the impact of belatacept on preexisting HLA antibodies was assessed. Of the 163 candidates, 72 underwent transplantation between December 4, 2014 and April 15, 2017; 60 of these transplanted patients remained on belatacept consecutively for at least 6 months. We observed a decrease in the breadth and/or strength of HLA class I antibodies as assessed by FlowPRA in belatacept-treated patients compared to controls who did not receive belatacept. Specifically, significant HLA antibody reduction was evident for class I (P < .0009). Posttransplant belatacept-treated patients also had a clinically significant reduction in their cPRA compared to controls (P < .01). Collectively, these findings suggest belatacept can reduce HLA class I antibodies in a significant proportion of highly sensitized recipients and could be an option to improve pretransplant compatibility with organ donors., (© 2019 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Donor-specific antibodies (DSAs) contribute to renal allograft loss. However, biomarkers to guide clinical management of DSA posttransplant or detect humoral alloimmune responses before alloantibodies develop are not available. Circulating T follicular helper (cTfh) cells are CD4 + CXCR5 + Tfh-like cells in the blood that have been associated with alloantibodies in transplant recipients, but whether they precede antibody formation for their evaluation as a predictive biomarker in transplant is unknown. To evaluate the ability of cTfh cells to predict DSA, we used murine transplant models to determine the temporal relationship between cTfh cells, germinal center formation, and DSA development. We observed that donor-reactive CD4 + CXCR5 + cTfh cells expand after allotransplant. These cTfh cells were equivalent to graft-draining lymph node-derived Tfh cells in their ability to provide B cell help for antibody production. cTfh cell expansion and differentiation into ICOS + PD-1 + cells temporally correlated with germinal center alloreactivity and preceded the generation of DSAs in instances of modified and unmodified alloantibody formation. Importantly, delayed costimulation blockade initiated after the detection of ICOS + PD-1 + cTfh cells prevented DSAs. These findings suggest that cTfh cells could serve as a biomarker for humoral alloreactivity before the detection of alloantibodies and inform therapeutic approaches to prevent DSAs., (© 2019 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Renal injury almost always accompanies the multisystem organ failure that precedes cardiac transplantation and renal function is further compromised by the nephrotoxicity of calcineurin inhibitors posttransplant. Renal dysfunction in turn causes significant morbidity and mortality. The development of belatacept was motivated by need for an alternative to calcineurin-based immunosuppression, particularly in renal transplantation where the nephrotoxicity of calcineurin inhibitors reduce graft longevity and adverse cardiovascular effects of calcineurin inhibitors increase overall mortality. In 2011, the FDA approved belatacept for use in renal transplantation. Seven-year data from the multicenter randomized phase III BENEFIT trial, which compared belatacept with cyclosporine in renal transplant recipients, show belatacept therapy offers both improved renal function and 43% risk reduction for the combined endpoint of graft loss and death. At present, belatacept use is predominantly confined to renal transplant recipients; however, reports of belatacept use in other transplant settings are emerging. Here, we describe successful long-term use of belatacept in a kidney-after-heart transplant recipient and review use of belatacept in cardiothoracic and other nonrenal transplant settings., Competing Interests: C.P.L. is an investigator on BMS sponsored clinical trials. The other authors declare no conflicts of interest., (Copyright © 2019 The Author(s). Transplantation Direct. Published by Wolters Kluwer Health, Inc.)

Purpose of Review: Highlight developments in T and B cell biology that are helping elucidate the mechanisms underlying CD28 pathway blockade-mediated inhibition of alloantibodies in transplantation, and discuss recent clinical observations on the impact of belatacept on de novo and established HLA antibodies., Recent Findings: The identification of T follicular helper cells as the CD4+ T cell subset required for optimal humoral immunity, along with newly identified roles for CD28 and the B7 molecules on B cell lineage cells has begun to pave the way for improved understanding and discovery of the mechanisms of CD28 costimulation blockade-mediated antibody inhibition. There has been resurgent clinical interest in the ability of belatacept to attenuate alloantibody responses. New reports have continued to document its ability to prevent de novo antibody responses, and more recent studies have surfaced exploring its potential to control nascent or pre-existing HLA antibodies., Summary: A growing understanding of the mechanisms of anti-CD28-mediated alloantibody inhibition and continued clinical successes will guide the clinical optimization of belatacept and next generation CD28 blockers to prevent and reduce alloantibodies over the long-term., Competing Interests: Conflict of Interest Raul Badell reports grants from NIH/NIAID (K08 AI132747), during the conduct of the study. Ronald F. Parsons, Christian P. Larsen, and Thomas C. Pearson declare no conflict of interest.

A majority of kidney transplant recipients receive calcineurin inhibitor-based immunosuppression. However, some do not tolerate calcineurin inhibitors and require other immunosuppressive strategies. Until recently, alternative approaches have been associated with inferior outcomes, but recent methods have effectively utilized belatacept in calcineurin inhibitor-intolerant patients. Though promising, belatacept uptake has been limited by higher acute rejection rates, unavailability due to production shortages, and logistical challenges as a result of intravenous infusion requirements. Interestingly, its predecessor abatacept is clinically available in subcutaneous formulation to treat autoimmune disorders but has not been used in clinical transplantation. Here we report on a series of 9 calcineurin inhibitor-intolerant transplant recipients converted to abatacept early after transplant as rescue immunosuppression during periods of belatacept unavailability. Retrospective review revealed successful allograft salvage and 100% patient and graft survival (median 115 months) after conversion to abatacept. Patients received abatacept for a median duration of 82 months with stable, long-term renal allograft function, a single cellular rejection episode, and no clinically apparent protective immunity concerns. Hence our findings suggest that future clinical studies utilizing abatacept either de novo or as conversion therapy in transplant recipients should be considered., (© 2019 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Background: Xenogeneic donors would provide an unlimited source of islets for the treatment of type 1 diabetes (T1D). The goal of this study was to assess the function of microencapsulated adult porcine islets (APIs) transplanted ip in streptozotocin (STZ)-diabetic non-human primates (NHPs) given targeted immunosuppression., Methods: APIs were encapsulated in: (a) single barium-gelled alginate capsules or (b) double alginate capsules with an inner, islet-containing compartment and a durable, biocompatible outer alginate layer. Immunosuppressed, streptozotocin-diabetic NHPs were transplanted ip with encapsulated APIs, and graft function was monitored by measuring blood glucose, %HbA1c, and porcine C-peptide. At graft failure, explanted capsules were assessed for biocompatibility and durability plus islet viability and functionality. Host immune responses were evaluated by phenotyping peritoneal cell populations, quantitation of peritoneal cytokines and chemokines, and measurement of anti-porcine IgG and IgM plus anti-Gal IgG., Results: NHP recipients had reduced hyperglycemia, decreased exogenous insulin requirements, and lower percent hemoglobin A1c (%HbA1c) levels. Porcine C-peptide was detected in plasma of all recipients, but these levels diminished with time. However, relatively high levels of porcine C-peptide were detected locally in the peritoneal graft site of some recipients at sacrifice. IV glucose tolerance tests demonstrated metabolic function, but the grafts eventually failed in all diabetic NHPs regardless of the type of encapsulation or the host immunosuppression regimen. Explanted microcapsules were intact, "clean," and free-floating without evidence of fibrosis at graft failure, and some reversed diabetes when re-implanted ip in diabetic immunoincompetent mice. Histology of explanted capsules showed scant evidence of a host cellular response, and viable islets could be found. Flow cytometric analyses of peritoneal cells and peripheral blood showed similarly minimal evidence of a host immune response. Preformed anti-porcine IgG and IgM antibodies were present in recipient plasma, but these levels did not rise post-transplant. Peritoneal graft site cytokine or chemokine levels were equivalent to normal controls, with the exception of minimal elevation observed for IL-6 or IL-1β, GRO-α, I-309, IP-10, and MCP-1. However, we found central necrosis in many of the encapsulated islets after graft failure, and explanted islets expressed endogenous markers of hypoxia (HIF-1α, osteopontin, and GLUT-1), suggesting a role for non-immunologic factors, likely hypoxia, in graft failure., Conclusions: With donor xenoislet microencapsulation and host immunosuppression, APIs corrected hyperglycemia after ip transplantation in STZ-diabetic NHPs in the short term. The islet xenografts lost efficacy gradually, but at graft failure, some viable islets remained, substantial porcine C-peptide was detected in the peritoneal graft site, and there was very little evidence of a host immune response. We postulate that chronic effects of non-immunologic factors, such as in vivo hypoxic and hyperglycemic conditions, damaged the encapsulated islet xenografts. To achieve long-term function, new approaches must be developed to prevent this damage, for example, by increasing the oxygen supply to microencapsulated islets in the ip space., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Memory T cells pose a significant problem to successful therapeutic control of unwanted immune responses during autoimmunity and transplantation, as they are differentially controlled by cosignaling receptors such as CD28 and CTLA-4. Treatment with abatacept and belatacept impede CD28 signaling by binding to CD80 and CD86, but they also have the unintended consequence of blocking the ligands for CTLA-4, a process that may inadvertently boost effector responses. Here, we show that a potentially novel anti-CD28 domain antibody (dAb) that selectively blocks CD28 but preserves CTLA-4 coinhibition confers improved allograft survival in sensitized recipients as compared with CTLA-4 Ig. However, both CTLA-4 Ig and anti-CD28 dAb similarly and significantly reduced the accumulation of donor-reactive CD8+ memory T cells, demonstrating that regulation of the expansion of CD8+ memory T cell populations is controlled in part by CD28 signals and is not significantly impacted by CTLA-4. In contrast, selective CD28 blockade was superior to CTLA-4 Ig in inhibiting IFN-γ, TNF, and IL-2 production by CD8+ memory T cells, which in turn resulted in reduced recruitment of innate CD11b+ monocytes into allografts. Importantly, this superiority was CTLA-4 dependent, demonstrating that effector function of CD8+ memory T cells is regulated by the balance of CD28 and CTLA-4 signaling.

Donor-specific antibodies (DSAs) are a barrier to improved long-term outcomes after kidney transplantation. Costimulation blockade with CTLA4-Ig has shown promise as a potential therapeutic strategy to control DSAs. T follicular helper (Tfh) cells, a subset of CD4 + T cells required for optimal antibody production, are reliant on the CD28 costimulatory pathway. We have previously shown that selective CD28 blockade leads to superior allograft survival through improved control of CD8 + T cells relative to CTLA4-Ig, but the impact of CD28-specific blockade on CD4 + Tfh cells is unknown. Thus, we identified and characterized donor-reactive Tfh cells in a murine skin transplant model and then used this model to evaluate the impact of selective CD28 blockade with an anti-CD28 domain antibody (dAb) on the donor-specific Tfh cell-mediated immune response. We observed that the anti-CD28 dAb led to superior inhibition of donor-reactive CXCR5 + PD-1 high Tfh cells, CD95 + GL7 + germinal center B cells and DSA formation compared with CTLA4-Ig. Interestingly, donor-reactive Tfh cells differentially upregulated CTLA4 expression, suggesting an important role for CTLA4 in mediating the superior inhibition observed with the anti-CD28 dAb. Therefore, selective CD28 blockade as a novel approach to control Tfh cell responses and prevent DSA after kidney transplantation warrants further study., (© 2017 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Infectious pseudoaneurysm (IPA) is a rare but devastating complication following renal transplantation that typically leads to graft loss and occasionally patient death. IPAs following kidney transplantation are most often mycotic in etiology, but have been sporadically reported to result from Pseudomonas aeruginosa infection. These IPAs occur at various anatomic sites, most commonly at the vascular anastomosis or iliac artery, and very rarely in the transplanted renal artery or hilum. Here we report the occurrence of single donor-derived P aeruginosa IPAs in two kidney transplant recipients with divergent allograft outcomes. Both recipients manifested Pseudomonas infections and early, hemodynamically relevant postoperative hemorrhage as a result of pseudoaneurysm rupture. One recipient required allograft nephrectomy during emergent operative exploration due to rupture of a pseudoaneurysm at the vascular anastomosis. Conversely, the other recipient's allograft was salvaged by endovascular stenting of a pseudoaneurysm unusually located in the main donor renal artery. To the best of our knowledge, this is the first case of a ruptured IPA occurring in the transplanted renal artery with successful allograft salvage via endovascular technique. In this report, we discuss details of the two cases, relevant literature, and possible clinical implications., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Purpose of Review: T follicular helper (Tfh) cells are an increasingly relevant CD4 T cell subset responsible for the provision of help to B cells for the generation of an effective humoral immune response. Here we review recent studies that have provided critical insights into the mechanisms of Tfh cell differentiation and function, and introduce newly identified roles for Tfh cells in human disease., Recent Findings: Novel molecular regulators of the Tfh cell differentiation program along with new found roles for the costimulatory and coinhibitory inducible T cell costimulator (ICOS), programmed death 1, and cytotoxic T lymphocyte antigen 4 pathways on Tfh cell function have been appreciated. Although circulating Tfh and Tfh-like subset signatures have been linked to numerous immune conditions, extrapolation of these findings to organ transplantation is just beginning., Summary: The combination of recent progress with regard to Tfh cell biology at the basic science and clinical levels is guiding the elucidation of the role of Tfh cells in the alloimmune response. Application of this knowledge toward the development of novel therapeutic strategies for use in transplantation is imminent.

Recent studies have shown that the quantity of donor-reactive memory T cells is an important factor in determining the relative heterologous immunity barrier posed during transplantation. Here, we hypothesized that the quality of T cell memory also potently influences the response to costimulation blockade-based immunosuppression. Using a murine skin graft model of CD8(+) memory T cell-mediated costimulation blockade resistance, we elicited donor-reactive memory T cells using three distinct types of pathogen infections. Strikingly, we observed differential efficacy of a costimulation and integrin blockade regimen based on the type of pathogen used to elicit the donor-reactive memory T cell response. Intriguingly, the most immunosuppression-sensitive memory T cell populations were composed primarily of central memory cells that possessed greater recall potential, exhibited a less differentiated phenotype, and contained more multi-cytokine producers. These data, therefore, demonstrate that the memory T cell barrier is dependent on the specific type of pathogen infection via which the donor-reactive memory T cells are elicited, and suggest that the immune stimulation history of a given transplant patient may profoundly influence the relative barrier posed by heterologous immunity during transplantation., (© Copyright 2015 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Mounting evidence in models of both autoimmunity and chronic viral infection suggests that the outcome of T cell activation is critically impacted by the constellation of co-stimulatory and co-inhibitory receptors expressed on the cell surface. Here, we identified a critical role for the co-inhibitory SLAM family member 2B4 (CD244) in attenuating primary antigen-specific CD8(+) T cell responses in the presence of immune modulation with selective CD28 blockade. Our results reveal a specific up-regulation of 2B4 on antigen-specific CD8(+) T cells in animals in which CD28 signaling was blocked. However, 2B4 up-regulation was not observed in animals treated with CTLA-4 Ig (abatacept) or CD28 blockade in the presence of anti-CTLA-4 mAb. 2B4 up-regulation after CD28 blockade was functionally significant, as the inhibitory impact of CD28 blockade was diminished when antigen-specific CD8(+) T cells were deficient in 2B4. In contrast, 2B4 deficiency had no effect on CD8(+) T cell responses during unmodified rejection or in the presence of CTLA-4 Ig. We conclude that blockade of CD28 signals in the presence of preserved CTLA-4 signals results in the unique up-regulation of 2B4 on primary CD8(+) effectors, and that this 2B4 expression plays a critical functional role in controlling antigen-specific CD8(+) T cell responses.

Background: Improved outcomes have expanded the indications for liver transplantation, thus aggravating the already limited supply of donor organs. Domino liver transplantation (DLT) has been one strategy to augment the supply of donor organs in cases of inborn errors of metabolism. One such disease is maple syrup urine disease (MSUD), an inherited disorder of branched-chain amino acid (BCAA) metabolism., Methods: We report on the transplantation of a deceased donor liver into a patient with MSUD, and the sequential transplantation of the explanted liver into a patient with hemophilia A, HIV, hepatitis C, and a low priority on the transplant waiting list., Results: At 30 months, the MSUD recipient has had significant correction of BCAA metabolism on a protein-unrestricted diet and no progression of neuropsychiatric symptoms. The DLT recipient has been cured of hemophilia and has normal BCAA homeostasis. This case provides further evidence that elective orthotopic liver transplantation for MSUD attenuates the disease with restoration of BCAA metabolism, and that DLT in this setting can achieve excellent results in ESLD patients., Conclusion: It is possible that domino grafts from patients with MSUD could be used in more conventional recipients, but additional studies and longer-term outcomes are needed to determine the validity of DLT in MSUD., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Calcineurin inhibitors (CNI) and steroids are known to promote insulin resistance, and their avoidance after islet transplantation is preferred from a metabolic standpoint. Belatacept, a B7-specific mediator of costimulation blockade (CoB), is clinically indicated as a CNI alternative in renal transplantation, and we have endeavored to develop a clinically translatable, belatacept-based regimen that could obviate the need for both CNIs and steroids. Based on the known synergy between CoB and mTOR inhibition, we studied rhesus monkeys undergoing MHC-mismatched islet allotransplants treated with belatacept and the mTOR inhibitor, sirolimus. To extend prior work on CoB-resistant rejection, some animals also received CD2 blockade with alefacept (LFA3-Ig). Nine rhesus macaques were rendered diabetic with streptozotocin and underwent islet allotransplantation. All received belatacept and sirolimus; six also received alefacept. Belatacept and sirolimus significantly prolonged rejection-free graft survival (median 225 days compared to 8 days in controls receiving basiliximab and sirolimus; p = 0.022). The addition of alefacept provided no additional survival benefit, but was associated with Cytomegalovirus reactivation in four of six animals. No recipients produced donor-specific alloantibodies. The combination of belatacept and sirolimus successfully prevents islet allograft survival in rhesus monkeys, but induction with alefacept provides no survival benefit and increases the risk of viral reactivation., (© Copyright 2012 The American Society of Transplantation and the American Society of Transplant Surgeons.)

The importance of CD40/CD154 costimulatory pathway blockade in immunosuppression strategies is well-documented. Efforts are currently focused on monoclonal antibodies specific for CD40 because of thromboembolic complications associated with monoclonal antibodies directed towards CD154. Here we present the rational development and characterization of a novel antagonistic monoclonal antibody to CD40. Rhesus macaques were treated with the recombinant anti-CD40 mAb, 2C10, or vehicle before immunization with keyhole limpet hemocyanin (KLH). Treatment with 2C10 successfully inhibited T cell-dependent antibody responses to KLH without significant peripheral B cell depletion. Subsequently, MHC-mismatched macaques underwent intraportal allogeneic islet transplantation and received basiliximab and sirolimus with or without 2C10. Islet graft survival was significantly prolonged in recipients receiving 2C10 (graft survival time 304, 296, 265, 163 days) compared to recipients receiving basiliximab and sirolimus alone (graft survival time 8, 8, 10 days). The survival advantage conferred by treatment with 2C10 provides further evidence for the importance of blockade of the CD40/CD154 pathway in preventing alloimmune responses. 2C10 is a particularly attractive candidate for translation given its favorable clinical profile., (© Copyright 2012 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Immunosuppressive therapies that block the CD40/CD154 costimulatory pathway have proven to be uniquely effective in preclinical xenotransplant models. Given the challenges facing clinical translation of CD40/CD154 pathway blockade, we examined the efficacy and tolerability of CD40/CD154 pathway-sparing immunomodulatory strategies in a pig-to-nonhuman primate islet xenotransplant model. Rhesus macaques were rendered diabetic with streptozocin and given an intraportal infusion of ≈ 50 000 islet equivalents/kg wild-type neonatal porcine islets. Base immunosuppression for all recipients included maintenance therapy with belatacept and mycophenolate mofetil plus induction with basiliximab and LFA-1 blockade. Cohort 1 recipients (n = 3) were treated with the base regimen alone; cohort 2 recipients (n = 5) were additionally treated with tacrolimus induction and cohort 3 recipients (n = 5) were treated with alefacept in place of basiliximab, and more intense LFA-1 blockade. Three of five recipients in both cohorts 2 and 3 achieved sustained insulin-independent normoglycemia (median rejection-free survivals 60 and 111 days, respectively), compared to zero of three recipients in cohort 1. These data show that CD40/CD154 pathway-sparing regimens can promote xenoislet survival. Further optimization of these strategies is warranted to aid the clinical translation of islet xenotransplantation., (© Copyright 2012 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Islet transplantation to treat type 1 diabetes has been limited in part by toxicities of current immunosuppression and recipient humoral sensitization. Blockade of the CD28/CD80/86 and CD40/CD154 pathways has shown promise to remedy both these limitations, but translation has been hampered by difficulties in translating CD154-directed therapies. Prior CD40-directed regimens have led to prolonged islet survival, but fail to prevent humoral allosensitization. We therefore evaluated the addition of CTLA4Ig to a CD40 blockade-based regimen in nonhuman primate (NHP) alloislet transplantation. Diabetic rhesus macaques were transplanted allogeneic islets using the CD40-specific antibody 3A8, basiliximab induction, and sirolimus with or without CTLA4Ig maintenance therapy. Allograft survival was determined by fasting blood glucose levels and flow cytometric techniques were used to test for donor-specific antibody (DSA) formation. CTLA4Ig plus 3A8, basiliximab and sirolimus was well tolerated and induced long-term islet allograft survival. The addition of CTLA4Ig prevented DSA formation, but did not facilitate withdrawal of the 3A8-based regimen. Thus, CTLA4Ig combines with a CD40-specific regimen to prevent DSA formation in NHPs, and offers a potentially translatable calcineurin inhibitor-free protocol inclusive of a single investigational agent for use in clinical islet transplantation without relying upon CD154 blockade., (© Copyright 2012 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Costimulation blockade of the CD40/CD154 pathway has been effective at preventing allograft rejection in numerous transplantation models. This strategy has largely depended on mAbs directed against CD154, limiting the potential for translation due to its association with thromboembolic events. Though targeting CD40 as an alternative to CD154 has been successful at preventing allograft rejection in preclinical models, there have been no reports on the effects of CD40-specific agents in human transplant recipients. This delay in clinical translation may in part be explained by the presence of cellular depletion with many CD40-specific mAbs. As such, the optimal biologic properties of CD40-directed immunotherapy remain to be determined. In this report, we have characterized 3A8, a human CD40-specific mAb and evaluated its efficacy in a rhesus macaque model of islet cell transplantation. Despite partially agonistic properties and the inability to block CD40 binding of soluble CD154 (sCD154) in vitro, 3A8-based therapy markedly prolonged islet allograft survival without depleting B cells. Our results indicate that the allograft-protective effects of CD40-directed costimulation blockade do not require sCD154 blockade, complete antagonism or cellular depletion, and serve to support and guide the continued development of CD40-specific agents for clinical translation., (©Copyright 2011 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Significant deficiencies in understanding of xenospecific immunity have impeded the success of preclinical trials in xenoislet transplantation. Although galactose-α1,3-galactose, the gal epitope, has emerged as the principal target of rejection in pig-to-primate models of solid organ transplant, the importance of gal-specific immunity in islet xenotransplant models has yet to be clearly demonstrated. Here, we directly compare the immunogenicity, survival and function of neonatal porcine islets (NPIs) from gal-expressing wild-type (WT) or gal-deficient galactosyl transferase knockout (GTKO) donors. Paired diabetic rhesus macaques were transplanted with either WT (n = 5) or GTKO (n = 5) NPIs. Recipient blood glucose, transaminase and serum xenoantibody levels were used to monitor response to transplant. Four of five GTKO versus one of five WT recipients achieved insulin-independent normoglycemia; transplantation of WT islets resulted in significantly greater transaminitis. The WT NPIs were more susceptible to antibody and complement binding and destruction in vitro. Our results confirm that gal is an important variable in xenoislet transplantation. The GTKO NPI recipients have improved rates of normoglycemia, likely due to decreased susceptibility of xenografts to innate immunity mediated by complement and preformed xenoantibody. Therefore, the use of GTKO donors is an important step toward improved consistency and interpretability of results in future xenoislet studies., (©Copyright 2011 The American Society of Transplantation and the American Society of Transplant Surgeons.)

The widespread clinical implementation of alloislet transplantation as therapy for type 1 diabetes has been hindered by the lack of suitable islet donors. Pig-to-human islet xenotransplantation is one strategy with potential to alleviate this shortage. Long-term survival of porcine islets has been achieved using CD154-specific antibodies to interrupt the CD40/CD154 costimulation pathway; however, CD154-specific antibodies seem unlikely candidates for clinical translation. An alternative strategy for CD40/CD154 pathway interruption is use of CD40-specific antibodies. Herein, we evaluate the ability of a chimeric CD40-specific monoclonal antibody (Chi220) to protect islet xenografts. Neonatal porcine islets (~50,000 IEQ/kg) were transplanted intraportally into pancreatectomized diabetic macaques. Immunosuppression consisted of induction therapy with Chi220 and the IL-2 receptor-specific antibody basiliximab, and maintenance therapy with sirolimus and the B7-specific fusion protein belatacept. Chi220 effectively promoted xenoislet engraftment and survival, with five of six treated recipients achieving insulin-independent normoglycemia (median rejection-free survival 59 days; mean 90.8 days, maximum 203 days). No thromboembolic phenomena were observed. CD40 represents a promising alternative to CD154 as a therapeutic target, and the efficacy of CD40-specific antibodies in islet xenotransplantation warrants further investigation., (©2011 The Authors Journal compilation©2011 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Outcomes in transplantation have been limited by suboptimal long-term graft survival and toxicities associated with current immunosuppressive approaches. T cell costimulation blockade has shown promise as an alternative strategy to avoid the side effects of conventional immunosuppressive therapies, but targeting CD28-mediated costimulation alone has proven insufficient to prevent graft rejection in primates. Donor-specific memory T (TM) cells have been implicated in costimulation blockade-resistant transplant rejection, due to their enhanced effector function and decreased reliance on costimulatory signaling. Thus, we have tested a potential strategy to overcome TM cell-driven rejection by targeting molecules preferentially expressed on these cells, such as the adhesion molecule lymphocyte function-associated antigen 1 (LFA-1). Here, we show that short-term treatment (i.e., induction therapy) with the LFA-1-specific antibody TS-1/22 in combination with either basiliximab (an IL-2Rα-specific mAb) and sirolimus (a mammalian target of rapamycin inhibitor) or belatacept (a high-affinity variant of the CD28 costimulation-blocker CTLA4Ig) prolonged islet allograft survival in nonhuman primates relative to control treatments. Moreover, TS-1/22 masked LFA-1 on TM cells in vivo and inhibited the generation of alloproliferative and cytokine-producing effector T cells that expressed high levels of LFA-1 in vitro. These results support the use of LFA-1-specific induction therapy to neutralize costimulation blockade-resistant populations of T cells and further evaluation of LFA-1-specific therapeutics for use in transplantation.

Islet transplantation is an experimental therapy for selected patients with type 1 diabetes (T1DM). It remains limited by immunosuppressive drug toxicity, progressive loss of insulin independence, allosensitization and the need for multiple islet donors. We describe our experience with an efalizumab-based immunosuppressive regimen as compared to the prevailing standard regimen, the Edmonton protocol. Twelve patients with T1DM received islet transplants: eight were treated with the Edmonton protocol; four were treated with daclizumab induction, a 6-month course of tacrolimus, and maintenance with efalizumab and mycophenolate mofetil. The primary endpoint was insulin independence after one islet infusion. Only two Edmonton protocol treated patients achieved the primary endpoint; six required islets from multiple donors, and all experienced leukopenia, mouth ulcers, anemia, diarrhea and hypertransaminasemia. Four became allosensitized. All patients treated with the efalizumab-based regimen achieved insulin independence with normal hemoglobin A1c after a single islet cell infusion and remained insulin independent while on efalizumab. These patients experienced significantly fewer side effects and none became allosensitized. Trial continuation was terminated by withdrawal of efalizumab from the market. These data suggest that this efalizumab-based regimen prevents islet rejection, is well tolerated, and allows for single donor islet transplantation., (© 2010 The Authors Journal compilation © 2010 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Postmenopausal osteoporosis and rheumatoid joint destruction result from increased osteoclast formation and bone resorption induced by receptor activator of NF-kappaB ligand (RANKL) and tumor necrosis factor (TNF). Osteoclast formation induced by these cytokines requires NF-kappaB p50 and p52, c-Fos, and NFATc1 expression in osteoclast precursors. c-Fos induces NFATc1, but the relationship between NF-kappaB and these other transcription factors in osteoclastogenesis remains poorly understood. We report that RANKL and TNF can induce osteoclast formation directly from NF-kappaB p50/p52 double knockout (dKO) osteoclast precursors when either c-Fos or NFATc1 is expressed. RANKL- or TNF-induced c-Fos up-regulation and activation are abolished in dKO cells and in wild-type cells treated with an NF-kappaB inhibitor. c-Fos expression requires concomitant RANKL or TNF treatment to induce NFATc1 activation in the dKO cells. Furthermore, c-Fos expression increases the number and resorptive capacity of wild-type osteoclasts induced by TNF in vitro. We conclude that NF-kappaB controls early osteoclast differentiation from precursors induced directly by RANKL and TNF, leading to activation of c-Fos followed by NFATc1. Inhibition of NF-kappaB should prevent RANKL- and TNF-induced bone resorption.

Chronic inflammatory bone diseases, such as rheumatoid arthritis, periodontal disease and aseptic periprosthetic osteolysis, are characterized by bone loss around affected joints and teeth caused by increased osteoclastic bone resorption. This resorption is mediated largely by the increased local production of pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNFa). These cytokines may induce resorption indirectly by affecting the production of the essential osteoclast differentiation factor, receptor activator of NF-kB ligand, and/or its soluble decoy receptor, osteoprotegerin, by osteoblast/stromal cells or directly by enhancing proliferation and/or activity of cells in the osteoclast lineage. The importance of TNFa in the pathogenesis of various forms of bone loss is supported by both experimental and clinical evidence. However, TNFa is not absolutely required for osteoclastogenesis, erosive arthritis, or osteolysis, as all these events could occur in the absence of TNFa and whether TNFa promotes osteoclast formation independently of RANK signaling is still a topic of debate. Here we review our current understanding of the mechanisms whereby TNFa increases osteoclastogenesis in vitro and in vivo.