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ObjectiveWe describe the characteristics of the 15 patients with fingolimod-associated progressive multifocal leukoencephalopathy (PML) identified from the Novartis data safety base and provide risk estimates for the disorder.MethodsThe Novartis safety database was searched for PML cases with a data lock point of August 31, 2017. PML classification was based on previously published criteria. The risk and incidence were estimated using the 15 patients with confirmed PML and the overall population of patients treated with fingolimod.ResultsAs of August 31, 2017, 15 fingolimod-treated patients had developed PML in the absence of natalizumab treatment in the preceding 6 months. Eleven (73%) were women and the mean age was 53 years (median: 53 years). Fourteen of the 15 patients were treated with fingolimod for >2 years. Two patients had confounding medical conditions. Two patients had natalizumab treatment. This included one patient whose last dose of natalizumab was 3 years and 9 months before the diagnosis of PML. The second patient was receiving fingolimod for 4 years and 6 months, which was discontinued to start natalizumab and was diagnosed with PML 3 months after starting natalizumab. Absolute lymphocyte counts were available for 14 of the 15 patients and none exhibited a sustained grade 4 lymphopenia (≤200 cells/μL).ConclusionsThe risk of PML with fingolimod in the absence of prior natalizumab treatment is low. The estimated risk was 0.069 per 1,000 patients (95% confidence interval: 0.039–0.114), and the estimated incidence rate was 3.12 per 100,000 patient-years (95% confidence interval: 1.75–5.15). Neither clinical manifestations nor radiographic features suggested any unique features of fingolimod-associated PML.

BACKGROUND The prevention of recurrent autoimmunity is a prerequisite for successful islet transplantation in patients with type I diabetes. Therapies effective in preserving pancreatic beta-cell mass in patients with newly diagnosed diabetes are good candidates for achieving this goal. Anti-CD3 monoclonal antibody (mAb) and antilymphocyte antisera are the only therapies to date that have cured early diabetic disease in the nonobese diabetic (NOD) mouse. We investigated whether other immunosuppressive therapies, including short-term depleting anti-CD4 mAb or costimulation blockade, would affect the disease progression in recently diabetic NOD mice. We also evaluated the effect of the anti-CD4 mAb on syngeneic and allogeneic graft survival in diabetic NOD recipients. METHODS AND RESULTS We demonstrate that a short course of anti-CD4 mAb early after hyperglycemia onset cured diabetes. Normal islets and islets with CD4+ and CD8+ T-cell peri-insulitic infiltrate were found in the pancreata of cured NOD mice. A similar regimen prevented the recurrence of autoimmune diabetes in NOD/severe combined immunodeficient disease (SCID) islet isografts and delayed the rejection of allogeneic C57BL/6 islet allografts in diabetic female NOD mice. The co-transfer of diabetogenic splenocytes with splenocytes from anti-CD4 mAb-treated and cured NOD mice into 7-week-old, irradiated, NOD male mice was not able to protect from diabetes occurrence. This indicates that an anti-CD4-mediated cure of diabetes is independent of the induction of immunoregulatory T cells. Anti-CD154 mAb and cytotoxic T-lymphocyte antigen 4 immunoglobulin were ineffective in early-onset diabetes. CONCLUSION Our results provide the first evidence that newly established autoimmune islet destruction in NOD mice responds to a short course of anti-CD4 mAb. In contrast, costimulation blockade is ineffective in this clinically relevant model.

The prevalence of hypertension is greater in African Americans, and management of this condition presents challenges for practicing physicians.The effectiveness and safety of perindopril was evaluated in hypertensive African-American patients (n = 1412) and hypertensive white patients (n = 7745) who had participated in a large United States community trial. Patients received perindopril 4 mg once daily for 6 weeks. Based on physicians' clinical judgment at week 6, the dose was either maintained or increased to 8 mg for an additional 6 weeks.Reduction of blood pressure (BP) was significant with perindopril monotherapy (4 to 8 mg once daily) in African Americans and whites (P.001). The magnitude of BP reduction was significantly more in whites (P.001). Up-titration of perindopril achieved additional BP reduction in both ethnic groups (P.001). Control of BP (140/90 mm Hg) in elderly (65 years of age) and diabetic African-Americans subgroups was achieved in 32.1% and 31.6%, respectively. Perindopril was safe and well tolerated.Perindopril monotherapy is effective and is a viable initial therapeutic option as an antihypertensive agent in African-American individuals with hypertension.

The CD134-CD134 ligand (CD134L) costimulatory pathway has been shown to be critical for both T and B cell activation; however, its role in regulating the alloimmune response remains unexplored. Furthermore, its interactions with other costimulatory pathways and immunosuppressive agents are unclear. We investigated the effect of CD134-CD134L pathway blockade on allograft rejection in fully MHC-mismatched rat cardiac and skin transplantation models. CD134L blockade alone did not prolong graft survival compared with that of untreated recipients, and in combination with donor-specific transfusion, cyclosporine, or rapamycin, was less effective than B7 blockade in prolonging allograft survival. However, in combination with B7 blockade, long-term allograft survival was achieved in all recipients (>200 days). Moreover, this was synergistic in reducing the frequency of IFN-γ-producing alloreactive lymphocytes and inhibiting the generation of activated/effector lymphocytes. Most impressively, this combination prevented rejection in a presensitized model using adoptive transfer of primed lymphocytes into athymic heart transplant recipients. In comparison to untreated recipients (mean survival time (MST): 5.3 ± 0.5 days), anti-CD134L mAb alone modestly prolonged allograft survival (MST: 14 ± 2.8 days) as did CTLA4Ig (MST: 21.5 ± 1.7 days), but all grafts were rejected within 24 days. Importantly, combined blockade further and significantly prolonged allograft survival (MST: 75.3 ± 12.7 days) and prevented the expansion and/or persistence of primed/effector alloreactive T cells. Our data suggest that CD134-CD134L is a critical pathway in alloimmune responses, especially recall/primed responses, and is synergistic with CD28-B7 in mediating T cell effector responses during allograft rejection. Understanding the mechanisms of collaboration between these different pathways is important for the development of novel strategies to promote long-term allograft survival.

Background. Immune-mediated injury to the graft has been implicated in the pathogenesis of chronic rejection. However, little is known regarding the nature of the antigen(s) involved in this immune process. We demonstrated that cardiac transplantation in mice induces an autoimmune T-cell response to a heart tissue-specific protein, cardiac myosin (CM). This response contributes to transplant rejection in that its modulation affects cardiac graft survival. This study investigates whether anti-CM T cells undergo activation and expansion in mice with chronic cardiac allograft rejection. Methods. The frequency of CM- and donor major histocompatibility complex (MHC)-specific interferon (IFN)-γ–producing T cells were assessed by ELISPOT in BALB/c mice, which were injected with anti-CD40L (MR1) mAb (chronic rejection group) or CTLA4Ig fusion protein (tolerant group) and transplanted with C57BL/6 cardiac allografts. Results and Conclusions. MR1-treated BALB/c recipients of C57BL/6 hearts with chronic rejection displayed a high frequency of activated CM-specific T cells, whereas the frequency of activated alloreactive T cells were similar to naive, nontransplanted mice. In contrast, no activation of CM-reactive T cells was detected in tolerant recipients after CTLA4Ig treatment. Therefore, in the absence of alloimmunity, chronic rejection is associated with persistence of a T-cell response against CM. Our data indicate that anti-CM autoimmunity may be involved in the immune mechanisms of chronic rejection and suggest that tolerance strategies should target both allo- and autoimmune responses to prevent this process.

Regulatory functions of alloreactive Th2 clones in human renal transplant recipients. Background Chronic allograft rejection is the major clinical problem in organ transplantation. There is evidence that indirect T cell recognition of donor-specific HLA peptides may play an important role in the immunopathogenesis of chronic allograft rejection. We have recently shown that HLA allopeptide-specific T cell clones generated from renal transplant recipients with chronic allograft nephropathy are of the Th1 phenotype, while those from stable patients are Th2. There is evidence in experimental animal models of autoimmunity and transplantation that Th2 cells may function to regulate immune responses, but the biological relevance of these observations in humans has not been reported. Methods The purpose of this study was to investigate the putative regulatory functions of alloreactive human Th2 clones. HLA-DR allopeptide-specific Th1 and Th2 cell clones were generated from peripheral blood lymphocytes of human renal allograft recipients with chronic allograft nephropathy (CAN) or with stable renal function (SRF), respectively. Results An in vitro co-culture system showed that the proliferative responses of Th1 clones from patients with CAN were significantly inhibited by the Th2 clones in response to the donor-derived HLA allopeptides. In addition, co-culture of the Th2 clones inhibited cytokine production (IFN-γ) by the Th1 clones in response to the donor-specific peptides. The regulatory functions of Th2 clones were antigen-specific since they only occurred when both the Th1 and Th2 clones were reactive to the same HLA-DR allopeptide, and were mediated by IL-4 and IL-10. Conclusions This is the first demonstration, to our knowledge, indicating that Th2 cells may function to regulate indirect Th1 alloimmune responses that are critical for the progression of CAN in humans.

The role of immune response to tissue-specific Ags in transplant rejection is poorly defined. We have previously reported that transplantation of cardiac allografts triggers a CD4+ Th1 cell response to cardiac myosin (CM), a major contractile protein of the heart, and that pretransplant activation of proinflammatory CM-specific T cells accelerates rejection. In this study, we show that administration of CM together with IFA (CM/IFA) can prevent acute rejection of an allogeneic heart transplant. Prolongation of cardiac graft survival is associated with activation of CM- and allo-specific T cells secreting type 2 cytokines (IL-4, IL-5) and reduction of the frequency of proinflammatory IFN-γ-secreting (type 1) alloreactive T cells. Blocking of IL-4 cytokine with Abs abrogates the prolongation. CM/IFA treatment prevents acute rejection of MHC class I-mismatched, but not fully mismatched grafts. However, if donor heart is devoid of MHC class II expression, CM-IFA administration delays rejection of fully allogeneic cardiac transplants. This finding suggests that the effect of CM modulation depends on the type (direct vs indirect) and strength of recipient’s CD4+ T cell alloresponse. Our results underscore the important role of host immunity to tissue-specific Ags in the rejection of an allograft. This study demonstrates that modulation of the immune response to a tissue-specific Ag can significantly prolong cardiac allograft survival, an observation that may have important implications for the development of novel selective immune therapies in transplantation.

Background. The CD40-CD154 interaction is critically important in the cell-mediated immune responses. Blockade of this costimulatory pathway has been shown to prevent acute allograft rejection in murine, as well as nonhuman primate models. However, the role of the CD40-CD154 pathway in the development of chronic rejection and the effects of CD154 targeting on progression of chronic rejection have not been evaluated. Methods. We examined the effect of AH.F5, a new hamster anti-rat CD154 monoclonal antibody, in a fully allogeneic acute u ) into Lewis [LEW] (RT1 1 )and chronic [WF.1L (RT1 I ) into LEW (RT1 1 )] vascularized cardiac allograft rejection model. In the chronic model, the antibody was evaluated for prevention (starting day of transplant) and interruption of progression (starting day 30 or 60 after transplant) of chronic vasculopathy. Graft survival, morphology, and immunohistology were evaluated. Results. In the acute rejection model, anti-CD154 therapy alone prevented acute allograft rejection and resulted in 50% long-term allograft survival (>200 days) and donor-specific tolerance. In recipients treated with anti-CD154 monoclonal antibody in combination with a short course of cyclosporine, 100% of allografts survived long-term and all recipients achieved donor-specific tolerance. In the chronic rejection model, allografts from animals treated with the anti-CD154 antibody had a statistically significant lower score of graft arteriosclerosis and fibrosis in both the prevention and 30-day interruption groups when compared with control allografts. In addition, immunohistochemistry showed a decrease in intragraft mononuclear cell infiltration and activation. Conclusion. A new anti-CD154 antibody not only prevents acute allograft rejection, but also inhibits and interrupts the development of chronic rejection. In the acute rejection model cyclosporine acts synergistically with anti-CD154 therapy to prolong allograft survival and induce tolerance. In the chronic rejection model relatively early initiation of therapy is essential to prevent progression of chronic allograft vasculopathy and fibrosis.

There is increasing evidence that ongoing T-cell recognition of alloantigen and activation are key mediators of chronic allograft rejection. The CD28-B7 pathway is unique among costimulatory pathways in that two alternate ligands for B7 exist: CD28 and CTLA4. Recently, it has been suggested that CTLA4 negative signaling may be required for induction of acquired tolerance in vivo. A strategy by which the T cell is targeted at the CD28 receptor rather than its ligands would theoretically allow the inhibitory functions of the CTLA4-B7-1/2 axis to remain intact. Using a rat-specific monoclonal antibody, we investigated the effect of targeting CD28 in a model of chronic rejection without the confounding variable of immunosuppression. We also used an acute cardiac allograft rejection model to investigate CD28 stimulation-based strategies to induce donor-specific tolerance. We demonstrated that anti-CD28 monoclonal antibody was as effective as CTLA4 immunoglobulin in protecting against chronic allograft vasculopathy. In addition, a short course of cyclosporine therapy synergized with either anti-CD28 monoclonal antibody or CTLA4 immunoglobulin, suggesting that it may be clinically relevant to combine low-dose calcineurin inhibitors with CTLA4 immunoglobulin or anti-B7 antibodies. Finally, we report on the potential mechanisms of action of targeting CD28 in vivo.

Experimental autoimmune encephalomyelitis (EAE) is a T cell-mediated disease initiated by antigen-specific CD4(+) T cells. Signaling through CD28 is a critical second signal for activation of T cells, and CD28 knockout (CD28KO) mice have been reported to be resistant to induction of EAE. We now report that CD28KO mice have no intrinsic defect in mediating disease, because they developed EAE after passive transfer of primed T cells. After immunization, peripheral T cells from CD28KO mice were primed and developed memory phenotype, but had decreased antigen-specific IFN-gamma production as compared with cells from wild-type (WT) animals. Reimmunization of CD28KO mice brought out clinical disease and increased IFN-gamma production in vitro. Pathologically, there were cellular infiltrates in the central nervous system, in contrast to single-immunized mice. We show furthermore that blocking B7-1 or CTLA4, but not B7-2, in CD28KO mice induces disease after a single immunization. Thus, EAE can be induced in animals lacking CD28-dependent costimulation, suggesting that alternative costimulatory pathways were used. Blocking the OX40-OX40L costimulatory pathway differentially affected disease induction in CD28KO mice as compared with WT controls. Our data show that EAE may develop in the absence of CD28 T-cell costimulation. These findings have implications for therapies aimed at blocking costimulatory signals in autoimmune diseases.

Abscesses are a classic host response to infection by many pathogenic bacteria. The immunopathogenesis of this tissue response to infection has not been fully elucidated. Previous studies have suggested that T cells are involved in the pathologic process, but the role of these cells remains unclear. To delineate the mechanism by which T cells mediate abscess formation associated with intra-abdominal sepsis, the role of T-cell activation and the contribution of antigen-presenting cells via CD28-B7 costimulation were investigated. T cells activated in vitro by zwitterionic bacterial polysaccharides (Zps) known to induce abscess formation required CD28-B7 costimulation and, when adoptively transferred to the peritoneal cavity of naı̈ve rats, promoted abscess formation. Blockade of T-cell activation via the CD28-B7 pathway in animals with CTLA4Ig prevented abscess formation following challenge with different bacterial pathogens, includingStaphylococcus aureus,Bacteroides fragilis, and a combination ofEnterococcus faeciumandBacteroides distasonis. In contrast, these animals had an increased abscess rate following in vivo T-cell activation via CD28 signaling. Abscess formation in vivo and T-cell activation in vitro required costimulation by B7-2 but not B7-1. These results demonstrate that abscess formation by pathogenic bacteria is under the control of a common effector mechanism that requires T-cell activation via the CD28–B7-2 pathway.

T-cell costimulation is critical for all T-cell-mediated immune responses that are responsible for endpoints such as allograft rejection or autoimmune disease. Recent experimental data elucidate specific pathways for T-cell activation and negative regulatory mechanisms via unique costimulatory molecules. These data have implications for the development of novel therapeutic strategies to prevent graft rejection and improve long-term graft survival in transplant patients, and to treat autoimmune diseases in humans.

Background. There is speculation that coronary angiography may be overused for the assessment of coronary artery disease. Because of its proved ability to differentiate high- and low-risk subsets of patients with coronary artery disease, myocardial perfusion scintigraphy should be an effective strategy in patient selection for angiography. This retrospective clinical study analyzed the relation between scintigraphic findings and subsequent angiography. Methods and Results. Coronary angiographic rates were determined by following up on all stress single-photon emission computed tomographic (SPECT) myocardial perfusion studies performed in a cardiology practice nuclear laboratory during a 26-month interval. All patients were followed up for at least 3 months; mean follow-up was 8.9 months. Subsequent angiography was determined from catheterization laboratory logs, medical records, and telephone contact. Scintigraphic studies were graded according to presence or absence of reversible perfusion defects, affected coronary territories, and lung uptake of 201Tl. Scans were categorized high risk if more than two of the following three criteria were met: reversibility of left anterior descending or multivessel distributions or abnormal lung uptake of thallium. Of 4162 studies, 60% had reversible perfusion defects. Of such studies, 32% were followed up by angiography versus 3.5% without reversible defects. Among studies with reversible defects, the subsequent angiography rate was 60% for those showing high-risk reversibility, compared with 9% for all other studies demonstrating reversibility. Multivariate logistic regression identified high-risk reversibility (odds ratio 20.96) and any reversibility (odds ratio 8.22) as the strongest predictors of angiography. Other correlates of lesser statistical significance were angina and absence of prior infarction or coronary bypass. Conclusions. In this large retrospective study, the results of SPECT scintigraphy overpowered all other clinical and treadmill characteristics in determining the likelihood of subsequent coronary angiography. Only rarely were patients categorized as relatively low risk by scintigraphy referred for angiography. As such, SPECT scintigraphy as used in a private practice, self-referral environment appears to be effective in stratifying potential candidates for coronary angiography.

Focal segmental glomerulosclerosis and progressive renal failure have been reported in reduced renal mass models. However, these findings are not consistent across all species. Increased intracapillary pressure and chronic glomerular hyperfiltration have been shown to contribute to this progression. Studies also have shown that a strong correlation exists between higher glomerular size and the degree of glomerular sclerosis that develops following loss of functioning nephrons.To evaluate the relationship between glomerular size and long-term renal function following massive renal mass removal we describe 9 patients with less than 50% renal mass. All patients had undergone initial nephrectomy followed by partial second nephrectomy, with 30% to 50% of the second kidney excised for localized tumor.In 3 patients moderate to severe renal failure developed. Mean planar glomerular area (MPA) was 29.45 +/- 2.2 mm3 for all patients. Multivariate linear regression analysis demonstrated a significant association between MPA 30 mm3 or greater and increased serum creatinine. For patients with MPA 30 mm3 or greater the predicted delta creatinine was 2.43 mg/dl higher than that in patients with MPA less than 30 mm3 (p = 0.0028). Age was the only other covariate significantly associated with outcome in multivariate analysis.Our data indicate an individual susceptibility toward developing renal failure after significant renal mass reduction. We suggest that patients with less than 50% renal mass and higher MPA are at greater risk for progressive renal failure. MPA may be used as a marker in high risk patients with substantial loss of renal mass to predict long-term renal function.

The contribution of direct and indirect alloresponses by CD4(+) Th1 and Th2 cells in acute and chronic rejection of allogeneic transplants remains unclear. In the present study, we addressed this question using a transplant model in a single MHC class I-disparate donor-recipient mouse combination. BALB/c-dm2 (dm2) mutant mice do not express MHC class I L(d) molecules and reject acutely L(d+) skin grafts from BALB/c mice. In contrast, BALB/c hearts placed in dm2 mice are permanently accepted in the absence of chronic allograft vasculopathy. In this model, CD4(+) T cells are activated following recognition of a donor MHC class I determinant, L(d) 61-80, presented by MHC Class II A(d) molecules on donor and recipient APC. Pre-transplantation of recipients with L(d) 61-80 peptide emulsified in complete Freund's adjuvant induced a Th1 response, which accelerated the rejection of skin allografts, but it had no effect on cardiac transplants. In contrast, induction of a Th2 response to the same peptide abrogated the CD8(+) cytotoxic T cells response and markedly delayed the rejection of skin allografts while it induced de novo chronic rejection of heart transplants. This shows that Th2 cells activated via indirect allorecognition can exert dual effects on acute and chronic rejection of allogeneic transplants.

The authors evaluated, in a community-based open-label trial, the effectiveness and safety of perindopril in 13,220 US hypertensive patients and studied how physicians adhere to hypertension treatment guidelines. Patients received perindopril 4 mg q.d. for 6 weeks. Based on physicians' perception of blood pressure response, the patient was either maintained on 4 mg or the dose was increased to 8 mg for an additional 6 weeks. From baseline to week 12, the mean sitting blood pressure significantly declined from 156.9/94.5 mm Hg to 139.2/84.0 mm Hg. Further dose titration resulted in a clinically significant reduction in blood pressure in all patients with inadequate response on 4 mg at week 6. Blood pressure control (

Chemokines are a group of small, pro-inflammatory molecules first described for their pivotal role in the mobilization of specific leukocyte subsets towards sites of inflammation and their activation once they arrive. They have now emerged as key regulators in the development, differentiation and anatomic distribution of inflammatory cells. Chemokines also orchestrate both the innate immune response and antigen specific immunity through their coordination of dendritic cells and lymphocytes. Due to their vast functional responsibilities, they are linked to the pathogenesis of many seemingly unrelated diseases that include HIV infection, cancer, atherosclerosis, autoimmune diseases, graft rejection and dermatological disorders. This review focuses on the physiology of chemokines and their significant roles in the pathogenesis and progression of major diseases.

Blocking T-cell costimulatory activation pathways is an effective strategy in preventing allograft rejection, promoting long-term survival, and inducing tolerance in some experimental transplant models. 1–8 The mechanisms of action of T-cell costimulatory blockade in vivo include induction of T-cell anergy, apoptosis, regulatory cells, and immune deviation. 9,10 Recent studies also demonstrated the efficacy of CD28-B7 and CD154-CD40 blockade in prolonging primate renal and islet allograft survival as relevant preclinical models for future translation to humans. 11–15 Furthermore, costimulatory blockade has been extensively studied as a promising therapeutic strategy not only in transplantation but also in autoimmunity, allergy, and infections. 9,16 Indeed, the efficacy of CTLA4Ig therapy has already been proven clinically in the autoimmune disease psoriasis vulgaris. 17–19 Phase I-II studies are underway with CTLA4Ig, humanized anti-B7, and anti-CD154 monoclonal antibodies (mAbs) in transplantation and autoimmunity. One of the major challenges to developing T-cell costimulatory blockade strategies for the clinic, especially in the transplant setting, is understanding the interactions between agents that block T-cell costimulation and conventional immunosuppressive drugs currently in clinical use. 20 This is an extremely important and clinically relevant issue since immunosuppressive drugs may abrogate, synergize with, or not affect the functions of such agents. Previous reports showed that cyclosporine but not rapamycin abrogated the effect of combined blockade of CD28-B7 (by CTLA4Ig) and CD154-CD40 (by anti-CD154 mAb) costimulatory pathways in rodent transplantation models. 6,21,22 Smiley et al. also reported the distinct effects of some immunosuppressive drugs on anti-CD154 mAb therapy and showed that cyclosporine and steroids but not rapamycin abrogated the effect of anti-CD154 mAb plus concomitant administration of donor cells in promoting long-term allograft survival in a mouse heart transplant model. 23 The effect of the immunosuppressive drugs on CD154 mAb therapy alone was not investigated in that study. Kirk et al. recently reported that the additional use of steroids or tacrolimus to humanized anti-CD154 mAb might have a detrimental effect on graft survival in a primate renal transplant model. 13 Addition of cyclosporine or rapamycin to CTLA4Ig was reported to enhance allograft survival in a class I MHC-mismatched skin transplant model. 24 In this study, we investigated systematically the interactions between T-cell costimulatory blockade (CTLA4Ig to block CD28-B7 or MR1 to block CD154-CD40) and the immunosuppressive agents cyclosporine, tacrolimus, rapamycin, steroids, and IL-2R mAb in vivo. We used a model of vascularized cardiac transplantation in a fully allogeneic mouse strain combination, C57BL/6 into BALB/c. Our data highlight the complex interactions between B7 or CD154 blockade on the one hand and immunosuppressive drugs on the other in acute and chronic rejection, and provide clinically relevant novel data to translate to large animals and humans.

Immune-mediated injury to the graft has been implicated in the pathogenesis of chronic rejection. However, little is known regarding the nature of the antigen(s) involved in this immune process. We demonstrated that cardiac transplantation in mice induces an autoimmune T-cell response to a heart tissue-specific protein, cardiac myosin (CM). This response contributes to transplant rejection in that its modulation affects cardiac graft survival. This study investigates whether anti-CM T cells undergo activation and expansion in mice with chronic cardiac allograft rejection.The frequency of CM- and donor major histocompatibility complex (MHC)-specific interferon (IFN)-gamma-producing T cells were assessed by ELISPOT in BALB/c mice, which were injected with anti-CD40L (MR1) mAb (chronic rejection group) or CTLA4Ig fusion protein (tolerant group) and transplanted with C57BL/6 cardiac allografts.MR1-treated BALB/c recipients of C57BL/6 hearts with chronic rejection displayed a high frequency of activated CM-specific T cells, whereas the frequency of activated alloreactive T cells were similar to naïve, nontransplanted mice. In contrast, no activation of CM-reactive T cells was detected in tolerant recipients after CTLA4Ig treatment. Therefore, in the absence of alloimmunity, chronic rejection is associated with persistence of a T-cell response against CM. Our data indicate that anti-CM autoimmunity may be involved in the immune mechanisms of chronic rejection and suggest that tolerance strategies should target both allo- and autoimmune responses to prevent this process.

Angioedema and cough are known side effects of angiotensin-converting enzyme (ACE) inhibitors. Angiotensin-converting enzyme is a potent inhibitor of kinase II, which facilitates the breakdown of bradykinin. An increase in bradykinin levels results in continued prostaglandin E2 synthesis, vasodilation, increased vascular permeability, and increased interstitial fluid. In contrast, the angiotensin II receptor blockers (ARBs) do not increase bradykinin levels. Angioedema as a complication of ACE inhibitor therapy is not widely recognized; this complication is even less recognized with second-line ARBs. We report angioedema associated with losartan (an ARB) in a patient who had experienced angioedema secondary to enalapril (an ACE inhibitor). Almost half of patients with ARB-associated angioedema also had developed angioedema while receiving ACE inhibitor therapy. Clinicians should exercise caution when using ARBs in patients with a history of angioedema secondary to ACE inhibitors.

The CD40-CD154 interaction is critically important in the cell-mediated immune responses. Blockade of this costimulatory pathway has been shown to prevent acute allograft rejection in murine, as well as nonhuman primate models. However, the role of the CD40-CD154 pathway in the development of chronic rejection and the effects of CD154 targeting on progression of chronic rejection have not been evaluated.We examined the effect of AH.F5, a new hamster anti-rat CD154 monoclonal antibody, in a fully allogeneic acute(u) into Lewis [LEW] (RT11) and chronic [WF.1L (RT1l) into LEW (RT1l)] vascularized cardiac allograft rejection model. In the chronic model, the antibody was evaluated for prevention (starting day of transplant) and interruption of progression (starting day 30 or 60 after transplant) of chronic vasculopathy. Graft survival, morphology, and immunohistology were evaluated.In the acute rejection model, anti-CD154 therapy alone prevented acute allograft rejection and resulted in 50% long-term allograft survival (200 days) and donor-specific tolerance. In recipients treated with anti-CD154 monoclonal antibody in combination with a short course of cyclosporine, 100% of allografts survived long-term and all recipients achieved donor-specific tolerance. In the chronic rejection model, allografts from animals treated with the anti-CD154 antibody had a statistically significant lower score of graft arteriosclerosis and fibrosis in both the prevention and 30-day interruption groups when compared with control allografts. In addition, immunohistochemistry showed a decrease in intragraft mononuclear cell infiltration and activation.A new anti-CD154 antibody not only prevents acute allograft rejection, but also inhibits and interrupts the development of chronic rejection. In the acute rejection model cyclosporine acts synergistically with anti-CD154 therapy to prolong allograft survival and induce tolerance. In the chronic rejection model relatively early initiation of therapy is essential to prevent progression of chronic allograft vasculopathy and fibrosis.

The effects of a signaling anti-CD28 mAb (JJ319), which interferes with the CD28-B7 T cell costimulation pathway thought to be involved in the development of chronic rejection of organ transplants, was investigated. Functional, morphologic, and molecular changes in rat renal allografts were examined up to 24 wk after placement. Control Lewis rats, recipients of F344 kidneys, received a single dose of a nonspecific mouse mAb intravenously on the day of transplantation (group 1). Group 2 animals were given anti-CD28 mAb in similar fashion. Group 3 animals were treated with a short course of cyclosporin A (CsA), and group 4 received both anti-CD 28 mAb and CsA. The majority (>95%) of animals in groups 2, 3, and 4 survived throughout the follow-up, compared with 28% in group 1 (P < 0.001). Group 2 and 4 recipients produced negligible proteinuria, whereas group 1 controls developed progressively increasing proteinuria after 4 wk and group 3 animals developed proteinuria by 24 wk. Allografts in groups 2 and 4 were morphologically unremarkable at 24 wk. Kidneys of group 1 animals rapidly developed changes of acute rejection, and those that survived long-term showed extensive glomerulosclerosis and interstitial fibrosis. Changes of early chronic rejection were noted in group 3 grafts. By reverse transcriptase-PCR, expression of representative inflammatory factors interferon-gamma and interleukin-10 were significantly elevated at 24 wk only in the surviving group 1 animals. A single dose of a signaling anti-CD28 mAb administered at transplantation or in combination with a short course of CsA significantly prolonged recipient survival, normalized function, and preserved the morphology of renal allografts in an established model of chronic rejection. These data support an important role for T cell costimulation in the evolution of the chronic process.

T cell costimulation by B7 molecules plays an important role in the regulation of alloimmune responses. Although both B7-1 and B7-2 bind CD28 and CTLA-4 on T cells, the role of B7-1 and B7-2 signaling through CTLA-4 in regulating alloimmune responses is incompletely understood. To address this question, we transplanted CD28-deficient mice with fully allogeneic vascularized cardiac allografts and studied the effect of selective blockade of B7-1 or B7-2. These mice reject their grafts by a mechanism that involves both CD4+ and CD8+ T cells. Blockade of CTLA-4 or B7-1 significantly accelerated graft rejection. In contrast, B7-2 blockade significantly prolonged allograft survival and, unexpectedly, reversed the acceleration of graft rejection caused by CTLA-4 blockade. Furthermore, B7-2 blockade prolonged graft survival in recipients that were both CD28 and CTLA-4 deficient. Our data indicate that B7-1 is the dominant ligand for CTLA-4-mediated down-regulation of alloimmune responses in vivo and suggest that B7-2 has an additional receptor other than CD28 and CTLA-4 to provide a positive costimulatory signal for T cells.

We used signal transducer and activator of transcription 4 (STAT4) and STAT6 gene knockout (-/-) mice as recipients of fully mismatched cardiac allografts to study the role of T-cell costimulatory pathways in regulating allogeneic T-helper 1 (Th1) versus Th2 responses in vivo. STAT4(-/-) mice have impaired Th1 responses, whereas STAT6(-/-) mice do not generate normal Th2 responses. Cardiac allografts from C57BL/6 mice were transplanted into normal wild-type (WT), STAT4(-/-), and STAT6(-/-) BALB/c recipients. STAT4(-/-) and STAT6(-/-) mice rejected their grafts with the same tempo as untreated WT recipients. CD28-B7 blockade by a single injection of CTLA4Ig induced long-term engraftment and donor-specific tolerance in all three groups of recipients. CD154 blockade by a single injection of MR1 was effective in prolonging allograft survival and inducing tolerance in STAT4(-/-) mice but was only marginally effective in STAT6(-/-) recipients and WT controls. In addition, a similar protocol of MR1 was ineffective in prolonging graft survival in CD28(-/-) BALB/c recipients, suggesting that the lack of efficacy seen in WT and STAT6(-/-) mice is not due to the presence of a functional CD28-B7 pathway. Furthermore, there was a similar differential effect of CD28-B7 versus CD154-CD40 blockade in inhibiting immune responses in animals immunized with ovalbumin and complete Freund's adjuvant. These novel data indicate that Th1 and Th2 cells are differentially regulated by CD28-B7 versus CD154-CD40 costimulation pathways in vivo and may have potential implications for the development of therapeutic strategies such as T-cell costimulatory blockade in humans.

Allogeneic bone marrow transplantation (in immunocompetent adults) has always required cytoreductive treatment of recipients with irradiation or cytotoxic drugs to achieve lasting engraftment at levels detectable by non-PCR-based techniques ('macrochimerism' or 'mixed chimerism'). Only syngeneic marrow engraftment at such levels has been achieved in unconditioned hosts. This requirement for potentially toxic myelosuppressive host pre-conditioning has precluded the clinical use of allogeneic bone marrow transplantation for many indications other than malignancies, including tolerance induction. We demonstrate here that treatment of naive mice with a high dose of fully major histocompatibility complex-mismatched allogeneic bone marrow, followed by one injection each of monoclonal antibody against CD154 and cytotoxic T-lymphocyte antigen 4 immunoglobulin, resulted in multi-lineage hematopoietic macrochimerism (of about 15%) that persisted for up to 34 weeks. Long-term chimeras developed donor-specific tolerance (donor skin graft survival of more than 145 days) and demonstrated ongoing intrathymic deletion of donor-reactive T cells. A protocol of high-dose bone marrow transplantation and co-stimulatory blockade can thus achieve allogeneic bone marrow engraftment without cytoreduction or T-cell depletion of the host, and eliminates a principal barrier to the more widespread use of allogeneic bone marrow transplantation. Although efforts have been made to minimize host pre-treatment for allogeneic bone marrow transplantation for tolerance induction, so far none have succeeded in eliminating pre-treatment completely. Our demonstration that this can be achieved provides the rationale for a safe approach for inducing robust transplantation tolerance in large animals and humans.

Recent advances have enabled researchers to induce tolerance in animal transplant models. Although it has been relatively easy to do so in rodents, it has been much more difficult to translate such strategies into primates. Understanding the cellular and molecular mechanisms of the alloimmune response has prompted the development of novel strategies that may obviate the need for immunosuppression in humans. Mechanisms of tolerance and promising new therapies, as well as the inherent difficulties in bringing them into clinical practice, are reviewed.