Pretransplant risk stratification for early survival of renal allograft recipients.

BACKGROUND: Baseline comorbidities influence patient outcomes in renal transplantation. Identification of high-risk recipients for patient death and early allograft loss might lead to superior stratification. MATERIAL AND METHODS: In this retrospective study, risk stratification models were developed in a cohort of 392 kidney transplant recipients and validated in an independent cohort to predict short-term (2 year) outcomes. RESULTS: Peripheral arterial disease [OR 7·7 (95% confidence interval (CI): 2·45-24·60); P < 0·001], use of oral anticoagulation [OR 18·68 (95% CI: 3·77-92·46); P < 0·0001], smoking [OR 5·15 (95% CI: 1·67-15·84); P = 0·004], recipient age > 60 years [OR 7·28 (95% CI: 2·33-22·69; P = 0·001)], serum albumin < 40 g/L [OR 5·08 (95% CI: 1·82-14·19); P = 0·002], serum calcium ≥ 2·42 mM [OR 6·47 (95% CI: 1·37-30·58); P = 0·02] living donation [OR 2·95, (95% CI: 0·31-28·29); P = 0·34)] and previous haemodialysis [OR 3·33, (95% CI: 0·39-28·11); P = 0·27)] were included in the model. The validated model discriminated between low- (< 3 points) and high-risk recipients (> 8·5 points) with mortality rates of 0% vs. 54%. The comparison of the model with the Charlson comorbidity index (CCI) yielded significantly better receiver operating characteristic (ROC) areas (Novel Score ROC: 0·87 vs. CCI: 0·72, P = 0·0012). Early allograft loss was associated with presensitization [OR 3·02 (95% CI: 1·29-7·09); P = 0·011] and presence of hepatitis C antibodies [OR 2·42 (95% CI: 1·09-5·34); P = 0·029]. A risk model (ROC: 0·62) for allograft loss could not be developed. CONCLUSION: Risk stratification based on the novel score might identify high-risk recipients with disproportional risk of early patient death and lead to optimized strategies.

Clinicopathological relevance of granular C4d deposition in peritubular capillaries of kidney allografts.

While linear C4d staining in peritubular capillaries (PTC) is established as a marker of antibody-mediated rejection, the significance of a distinct granular C4d deposition pattern has not yet been clarified. In this study, 329 renal allograft recipients who underwent indication biopsies were analysed for immunohistochemical C4d staining characteristics. Fifty-six (17%) recipients showed granular C4d in PTC, without any relationship to conventional risk factors and morphological features of rejection. We found a strong association with long-term overall graft survival (7-year survival: 41% vs. 66% in granular C4d-negative subjects, P=0.001), which was mainly driven by a greater risk of mortality [hazard ratio: 3.12 (95% confidence interval: 1.23-7.94); P=0.02]. Granular C4d was associated with delayed graft function [39% vs. 22% (C4d-negative subjects), P=0.007], higher 1-year serum creatinine [median 2.1 (interquartile range: 1.7-2.6) mg/dl vs. 1.6 (1.3-2.0) mg/dl, P=0.001] and a trend towards worse death-censored graft survival (P=0.07). In support of a role of capillary immune complex formation, granular C4d was associated with electron-dense deposits in PTC basement membranes, which were occasionally accompanied by focally distributed capillary IgG deposits. In conclusion, our study suggests clinical relevance of detecting capillary granular C4d deposition. Our results point to a pathogenetic role of alloimmune-independent immune complex deposition.

Donor specific transplant tolerance is dependent on complement receptors.

The complement system has recently been described as a crucial component for transplant tolerance induction, but the underlying mechanisms are poorly understood. Using a rodent model of donor lymphocyte infusion-induced male histocompatibility antigen-specific transplant tolerance, we demonstrate that tolerance induction is dependent on the complement receptors decay accelerating factor, complement receptor 3, and complement component 3a receptor (C3aR). Furthermore, we have provided evidence that complement dependent tolerance is mediated through C3aR on infused donor splenocytes and on recipient cells. Ex vivo studies showed that C3aR deficiency leads to an imbalance between T regulatory and T effector cells. Increased numbers of antigen-specific CD8(+) cells in the blood and less T regulatory cells, with reduced suppressive function, in the spleen and in the skin grafts were detected in C3aR deficient compared to wild type mice. This imbalance might be explained by the requirement of complement for dendritic cells to generate T regulatory cells effectively. Our experiments suggest that multiple complement receptors play an important role in transplant tolerance induction providing new insights into the mechanisms of complement dependent tolerance.

Solid phase detection of C4d-fixing HLA antibodies to predict rejection in high immunological risk kidney transplant recipients.

Protocols for recipient desensitization may allow for successful kidney transplantation across major immunological barriers. Desensitized recipients, however, still face a considerable risk of antibody-mediated rejection (AMR), which underscores the need for risk stratification tools to individually tailor treatment. Here, we investigated whether solid phase detection of complement-fixing donor-specific antibodies (DSA) has the potential to improve AMR prediction in high-risk transplants. The study included 68 sensitized recipients of deceased donor kidney allografts who underwent peritransplant immunoadsorption for alloantibody depletion (median cytotoxic panel reactivity: 73%; crossmatch conversion: n = 21). Pre and post-transplant sera were subjected to detection of DSA-triggered C4d deposition ([C4d]DSA) applying single-antigen bead (SAB) technology. While standard crossmatch and [IgG]SAB testing failed to predict outcomes in our desensitized patients, detection of preformed [C4d]DSA (n = 44) was tightly associated with C4d-positive AMR [36% vs. 8%, P = 0.01; binary logistic regression: odds ratio: 10.1 (95% confidence interval: 1.6-64.2), P = 0.01]. Moreover, long-term death-censored graft survival tended to be worse among [C4d]DSA-positive recipients (P = 0.07). There were no associations with C4d-negative AMR or cellular rejection. [C4d]DSA detected 6 months post-transplantation were not related to clinical outcomes. Our data suggest that pretransplant SAB-based detection of complement-fixing DSA may be a valuable tool for risk stratification.

Lymphatic endothelial progenitor cells contribute to de novo lymphangiogenesis in human renal transplants.

De novo lymphangiogenesis influences the course of different human diseases as diverse as chronic renal transplant rejection and tumor metastasis. The cellular mechanisms of lymphangiogenesis in human diseases are currently unknown, and could involve division of local preexisting endothelial cells or incorporation of circulating progenitors. We analyzed renal tissues of individuals with gender-mismatched transplants who had transplant rejection and high rates of overall lymphatic endothelial proliferation as well as massive chronic inflammation. Donor-derived cells were detected by in situ hybridization of the Y chromosome. We compared these tissues with biopsies of essentially normal skin and intestine, and two rare carcinomas with low rates of lymphatic endothelial proliferation that were derived from individuals with gender-mismatched bone marrow transplants. Here, we provide evidence for the participation of recipient-derived lymphatic progenitor cells in renal transplants. In contrast, lymphatic vessels of normal tissues and those around post-transplant carcinomas did not incorporate donor-derived progenitors. This indicates a stepwise mechanism of inflammation-associated de novo lymphangiogenesis, implying that potential lymphatic progenitor cells derive from the circulation, transmigrate through the connective tissue stroma, presumably in the form of macrophages, and finally incorporate into the growing lymphatic vessel.

Prevention and treatment of alloantibody-mediated kidney transplant rejection.

Antibody-mediated rejection (AMR), which is commonly caused by preformed and/or de novo HLA alloantibodies, has evolved as a leading cause of early and late kidney allograft injury. In recent years, effective treatment strategies have been established to counteract the deleterious effects of humoral alloreactivity. One major therapeutic challenge is the barrier of a positive pretransplant lymphocytotoxic crossmatch. Several apheresis- and/or IVIG-based protocols have been shown to enable successful crossmatch conversion, including a strategy of peritransplant immunoadsorption for rapid crossmatch conversion immediately before deceased donor transplantation. While such protocols may increase transplant rates and allow for acceptable graft survival, at least in the short-term, it has become evident that, despite intense treatment, many patients still experience clinical or subclinical AMR. This reinforces the need for innovative strategies, such as complementary allocation programs to improve transplant outcomes. For acute AMR, various studies have suggested efficiency of plasmapheresis- or immunoadsorption-based protocols. There is, however, no established treatment for chronic AMR and the development of strategies to reverse or at least halt chronic active rejection remains a big challenge. Major improvements can be expected from studies evaluating innovative therapeutic concepts, such as proteasome inhibition or complement blocking agents.

Transplantation of the broadly sensitized patient: what are the options?

PURPOSE OF REVIEW: Recipient sensitization to a wide variety of human leukocyte antigens (HLA) represents a major barrier to transplantation. We discuss the options for the challenging group of broadly sensitized kidney transplant candidates. RECENT FINDINGS: Transplantation by way of kidney-paired donation (KPD) represents a preferable way to bypass immunological barriers. Recent data suggest that KPD programs can be optimized by the use of innovative serological techniques to define unacceptable HLA antigens, inclusion of altruistic donors or altruistic balanced paired kidney exchange, and complementary recipient desensitization. It has become evident that plasmapheresis and/or intravenous immunoglobulin (IVIG)-based crossmatch conversion may not completely prevent rejection and chronic injury. However, recent evidence suggests major improvements by sophisticated serological risk stratification and new innovative treatment principles. Wait-listed broadly sensitized patients were shown to benefit from specific allocation programs, such as priority allocation of organs via the Eurotransplant acceptable mismatch program. Recent studies have suggested a benefit from IVIG/rituximab-based desensitization on the waitlist, or apheresis-based protocols for rapid antibody removal immediately before transplantation. SUMMARY: A multifaceted repertoire of complementary strategies was shown to facilitate successful live and deceased donor kidney transplantation in high-risk patients. Recent promising developments can be expected to significantly improve outcomes.

Identification of Non-HLA antigens targeted by alloreactive antibodies in patients undergoing chronic hemodialysis.

The only treatment of end-stage renal disease patients undergoing chronic dialysis is kidney transplantation. However, about half of graft recipients encounter organ loss within ten years after renal transplantation. There is emerging evidence that the presence of alloreactive antibodies against non-HLA antigens in the serum of the recipient prior transplantation is associated with higher incidence of chronic rejection. However, the molecular identity of these antigens is largely unknown. To determine the most common non-HLA antigens, we tested lymphocytic extracts from 20 healthy volunteers with sera of 28 patients on the transplantation waiting list by Western blotting. There was a group of five proteins that was recognized by most sera. Using patient's own lymphocytes revealed that autoimmunity plays a minor role in this recognition. Two-dimensional Western blotting experiments followed by mass spectrometry identified the antigens as tubulin beta chain, vimentin, lamin-B1, and Rho GDP-dissociation inhibitor 2. A detailed analysis of vimentin expression revealed that the antigenic 60 kDa isoform is underrepresented in patient's lymphocytes in comparison to those of healthy volunteers. The study revealed that preformed alloreactive antibodies are directed against a small number of specific protein isoforms. Our findings could provide a basis for future improvement of donor-recipient matching.

Clinical relevance of preformed C4d-fixing and non-C4d-fixing HLA single antigen reactivity in renal allograft recipients.

Donor-specific alloantibodies (DSA), especially those fixing complement, may pose a particular immunologic risk to transplant recipients. To assess the clinical impact of C4d- or non-C4d-fixing (IgG) HLA sensitization, pretransplant sera obtained from 338 kidney allograft recipients prescreened by FlowPRA were retrospectively evaluated by Luminex single antigen (SA) testing using a novel fluorescent-labeled anti-C4d reagent for detection of antibody-triggered C4d deposition in addition to IgG binding. Recipients with [IgG]DSA (n = 39) showed a substantially higher rate of C4d positive rejection (33%) than 16 patients with [IgG] non-DSA (0%) or 283 antibody-negative patients (4%, multivariate analysis excluding retransplantation because of high co-linearity: P < 0.0001), and adversely affected 5-year death-censored graft survival (74% vs. 81% and 90%, respectively, multivariate model: P < 0.05). [C4d] DSA (n = 21) and [C4d] non-DSA (n = 25) increased rates of C4d positive rejections to a similar extent (24% and 28% vs. 4% in recipients without C4d-fixing reactivity; multivariate analysis: P

Ingested matter affects intestinal lesions in Crohn's disease.

BACKGROUND: Environmental factors of the modern Western lifestyle may trigger Crohn's disease (CD) in susceptible individuals. Because such factors could be part of ingested matter, we intended to improve intestinal Crohn's lesions by exclusion thereof. METHODS: At first we tested a highly restricted diet (based on spelt bread and red meat, both derived from intensively monitored organic farming) in 5 pilot cases. In a subsequent controlled trial, 18 patients with mild-to-moderate CD were randomly assigned to receive either this active diet or a control diet (low-fiber, low-fat, and high-carbohydrate). Mucosal improvement was assessed by magnetic resonance imaging (MRI) and endoscopy. Secondary endpoints included sonography, the Crohn's Disease Activity Index (CDAI), and the Inflammatory Bowel Disease Questionnaire (IBDQ). RESULTS: Four of 5 pilot patients showed significant improvement within 4 weeks. From 18 patients in the controlled trial, 8 were randomized to the active and 10 to the control group; 4 decided to quit immediately after dietary counseling, 3 in the active and 1 in the control group (P = 0.183). At 6 weeks MRI and endoscopy showed improvement of intestinal lesions in 3 of 4 assessable patients of the active group and 1 of 9 patients of the control group (P = 0.027). Sonography showed improvement in 4 of 5 patients of the active group and in 1 of 8 assessable patients of the control group (P = 0.016). CDAI and IBDQ improved in both groups to a similar extent. CONCLUSIONS: Ingested matter as part of the modern Western lifestyle may cause persistence of intestinal Crohn's lesions.

Antibodies, isotypes and complement in allograft rejection.

PURPOSE OF REVIEW: Classical complement activation is a key step in the process of antibody-mediated rejection. Emphasizing novel diagnostic strategies, this study will discuss recent studies highlighting the particular relevance of alloantibodies with complement-fixing ability. RECENT FINDINGS: Reinforcing the pivotal role of complement, numerous studies have shown tight associations of capillary C4d deposition, a 'footprint' of alloantibody-triggered complement activation, with the occurrence of allograft injury. Distribution patterns of immunoglobulin isotypes or subclasses, which strongly differ in their ability to activate complement, may not adequately reflect the actual pathogenetic relevance of detected allosensitization. This fact may be explained by the finding that other variables, such as antibody-binding density or a synergism of antibodies against different epitopes of the same antigen, may contribute to complement activation. An attractive approach to distinguish between complement-fixing and presumably less harmful noncomplement-fixing alloreactivities could be the detection of C4d deposition in vitro. Applying such techniques, recent studies have shown that human leukocyte antigen reactivity with C4d-fixing ability, in contrast to noncomplement-fixing sensitization, may strongly predict antibody-mediated rejection and inferior graft survival. SUMMARY: Considering the pivotal role of complement, technologies that uncover the complement-fixing ability of alloantibodies may be of particular interest for the selective detection of deleterious sensitization.

Determinants of the complement-fixing ability of recipient presensitization against HLA antigens.

BACKGROUND: The presence of preformed alloantibodies with the ability to activate complement may pose a particular risk for kidney allograft rejection. The aim of this study was to evaluate variables that determine the complement-fixing capability of human leukocyte antigen (HLA) sensitization. METHODS: Sixty-five sensitized patients with > or =10% pretransplant panel-reactive antibody (PRA) levels uncovered by immunoglobulin G [IgG]FlowPRA HLA class I and/or class II screening were included. Applying modified FlowPRA screening, sera were evaluated for patterns of alloreactive IgG subclasses and IgM, and, in parallel, for their complement-activating ability assessed by flow cytometric detection of human complement split product deposition ([C4d]FlowPRA). RESULTS: Approximately two-thirds (68%) of tested sera were found to contain complement-fixing alloreactivity (> or =10%[C4d]FlowPRA). IgG1 type panel reactivity was predominant (detectable HLA class I and II reactivity in 93% and 91% of IgG-positive sera), followed by IgG3 (49%/44%), IgG2 (44%/27%), and IgG4 (19%/11%). Applying partial correlation we found an independent correlation of both %[IgG1]FlowPRA and %[IgG3]FlowPRA with %[C4d]FlowPRA reactivities (P< or =0.01). In addition, for IgG1 a contribution of the amount of bound alloantibody to complement-fixation was observed. Complement-fixation was also favored by the simultaneous presence of alloreactive IgG1, IgG3, and IgM. Previous grafting, but not pregnancy and transfusion, was independently associated with complement-fixing sensitization (P<0.05), presumably due to increased IgG1 type reactivity. CONCLUSIONS: Anti-HLA antibody-triggered complement activation is dependent on both the pattern of Ig reactivities and the amount of bound antibody. Previous transplantation represents a major risk factor for the development of complement-fixing sensitization.

Prevalence and qualitative properties of circulating anti-human leukocyte antigen alloantibodies after pregnancy: no association with unexplained recurrent miscarriage.

In pregnant women, circulating alloantibodies, triggered by exposure to paternal HLA antigens, are frequently detectable. The finding of lower alloantibody levels in women who experience spontaneous abortion (miscarriage) has led to the speculation that antipaternal antibodies could favor maintenance of pregnancy, whereas their lack poses a risk of miscarriage. Postulating a role of alloantibodies in the pathogenesis of unexplained abortion, we examined whether different categories of recurrent miscarriage (RM) can be distinguished according to prevalence or distinct qualitative properties of anti-human leukocyte antigen (HLA) antibody patterns. Sera obtained from 167 women with RM were assessed for complement- and non-complement-fixing anti-HLA alloreactivity using Luminex-based bead array technology. Women with RM had less often detectable anti-HLA class I and/or II reactivity (19%) compared with a control group of 96 multiparous women without a history of miscarriage (49%). However, analysis of different categories of RM (unknown [n = 112] versus known cause [n = 55]; primary [n = 125] versus secondary RM [n = 42]) did not reveal any differences regarding antibody prevalence, number of targeted HLA single antigens, antigen specificity, binding density, or complement-fixing ability of detected alloantibodies. Our results do not support a link between anti-HLA antibody formation and RM, and argue against a diagnostic value of alloantibody detection in the diagnostic work-up of women with RM.

Significance of peritubular capillary, glomerular, and arteriolar C4d staining patterns in paraffin sections of early kidney transplant biopsies.

BACKGROUND: Although diffuse linear C4d deposition in peritubular capillaries (PTCs) is a well-established criterion of alloantibody-mediated kidney transplant rejection, the actual relevance of focal or granular C4d deposits or staining outside PTC (glomeruli and arterioles) has yet to be established. METHODS: This study was designed to evaluate the diagnostic significance of such nontypical C4d staining patterns. A total of 539 early indication biopsies (329 kidney transplants) were analyzed by immunohistochemistry using a polyclonal anti-C4d antibody. RESULTS: We found a close interrelationship between diffuse or focal linear C4d deposition in PTC, linear endothelial deposition in glomeruli, and arteriolar C4d. These specific patterns were also related to transplant glomerulitis and recipient presensitization. No such associations, however, were observed for other patterns, such as granular C4d in PTC. Detection of diffuse but not focal linear C4d in PTC was found to be associated with adverse allograft survival (5-year death-censored graft survival: 48% vs. 82%, 89%, or 84% in patients with focal, minimal, or no C4d, respectively; P<0.0001). Univariate analysis also revealed inferior graft survival in recipients with linear C4d in glomeruli (P=0.02). Applying multivariate Cox regression analysis, however, only diffuse linear PTC staining was found to be predictive of graft loss (hazard ratio 3.95 [95% confidence interval 1.62-9.60]; P=0.002). CONCLUSION: There might be a relationship between humoral alloimmunity and distinct less established staining patterns, such as focal linear C4d in PTC, endothelial C4d in glomeruli, or arteriolar C4d. Nevertheless, our results reemphasize the prognostic value of diffuse linear PTC staining.

Lipoxygenase mediates invasion of intrametastatic lymphatic vessels and propagates lymph node metastasis of human mammary carcinoma xenografts in mouse.

In individuals with mammary carcinoma, the most relevant prognostic predictor of distant organ metastasis and clinical outcome is the status of axillary lymph node metastasis. Metastases form initially in axillary sentinel lymph nodes and progress via connecting lymphatic vessels into postsentinel lymph nodes. However, the mechanisms of consecutive lymph node colonization are unknown. Through the analysis of human mammary carcinomas and their matching axillary lymph nodes, we show here that intrametastatic lymphatic vessels and bulk tumor cell invasion into these vessels highly correlate with formation of postsentinel metastasis. In an in vitro model of tumor bulk invasion, human mammary carcinoma cells caused circular defects in lymphatic endothelial monolayers. These circular defects were highly reminiscent of defects of the lymphovascular walls at sites of tumor invasion in vivo and were primarily generated by the tumor-derived arachidonic acid metabolite 12S-HETE following 15-lipoxygenase-1 (ALOX15) catalysis. Accordingly, pharmacological inhibition and shRNA knockdown of ALOX15 each repressed formation of circular defects in vitro. Importantly, ALOX15 knockdown antagonized formation of lymph node metastasis in xenografted tumors. Furthermore, expression of lipoxygenase in human sentinel lymph node metastases correlated inversely with metastasis-free survival. These results provide evidence that lipoxygenase serves as a mediator of tumor cell invasion into lymphatic vessels and formation of lymph node metastasis in ductal mammary carcinomas.

Prospects and limitations of post-transplantation alloantibody detection in renal transplantation.

Antibody-mediated immunity is generally accepted to be a major cause of kidney allograft injury and loss. Post-transplantation monitoring for circulating alloantibodies was proposed to represent a useful noninvasive diagnostic approach to assess individual immunologic risks and to guide the implementation of specific therapeutic measures. This was supported by the recent establishment of highly sensitive and specific solid phase immunoassays for detailed characterisation of reactivity patterns. Nevertheless, imperfect associations of serologic results with biopsy-based criteria of antibody-mediated rejection, as well as the disputed long-term significance of circulating alloantibody detected in recipients with normal graft function, necessitate a careful interpretation of monitoring results.

Tetrahydrobiopterin corrects Escherichia coli endotoxin-induced endothelial dysfunction.

Acute inflammation causes endothelial dysfunction, which is partly mediated by oxidant stress and inactivation of nitric oxide. The contribution of depletion of tetrahydrobiopterin (BH(4)), the cofactor required for nitric oxide generation, is unclear. In this randomized, double-blind, three-way crossover study, forearm blood flow (FBF) responses to ACh and glyceryltrinitrate (GTN) were measured before and 3.5 h after infusion of Escherichia coli endotoxin (LPS, 20 IU/kg iv) in eight healthy men. The effect of intra-arterial BH(4) (500 microg/min), placebo, or vitamin C (24 mg/min) was studied on separate days 3.5 h after LPS infusion. In addition, human umbilical vein endothelial cells were incubated for 24 h with vitamin C and LPS. ACh and GTN caused dose-dependent forearm vasodilation. The FBF response to ACh, which was decreased by 23 +/- 17% (P < 0.05) by LPS infusion, was restored to baseline reactivity by BH(4) and vitamin C. FBF responses to GTN were not affected by BH(4) or vitamin C. LPS increased leukocyte count, high-sensitivity C-reactive protein, IL-6, IL-1beta, IFN-gamma, monocyte chemoattractant protein-1, pulse rate, and body temperature and decreased platelet count and vitamin C concentration. Vitamin C increased forearm plasma concentration of BH(4) by 32% (P < 0.02). Incubation with LPS and vitamin C, but not LPS alone, increased intracellular BH(4) concentration in human umbilical vein endothelial cells. Impaired endothelial function during acute inflammation can be restored by BH(4) or vitamin C. Vitamin C may exert some of its salutary effects by increasing BH(4) concentration.