Interferon Gamma ELISPOT Testing as a Risk-Stratifying Biomarker for Kidney Transplant Injury: Results From the CTOT-01 Multicenter Study.

Previous studies suggest that quantifying donor-reactive memory T cells prior to kidney transplantation by interferon gamma enzyme-linked immunosorbent spot assay (IFNγELISPOT) can assist in assessing risk of posttransplant allograft injury. Herein, we report an analysis of IFNγELISPOT results from the multicenter, Clinical Trials in Organ Transplantation-01 observational study of primary kidney transplant recipients treated with heterogeneous immunosuppression. Within the subset of 176 subjects with available IFNγELISPOT results, pretransplant IFNγELISPOT positivity surprisingly did not correlate with either the incidence of acute rejection (AR) or estimated glomerular filtration rate (eGFR) at 6- or 12-month. These unanticipated results prompted us to examine potential effect modifiers, including the use of T cell-depleting, rabbit anti-thymocyte globulin (ATG). Within the no-ATG subset, IFNγELISPOT(neg) subjects had higher 6- and 12-month eGFRs than IFNγELISPOT(pos) subjects, independent of biopsy-proven AR, peak PRA, human leukocyte antigen mismatches, African-American race, donor source, and recipient age or gender. In contrast, IFNγELISPOT status did not correlate with posttransplant eGFR in subjects given ATG. Our data confirm an association between pretransplant IFNγELISPOT positivity and lower posttransplant eGFR, but only in patients who do not receive ATG induction. Controlled studies are needed to test the hypothesis that ATG induction is preferentially beneficial to transplant candidates with high frequencies of donor-reactive memory T cells.

Banff 2013 meeting report: inclusion of c4d-negative antibody-mediated rejection and antibody-associated arterial lesions.

The 12th Banff Conference on Allograft Pathology was held in Comandatuba, Brazil, from August 19-23, 2013, and was preceded by a 2-day Latin American Symposium on Transplant Immunobiology and Immunopathology. The meeting was highlighted by the presentation of the findings of several working groups formed at the 2009 and 2011 Banff meetings to: (1) establish consensus criteria for diagnosing antibody-mediated rejection (ABMR) in the presence and absence of detectable C4d deposition; (2) develop consensus definitions and thresholds for glomerulitis (g score) and chronic glomerulopathy (cg score), associated with improved inter-observer agreement and correlation with clinical, molecular and serological data; (3) determine whether isolated lesions of intimal arteritis ("isolated v") represent acute rejection similar to intimal arteritis in the presence of tubulointerstitial inflammation; (4) compare different methodologies for evaluating interstitial fibrosis and for performing/evaluating implantation biopsies of renal allografts with regard to reproducibility and prediction of subsequent graft function; and (5) define clinically and prognostically significant morphologic criteria for subclassifying polyoma virus nephropathy. The key outcome of the 2013 conference is defining criteria for diagnosis of C4d-negative ABMR and respective modification of the Banff classification. In addition, three new Banff Working Groups were initiated.

Multicenter validation of urinary CXCL9 as a risk-stratifying biomarker for kidney transplant injury.

Noninvasive biomarkers are needed to assess immune risk and ultimately guide therapeutic decision-making following kidney transplantation. A requisite step toward these goals is validation of markers that diagnose and/or predict relevant transplant endpoints. The Clinical Trials in Organ Transplantation-01 protocol is a multicenter observational study of biomarkers in 280 adult and pediatric first kidney transplant recipients. We compared and validated urinary mRNAs and proteins as biomarkers to diagnose biopsy-proven acute rejection (AR) and stratify patients into groups based on risk for developing AR or progressive renal dysfunction. Among markers tested for diagnosing AR, urinary CXCL9 mRNA (odds ratio [OR] 2.77, positive predictive value [PPV] 61.5%, negative predictive value [NPV] 83%) and CXCL9 protein (OR 3.40, PPV 67.6%, NPV 92%) were the most robust. Low urinary CXCL9 protein in 6-month posttransplant urines obtained from stable allograft recipients classified individuals least likely to develop future AR or a decrement in estimated glomerular filtration rate between 6 and 24 months (92.5-99.3% NPV). Our results support using urinary CXCL9 for clinical decision-making following kidney transplantation. In the context of acute dysfunction, low values can rule out infectious/immunological causes of injury. Absent urinary CXCL9 at 6 months posttransplant defines a subgroup at low risk for incipient immune injury.

Detection of clinical and subclinical tubulo-interstitial inflammation by the urinary CXCL10 chemokine in a real-life setting.

Urinary CXCL10 is a promising noninvasive biomarker for tubulo-interstitial allograft inflammation, but its diagnostic characteristics have not been assessed in a real-life setting. We investigated urinary CXCL10 in 213 consecutive renal allograft recipients having 362 surveillance biopsies at 3/6 months and 80 indication biopsies within the first year posttransplant. Allograft histology results were classified as (i) acute Banff score zero, (ii) interstitial infiltrates only, (iii) tubulitis t1, (iv) tubulitis t2-3 and (v) isolated vascular compartment inflammation. For clinical and subclinical pathologies, urinary CXCL10 correlated well with the extent of tubulo-interstitial inflammation. To determine diagnostic characteristics of urinary CXCL10, histological groups were separated into two categories: no relevant inflammation (i.e. acute Banff score zero and interstitial infiltrates only) versus all other pathologies (i.e. tubulitis t1-3 and isolated vascular compartment inflammation). For subclinical pathologies, AUC was 0.69 (sensitivity 61%, specificity 72%); for clinical pathologies, AUC was 0.74 (sensitivity 63%, specificity 80%). A urinary CXCL10-guided biopsy strategy would have reduced performance of surveillance and indication biopsies by 61% and 64%, respectively. Missed (sub)clinical pathologies were mostly tubulitis t1 and isolated vascular compartment lesions. In real life, urinary CXCL10 had clinically useful diagnostic properties making it a candidate biomarker to guide allograft biopsies.

Inflammation in areas of tubular atrophy in kidney allograft biopsies: a potent predictor of allograft failure.

The Banff scoring schema provides a common ground to analyze kidney transplant biopsies. Interstitial inflammation (i) and tubulitis (t) in areas of viable tissue are features in scoring acute rejection, but are excluded in areas of tubular atrophy (TA). We studied inflammation and tubulitis in a cohort of kidney transplant recipients undergoing allograft biopsy for new-onset late graft dysfunction (N = 337). We found inflammation ('iatr') and tubulitis ('tatr') in regions of fibrosis and atrophy to be strongly correlated with each other (p < 0.0001). Moreover, iatr was strongly associated with death-censored graft failure when compared to recipients whose biopsies had no inflammation, even after adjusting for the presence of interstitial fibrosis (Hazard Ratio = 2.31, [1.10-4.83]; p = 0.0262) or TA (hazard ratio = 2.42, [1.16-5.08]; p = 0.191), serum creatinine at the time of biopsy, time to biopsy and i score. Further, these results did not qualitatively change after additional adjustments for C4d staining or donor specific antibody. Stepwise regression identified the most significant markers of graft failure which include iatr score. We propose that a more global assessment of inflammation in kidney allograft biopsies to include inflammation in atrophic areas may provide better prognostic information. Phenotypic characterization of these inflammatory cells and appropriate treatment may ameliorate late allograft failure.

Histopathologic clusters differentiate subgroups within the nonspecific diagnoses of CAN or CR: preliminary data from the DeKAF study.

The nonspecific diagnoses 'chronic rejection''CAN', or 'IF/TA' suggest neither identifiable pathophysiologic mechanisms nor possible treatments. As a first step to developing a more useful taxonomy for causes of new-onset late kidney allograft dysfunction, we used cluster analysis of individual Banff score components to define subgroups. In this multicenter study, eligibility included being transplanted prior to October 1, 2005, having a 'baseline' serum creatinine < or =2.0 mg/dL before January 1, 2006, and subsequently developing deterioration of graft function leading to a biopsy. Mean time from transplant to biopsy was 7.5 +/- 6.1 years. Of the 265 biopsies (all with blinded central pathology interpretation), 240 grouped into six large (n > 13) clusters. There were no major differences between clusters in recipient demographics. The actuarial postbiopsy graft survival varied by cluster (p = 0.002). CAN and CNI toxicity were common diagnoses in each cluster (and did not differentiate clusters). Similarly, C4d and presence of donor specific antibody were frequently observed across clusters. We conclude that for recipients with new-onset late graft dysfunction, cluster analysis of Banff scores distinguishes meaningful subgroups with differing outcomes.

Pathological and clinical characterization of the 'troubled transplant': data from the DeKAF study.

We are studying two cohorts of kidney transplant recipients, with the goal of defining specific clinicopathologic entities that cause late graft dysfunction: (1) prevalent patients with new onset late graft dysfunction (cross-sectional cohort); and (2) newly transplanted patients (prospective cohort). For the cross-sectional cohort (n = 440), mean time from transplant to biopsy was 7.5 +/- 6.1 years. Local pathology diagnoses included CAN (48%), CNI toxicity (30%), and perhaps surprisingly, acute rejection (cellular- or Ab-mediated) (23%). Actuarial rate of death-censored graft loss at 1 year postbiopsy was 17.7%; at 2 years, 29.8%. There was no difference in postbiopsy graft survival for recipients with versus without CAN (p = 0.9). Prospective cohort patients (n = 2427) developing graft dysfunction >3 months posttransplant undergo 'index' biopsy. The rate of index biopsy was 8.8% between 3 and 12 months, and 18.2% by 2 years. Mean time from transplant to index biopsy was 1.0 +/- 0.6 years. Local pathology diagnoses included CAN (27%), and acute rejection (39%). Intervention to halt late graft deterioration cannot be developed in the absence of meaningful diagnostic entities. We found CAN in late posttransplant biopsies to be of no prognostic value. The DeKAF study will provide broadly applicable diagnostic information to serve as the basis for future trials.

Can protocol biopsy better inform our choices in renal transplantation?

The use of protocol biopsies has provided insights into the pathogenesis of many renal allograft diseases. It is now widely accepted that acute and chronic immune injury, as well as other pathologies associated with eventual graft loss, may occur initially in the absence of graft dysfunction. Indeed, renal transplant biopsies performed at the time of graft dysfunction may disclose advanced stages of renal injury that may not be amenable to treatment. However, protocol biopsies have several limitations that include their morbidity, cost, and potential for sampling error. Furthermore, the prevalence of early subclinical rejection is decreasing in the modern era of immunosuppression, which argues against their use in patients of low immunological risk. Conversely, the transplantation community has, perhaps unadvisedly, embarked upon protocols of immunosuppressive drug minimization. The consequences of these practices may result in late inflammation in the graft that could prove deleterious to its function in the long term. It is hoped that in the future "systems biology" techniques (eg, genomics, proteomics, and metabolomics) may guide clinicians regarding the safety of drug minimization protocols, inform them as to when a renal transplant biopsy should be procured, and perhaps one day replace the renal transplant biopsy altogether. Until such time, however, the renal biopsy remains an indispensable tool in the management of renal transplant recipients.

Urinary CXCL9 and CXCL10 levels correlate with the extent of subclinical tubulitis.

Subclinical tubulitis has been associated with the later development of interstitial fibrosis and tubular atrophy (IF/TA), leading to diminished allograft survival. The aim of this study was to investigate how concentrations of urinary CXC-receptor 3 (CXCR3) chemokines (i.e. CXCL4/9/10/11) and CCL2 relate to the extent of subclinical tubulitis. Using ELISA, urinary CXCR3 chemokines, CCL2 and tubular injury markers (i.e. urinary NGAL and alpha1-microglobulin [alpha1 m]) were measured in patients with stable estimated GFR >or=40 mL/min exhibiting normal tubular histology (n = 24), subclinical borderline tubulitis (n = 18) or subclinical tubulitis Ia/Ib (n = 22), as well as in patients with clinical tubulitis Ia/Ib (n = 17) or IF/TA (n = 10). CXCL9 and CXCL10 were significantly higher in subclinical tubulitis Ia/Ib than in subclinical borderline tubulitis (p

Lack of benefit of early protocol biopsies in renal transplant patients receiving TAC and MMF: a randomized study.

We conducted a randomized, multicenter study to determine whether treatment of subclinical rejection with increased corticosteroids resulted in beneficial outcomes in renal transplant patients receiving tacrolimus (TAC), mycophenolate mofetil (MMF) and prednisone. One hundred and twenty-one patients were randomized to biopsies at 0,1,2,3 and 6 months (Biopsy arm), and 119 to biopsies at 0 and 6 months only (Control arm). The primary endpoint of the study was the prevalence of the sum of the interstitial and tubular scores (ci + ct)> 2 (Banff) at 6 months. Secondary endpoints included clinical and subclinical rejection and renal function. At 6 months, 34.8% of the Biopsy and 20.5% of the Control arm patients had a ci + ct score >or= 2 (p = 0.07). Between months 0 and 6, clinical rejection episodes were 12 in 10 Biopsy arm patients and 8 in 8 Control arm patients (p = 0.44). Overall prevalence of subclinical rejection in the Biopsy arm was 4.6%. Creatinine clearance at 6 months was 72.9 +/- 21.7 in the Biopsy and 68.90 mL/min +/- 18.35 mL/min in the Control arm patients (p = 0.18). In conclusion, we found no benefit to the procurement of early protocol biopsies in renal transplant patients receiving TAC, MMF and prednisone, at least in the short term. This is likely due to their low prevalence of subclinical rejection.

Protocol biopsies in renal transplantation: insights into patient management and pathogenesis.

A 1-day symposium on the application of protocol biopsies in renal transplantation was held in Boston, 21 July 2006. Representatives from centers with extensive experience in the use of protocol biopsies for routine patient care and research reported results on the pathological findings and their value in patient management. The consensus was that protocol biopsies, in experienced hands, are a safe and valuable means of detecting subclinical disease that can benefit from modification of therapy. Furthermore, molecular studies reveal evidence of activity or progression not readily appreciated by histological techniques. Wider application is expected in multicenter clinical trials to predict and validate outcomes. The principal barrier to wider use of protocol biopsies is knowledge of the benefits of intervention.

Insights into subclinical rejection.

Several groups have performed graft biopsies at set times posttransplant ("protocol biopsies") and found unequivocal histologic criteria for acute rejection in a high proportion of patients with stable graft function. The significance of "subclinical" rejection remains controversial. Our group and others have shown that clinically silent infiltrates have inflammatory and cytotoxic potential. Furthermore, in a randomized trial, we demonstrated that treatment of subclinical rejection results in better graft histology and renal function. Although a decrease in the prevalence of subclinical rejection may reduce the rate of late graft losses, the risks and cost of protocol biopsies require that noninvasive methods for the diagnosis of subclinical rejection be developed.

Subclavian arterial injury associated with blunt trauma.

Blunt subclavian artery trauma is an uncommon but challenging surgical problem. The purpose of this study was to retrospectively review the management of blunt subclavian artery injuries treated by the Trauma and Vascular Surgery Services at the East Tennessee State University-affiliated hospitals between 1992 and 1998. Six patients with seven blunt subclavian artery injuries were identified. Physical signs indicating blunt subclavian artery injury were pain or contusion around the shoulder joint; fractures of the clavicle, scapula, or ribs; periclavicular hematomas; and ipsilateral pulse or neurologic deficits. Seven subclavian artery injuries were treated-two arterial transections, two pseudoaneurysms, and three intimal dissections. Associated injuries included four clavicle fractures, one humerus fracture, one combined rib and scapular fractures, and two pneumothoraxes. Vascular surgical treatment included three primary arterial repairs, two saphenous vein interposition grafts, and one polytetrafluoroethylene (PTFE) graft. One patient was treated nonoperatively with anticoagulation. No deaths occurred. Morbidity occurred in two patients with chronic upper extremity neuropathy producing prolonged disability from pain and weakness; one patient had reflex sympathetic dystrophy, and the other had a brachial plexus injury. In conclusion, blunt subclavian artery trauma can be successfully managed with early use of arteriography and prompt surgical correction by a variety of vascular techniques. Vascular morbidity is usually low, but long-term disability because of chronic neuropathy may result from associated brachial plexus nerve injury despite a successful arterial repair.

Durability of eversion carotid endarterectomy: comparison with primary closure and carotid patch angioplasty.

OBJECTIVES: Despite numerous studies in which various methods for arteriotomy closure after carotid endarterectomy (CEA) have been addressed, the optimum surgical technique to reduce complications and late carotid restenosis has yet to be firmly established. The purpose of this study was to prospectively compare the results of the eversion CEA technique with those of conventional CEA with either primary closure or carotid patch angioplasty, and to determine under clinical conditions whether eversion CEA influences the results and restenosis rate. PATIENTS AND METHODS: Over a 3-year period, 322 CEAs performed on 296 consecutive patients were concurrently evaluated. This study included 118 eversion CEAs, 97 CEAs with primary closure, and 107 CEAs with patch angioplasty. There were no differences in demographics, in surgical indications, or in the severity of carotid disease (not significant [NS]). The choice of CEA technique was not randomized because of technical considerations and surgeon preference. After entry into the protocol, no patients were excluded or withdrawn. Carotid restenosis was defined as a > 60% lumen reduction at the CEA site with established duplex ultrasonography criteria. RESULTS: The mean operative time for eversion CEA was 31 minutes, for CEA-primary closure it was 39 minutes, and for CEA-patch angioplasty it was 46 minutes (P <.01). The operative mortality rate for eversion CEA was 0.8% (1 patient), for CEA-primary closure it was 1.0% (1 patient), and for CEA-patch angioplasty it was 2.8% (3 patients) (NS). The postoperative stroke rate was 0.8% after eversion CEA, 1.0% after CEA-primary closure, and 2.8% after CEA-patch angioplasty (NS). The combined stroke and death rate in each group was thus 0.8% for eversion CEA (1 stroke-death), 1% for CEA with primary closure (1 stroke-death), and 5% for CEA with patch angioplasty (1 stroke-death, 2 fatal myocardial infarctions, and 2 nonfatal strokes) (NS). Transient ischemic attacks occurred in 2.5% after eversion CEA, in 5.2% after CEA-primary closure, and in 2.9% with CEA-patch angioplasty (NS). The mean clinical follow-up for all three groups was 23 months (range, 6-42 months) (NS). The restenosis rate was 1.7% after eversion CEA, 9.3% after CEA-primary closure, and 6.5% after CEA-patch angioplasty (P <.05). CONCLUSIONS: This prospective, nonrandomized clinical study indicates that eversion CEA is an effective surgical option comparable to conventional CEA with either primary arteriotomy closure or carotid patch angioplasty. No differences were found between eversion CEA and these more widely accepted CEA closure techniques with respect to operative morbidity and mortality. These data indicate, however, that eversion CEA has a lower restenosis rate than conventional CEA closure techniques and thus superior long-term durability.

Neointimal and tubulointerstitial infiltration by recipient mesenchymal cells in chronic renal-allograft rejection.

BACKGROUND: Tissue remodeling depends on mesenchymal cells (fibroblasts and myofibroblasts) and is a prominent feature of chronic renal-transplant rejection. It is not known whether the mesenchymal cells that participate in remodeling originate locally or from circulating precursor cells. METHODS: We obtained biopsy specimens of renal allografts from six male recipients of an allograft from a female donor, four female recipients of an allograft from a male donor, two male recipients of an allograft from a male donor, and two female recipients of an allograft from a female donor. All the allografts were undergoing chronic rejection. All but two specimens were obtained within six months after transplantation. We used immunohistochemical methods to identify mesenchymal cells with smooth-muscle alpha-actin and in situ hybridization to identify mesenchymal cells with Y-chromosome DNA. RESULTS: No Y-chromosome bodies were identified in the case of the two renal-allograft specimens in which both the donor and the recipient were female. In the case of the two renal-allograft specimens in which both the donor and the recipient were male, approximately 40 percent of mesenchymal cells contained a Y-chromosome body. In the case of the six specimens in which the donor was female and the recipient was male, a mean (+/-SD) of 34+/-16 percent of mesenchymal cells in the neointima, 38+/-12 percent of such cells in the adventitia, and 30+/-7 percent of such cells in the interstitium contained the Y-chromosomal marker, indicating that they originated from the recipient rather than the donor. In the case of the four renal-allograft specimens in which the donor was male and the recipient was female, the respective values were 24+/-15 percent, 33+/-9 percent, and 23+/-8 percent, indicating a persistent population of donor mesenchymal cells. CONCLUSIONS: The presence of mesenchymal cells of host origin in the vascular and interstitial compartments of renal allografts undergoing chronic rejection provides evidence that a circulating mesenchymal precursor cell has the potential to migrate to areas of inflammation.

Long-term allograft surveillance: the role of protocol biopsies.

The safety of the renal allograft biopsy and the standardization of allograft histopathology interpretation have renewed interest in the performance of protocol (surveillance) biopsies. Recent surveillance biopsy studies in the areas of pre-implantation and in the early and late post-transplant periods are discussed.

h-Caldesmon, a novel smooth muscle-specific antibody, distinguishes between cellular leiomyoma and endometrial stromal sarcoma.

The difficulty in distinguishing between smooth muscle and endometrial stromal-derived neoplasms of the uterine corpus is a notorious and clinically relevant problem in pathology of the female genital tract. Immunohistochemistry offers some aid in resolving this difficulty, because the expression of smooth muscle markers is reputed to indicate smooth muscle derivation. This expression, however, is not entirely specific, and difficult cases may still present in which immunohistochemistry is of little help. To explore this problem, the authors evaluated the expression of traditional muscle markers and high-molecular-weight caldesmon (h-cal), an actin and tropomyosin binding protein that has recently been described as a useful muscle marker, in uterine leiomyosarcoma (LMS), cellular leiomyomata (CL), and endometrial stromal sarcoma (ESS). Formalin-fixed and paraffin-embedded tissue sections from nine LMSs, 11 CLs, and 12 ESSs were evaluated with commercially available monoclonal antibodies against smooth muscle actin (SMA), desmin, and h-cal. Established morphologic criteria were used to classify the neoplasms. We found that there was, as expected, a significant difference in the expression of traditional smooth muscle markers (SMA and desmin) between tumors derived from smooth muscle and those derived from endometrial stroma (p = 0.005 for LMS and 0.013 for CL). We further found that h-cal was most useful in distinguishing between CL and ESS (p = 0.01). A significant difference between h-cal expression in LMS versus ESS was not found. Of note, one ESS expressed both SMA and desmin but lacked h-cal expression. Our findings confirm the most useful immunohistochemical data to date; smooth muscle neoplasms are generally distinguishable from endometrial stromal tumors by the expression of conventional muscle markers. We also report here that h-cal is useful more specifically in the differentiation of CL from ESS.

Timely surgery in intermittent and constant exotropia for superior sensory outcome.

PURPOSE: To determine whether time of strabismus surgery for patients with acquired intermittent exotropia and constant exotropia influences postoperative sensory outcome. METHODS: In a retrospective, cross-sectional study, 76 patients with acquired intermittent or constant exotropia and motor realignment were evaluated for postoperative sensory status. Age at surgery, duration of exotropia, and presence of intermittent or constant exotropia were correlated with postoperative sensory status. The 23 male and 53 female patients had an average age of 9.3 years at the time of surgery and a mean follow-up of 5.9 years. RESULTS: Patients had a significantly greater chance of having postoperative stereoacuity better than 60 seconds of arc (bifixation) if they were surgically aligned before 7 years of age (P <.01) or before 5 years of strabismus duration (P <.05), or with intermittent as compared with constant exotropia (P <.001). Patients with postoperative bifixation had earlier surgical intervention (P <.025) and shorter duration of exotropia (P <.025) than those with postoperative monofixation. CONCLUSIONS: Patients with intermittent or constant exotropia may achieve superior sensory outcome with motor realignment before age 7, before 5 years of strabismus duration, or while the deviation is intermittent.

Protocol biopsies in the management of renal allograft recipients.

The safety of the renal allograft biopsy and the standardization of allograft histopathology interpretation have renewed interest in the protocol biopsy. Recent studies in the areas of 'marginal' donors, surveillance of acute rejection, molecular biology and chronic rejection are discussed.