Antibody-Mediated Rejection in Kidney Transplantation Without Evidence of Anti-HLA Antibodies?

INTRODUCTION: The definition of antibody-mediated rejection (AMR) is based on serologic (presence and/or development of donor-specific anti-HLA antibodies [DSAs]) and histologic (C4d deposition and endothelial damage) criteria. However, several cases of AMR have been described without C4d deposition, and other cases of histologic AMR without DSAs, which could be driven by other non-HLA alloantibodies such as anti-MICA or anti-angiotensin II type I receptor (AT1R). Here we studied clinical and histologic humoral rejection in kidney transplant recipients without evidence of anti-HLA antibodies. MATERIALS AND METHODS: Fifteen kidney transplant recipients with AMR defined as C4d+ and/or histologic g+ptc without anti-HLA antibodies in screening test were studied. Sera at the moment of biopsy and 2 months earlier were studied for anti-HLA antibodies by Luminex, in neat, diluted 1/160, and sera after treatment with dithiothreitol (DTT) and confirmed by single-antigen test. The anti-AT1R was measured by enzyme-linked immunosorbent assay. RESULTS: A lack of anti-HLA and MICA antibodies was confirmed after anti-HLA screening test in all conditions (neat, diluted, and DTT-treated) and de novo development of AT1R antibodies was ruled out. Nevertheless, after single-antigen test, 3 patients were identified with a weak reaction against class I antigen and another 4 patients against class II antigen. Due to the lack of locus-C typing in the donors, the DSA assignment cannot be confirmed, whereas anti-HLA class II antigens were DSA. CONCLUSIONS: A low sensitivity in the screening of anti-HLA antibody testing was observed. Our results suggest performing single-antigen test in seronegative patients with clinical humoral rejection after screening to confirm the presence of DSA.

B-Cell-Activating Factor Levels Are Associated With Antibody-Mediated Histological Damage in Kidney Transplantation.

INTRODUCTION: Along with death engraftment, in recent years, antibody-mediated damage has been identified as the leading cause of loss of kidney transplants. Despite the recognition of the role of the B-lymphocyte subpopulation in the development of both tolerance and rejection, little is known about the trigger mechanisms and effectors of this humoral response. BACKGROUND: We analyzed the relationship between B lymphocyte subpopulations and levels of B-cell-activating factor (BAFF) with the histological findings in biopsies of renal transplantation. MATERIAL AND METHODS: We selected 35 patients whose kidney transplant biopsy was performed between January and November 2015. The biopsy specimens were classified according to Banff criteria. At the moment of the biopsy BAFF levels and B-lymphocyte subpopulations in blood were measured using enzyme-linked immunosorbent assay (ELISA) and using flow cytometry, respectively. RESULTS: Mean BAFF levels were 493 ± 245 pg/mL. The median performance of biopsy post-transplantation was 12.9 (11.7-23.9) months. BAFF levels correlated with pretransplantation antibodies (r = 0.523; P = .002) but not with kidney function. In biopsies performed more than 1 year after transplantation BAFF levels correlated with the severity of chronic glomerular (cg) involvement (r = 0.625; P = .003). Histological variables related to antibody-mediated injury selected by principal component analysis (glomerulitis, peritubular capillary, and chronic glomerulopathy) related to BAFF levels (B factor, 116; 95% confidence interval [CI], 12-220; P = .029). Biopsy specimens with transplant glomerulopathy (TG) showed lower levels of circulating naive CD19 + subpopulation, IgD+, and CD27- (32.7 ± 28.1 vs 87.9 ± 79.1; P = .017) compared with biopsy specimens without TG. CONCLUSIONS: Elevated levels of BAFF are associated with increased presence and severity of TG and a set of variables related to antibody-mediated histological damage. TG is associated with changes in circulating B-lymphocyte subpopulations that could contribute to its pathogenesis.

Serum Levels of Interleukin-34 During Acute Rejection in Liver Transplantation.

INTRODUCTION: Accumulating evidence indicates that interleukin (IL)-34 participates in T-cell homeostasis and tolerance due to the ability of IL-34 to trigger apoptosis of Th1, Th17, and Tc1 cells, but spare Th2 cells and Treg. In addition, IL-34 exerts anti-inflammatory effects by impairing leukocyte adhesion and transendothelial migration, and reducing the secretion of proinflammatory cytokines. The aim of our study was to investigate the time course of serum levels of IL-34 during hepatic allograft rejection. METHODS: Serum levels of IL-34 were determined in 20 healthy subjects and 45 hepatic transplant recipients. These patients were divided into 2 groups: group I was composed of 15 patients with acute rejection, and group II was composed of 30 patients without acute rejection. Samples were collected on days 1 and 7 after liver transplantation and on the day of liver biopsy. RESULTS: The concentrations of IL-34 were higher in the rejection group vs nonrejection group during the entire postoperative period. The whole transplant group, including those with stable graft function, showed higher IL-34 serum levels than the controls at all times after liver transplantation. CONCLUSIONS: Our preliminary results could be related to the recent finding that IL-34 may play an immune-suppressive role in liver transplantation. In our case, although we must be cautious with serum data, increased IL-34 would help to control alloresponse during rejection and protect from graft lost.

Activated Regulatory T Cells Expressing CD4(+)CD25(high)CD45RO(+)CD62L(+) Biomarkers Could Be a Risk Factor in Liver Allograft Rejection.

Activated regulatory T cells (aTregs) are nowadays a hot topic in organ transplantation to establish their role during acute rejection (AR) episodes. The aim of this multi-center study was to monitor the frequency of aTregs within the first year after transplantation in a cohort of first-time liver transplant recipients enrolled from 2010 to 2012. aTregs frequency was analyzed by means of flow cytometry. Patients who had AR showed higher levels of aTregs during first year after transplantation in comparison with patients who did not have higher levels. High levels of aTregs in liver recipients might be used as a biomarker of AR; however, further studies must be done to address the potential role of aTregs as biomarkers of AR in liver transplantation.

Frequencies of circulating B-cell subpopulations before kidney transplantation identify patients at risk of acute rejection.

The response mediated by B lymphocytes has a crucial impact on kidney transplantation due to the role of anti-human leukocyte antigen antibodies in rejection and the contradictory observation of high B-lymphocyte numbers in tolerant kidney transplant recipients. The basis of the contradiction could lay in the different function of B-cell subsets depending on their degree of differentiation. We ought to measure circulating B-lymphocyte percentages in patients with end-stage renal disease before kidney transplantation to identify those with a high risk of acute rejection. Eighty patients on the waiting list for kidney transplantation followed up in our center were recruited from 2010, and samples were taken just before kidney transplantation. Eleven of 80 patients presented an episode of acute rejection (13.75%) and had an increased frequency of switched (SW) B cells compared with the rejection-free group (median [interquartile range] 24.5% [18.6% to 39.6%] vs 15.1 [8.45% to 23.4%]; P = .025). Subsequently, the frequency of SW B cells was assessed as a predicting factor of acute rejection. A value higher than 18.4% predicted patients at risk of suffering an acute rejection episode with a sensitivity of 81.8% and a specificity of 60.9% and an area under the curve of 71.2%. Moreover, a decrease in naïve B-cell subsets was related to patients at risk of acute rejection. The percentage of circulating B-cell subsets before kidney transplantation could be used as biomarker of risk to suffer acute rejection. These unicenter data must be validated in multicenter studies.

Study of B-cell subpopulations in lung transplant recipients with posttransplant infection.

BACKGROUND: Posttransplant infection after lung transplantation is a common feature due to the immunodeficiency induced by the immunosuppressive load. AIM: To assess B-cell subsets in lung transplant recipients suffering at least one episode of infection within the first year posttransplantation. METHODS: Twenty-eight lung transplant recipients were enrolled in the study. Their overall mean age was 56.6 ± 10.7 years and 10 were women (35.7%). All recipients were treated with steroids, tacrolimus, and mycophenolate mofetil. B-cell subset levels were measured in peripheral blood before as well as 7, 14, 30, 60, 90, and 180 days posttransplantation. RESULTS: No difference in the absolute number of B-cell subsets was observed within the first year of follow-up. However, pre-germinal center-activated naïve B cells (Bm2'), defined as IgD(+)CD38(++), were increased among patients displaying infections within the first year. The increased Bm2' subset was accompanied by a decrease in the double negative (CD27(-)IgD(-)) B-cell population. CONCLUSION: Infections in lung transplant recipients were associated with an increase in the Bm2' subset even before transplantation. It is possible that Bm2' cells have a role in response to infection in lung transplantation.

Serum levels of interleukin-9 during acute rejection in liver transplantation.

INTRODUCTION: Interleukin-9 (IL-9) has been cast as a player in autoimmunity, but its role in liver transplantation remains to be clarified. The aim of our study was to investigate the time course of IL-9 serum levels during hepatic allograft rejection. METHODS: IL-9 serum levels were determined in 34 healthy subjects and 50 hepatic transplant recipients. The patients were divided into two groups: group I was composed of 15 patients with acute rejection episodes, and group II, 35 patients free of this problem. Samples were collected on days 1 and 7 after liver transplantation and on the day of liver biopsy. RESULTS: The concentrations of IL-9 were similar in the rejection and nonrejection groups over the entire postoperative period. The whole transplant group, including those with stable graft function, showed higher IL-9 serum levels than the controls at all times after liver transplantation. CONCLUSIONS: These preliminary results suggest a lack of participation of IL-9 in human liver allograft rejection.

Differentiation of autoimmune pancreatitis from pancreas cancer: utility of anti-amylase and anti-carbonic anhydrase II autoantibodies.

PURPOSE: To investigate the utility of different combinations of serum anti-carbonic anhydrase II antibodies (CA II Abs), anti-α amylase antibodies (AMY-α Abs) and IgG4 levels for the diagnosis of autoimmune pancreatitis (AIP). METHODS: We recruited 93 patients with clinical suspicion for AIP and 94 patients as control groups between June 2003 and October 2009. Serum antibodies were measured using homemade enzyme linked immunosorbent assay and IgG4 levels were determined by nephelometry. RESULTS: Both CA-II Abs and AMY-α Abs had the highest sensitivity (83%) although AMY-α Abs (89%) were more specific than CA-II Abs (75%). The presence of increased IgG4 levels was the most specific serological marker (94%), but it had the lowest sensitivity (58%). The combination of the three serological markers altogether had the highest specificity (99%) and positive predictive value (PPV) (86%), but they had a rather low sensitivity (50%). When we combined CA-II Abs and AMY-α Abs without IgG4 levels, we got the highest sensitivity (75%) and negative predictive value (98%) but the specificity and the PPV decreased to 93 and 50%, respectively. Importantly, AMY-α Abs were not detected in pancreas cancer. CONCLUSIONS: The presence of serum CA-II and AMY-α Abs with increased IgG4 is useful in the differential diagnosis of AIP from pancreatic cancer.

Quantitative assessment of serum free light chains in renal transplantation.

Plasma cell dyscrasias can cause renal disease. Sensitive methods have recently been introduced to quantify serum free light chains (sFLCs). Renal function may influence the variability of these methods, as shown in chronic kidney disease (CKD) patients, but this problem has not been widely addressed in renal transplant patients. Herein, we examined the association between polyclonal sFLC concentrations and renal function among a population of renal transplant patients. We studied 102 kidney allograft recipients and 53 CKD patients classified according to KDOQI (Kidney Disease Outcomes Quality Initiative) stages. None of them had been diagnosed with monoclonal gammopathy. sFLCs were quantified by nephelometry. Both serum κ and λ free light chain concentrations rose progressively through each stage of KDOQI among both transplant and nontransplant patients (P<.0001). In the former setting, sFLC concentrations significantly correlated, using a Spearman coefficient, with serum creatinine, and serum cystatin concentrations as well as estimated glomerular filtration rate: namely, 0.723, 0.797, and -0.711 for sκFLC and 0.705, 0.759, and -0.694 for sλFLC, respectively (P<.0001 in all cases). Spearman correlation coefficients in nontransplant patients were: 0.559, 0.848, and -0.766 for sκFLC and 0.702, 0.875, and -0.855 for sλFLC, respectively (P<.0001 in all cases). In conclusion, sFLCs must be interpreted cautiously due to their clear association with renal function. Therefore, renal transplantation did not produce changes that were different from those dependent on renal function.

Number of peripheral blood regulatory T cells and lymphocyte activation at 3 months after conversion to mTOR inhibitor therapy.

BACKGROUND: Mammalian target of rapamycin (mTOR) inhibitors are effective for induction and maintenance of regulatory T cells (Tregs). OBJECTIVE: To assess the effects of conversion from calcineurin inhibitors (CNIs) to mTOR on the number of circulating Tregs and lymphocyte activation. PATIENTS AND METHODS: In 18 renal transplant recipients receiving CNI therapy (cyclosporine in 9, and tacrolimus in 9), treatment was converted to mTOR inhibitors (everolimus in 14, and rapamycin in 4). Peripheral blood samples were obtained before and 3 months after conversion. The number of circulating Tregs was measured using flow cytometry, and defined as CD4+/CD25high/CD127low/CD27+/CD62L+/CD45RO+/Foxp3+. Lymphocyte activation was assessed indirectly according to production of intracellular adenosine triphosphate (iATP) on polyclonal activation using a phytohemaglutinin assay (Immuknow; Cylex, Inc, Columbia, Maryland). RESULTS: In 15 patients (83.3%), the absolute number of Tregs increased significantly (P=.001) after conversion (median, 16.35 cells/mm3; 95% confidence interval [CI], 13.97-21.94) vs 3 months after conversion (32.03 cells/mm3; 95% CI, 26.25-41.66). The iATP production decreased from 326 ng/mL (95% CI, 302-419) to 248 ng/mL (95% CI, 196-318; P=.02), and increased in 4 patients (22.22%). No significant correlation was demonstrated between Treg concentration and change in iATP production. No rejection episodes were reported during follow-up. CONCLUSIONS: Despite the small number of patients in whom therapy was converted from CNI inhibitors to mTOR inhibitors, the data suggest an increase in the absolute number of Tregs after conversion. In addition, the concentration of activated peripheral CD4+ T cells decreased to nearly that associated with risk of infection due to overimmunosuppression.

Diagnosis and management of immunodeficiencies in adults by allergologists.

Primary immunodeficiencies (PIDs) are genetic diseases that cause alterations in the immune response and occur with an increased rate of infection, allergy, autoimmune disorders, and cancer. They affect adults and children, and the diagnostic delay, morbidity, effect on quality of life, and socioeconomic impact are important. Therapy (gamma-globulin substitution in most cases) is highly effective. We examine adult PIDs and their clinical presentation and provide a sequential and directed framework for their diagnosis. Finally, we present a brief review of the most important adult PIDs, common variable immunodeficiency, including diagnosis, pathogenesis, clinical signs, and disease management.

Circulating cytokines in active polymyalgia rheumatica.

OBJECTIVE: To characterise the circulating cytokine profile and the cellular source of circulating cytokines in polymyalgia rheumatica (PMR). METHODS: The study included 34 patients with active untreated PMR and 17 age-matched healthy controls (HC). Circulating cytokines were measured by cytometric bead array and ELISA. Intracellular cytokines were assessed in CD3+ and CD14+ cells by flow cytometry. Cytokines in cell culture supernatants were also determined after polyclonal stimulation of patients' peripheral blood mononuclear cells. RESULTS: Circulating levels of interleukin-6 (IL6) were significantly higher in subjects with active PMR than in HC. Corticosteroid (CS) treatment was followed by a decrease in the level of IL6. Intracellular cytokine staining showed that circulating monocytes did not produce higher amounts of proinflammatory cytokines in patients with PMR than in HC. There was a discordance between serum levels and cytokine-producing monocyte and T cells, and it was not possible to demonstrate a Th1 bias in the peripheral compartment. CONCLUSIONS: Active PMR is characterised by increased serum levels of IL6, but not of other proinflammatory cytokines, that are rapidly suppressed by CS treatment. As circulating monocytes do not show increased production of proinflammatory cytokines, IL6 may be mainly produced in the inflamed tissue. A study of the circulating cytokine profile and its cellular source may provide a clue to new therapeutic options.

Severe course of community-acquired pneumonia in an adult patient who is heterozygous for Q481P in the perforin gene: are carriers of the mutation free of risk?

Most cases of autosomal recessive hemophagocytic lymphohistiocytosis (HLH) are associated with over 50 mutations in the perforin gene. Some of these mutations have no clear functional association. Only homozygous patients display a full-blown syndrome, whereas no severe disease has been described in heterozygous carriers of these mutations despite the presence of functional and phenotypic alterations in cytotoxic cells. We study the family of a child who died from HLH at 6 months of age due to a Q481P mutation in the perforin gene. The study is particularly interesting because the patient's heterozygous father experienced severe community-acquired pneumonia that could be attributed to deficient in vitro NK cell activity despite normal perforin expression. This case report suggests that impaired NK cell activity in a heterozygote can result in poorer initial control of infections with severe clinical expression.

T(H)17 versus Treg cells in renal transplant candidates: effect of a previous transplant.

INTRODUCTION: The T(H)1 and T(H)2 cells were described several years ago. However, this dichotomy has been disrupted by the description of other CD4(+) T cell subsets: the proinflammatory interleukin (IL)-17-producing T cells (T(H)17) and regulatory T cells (Tregs). The latter group inhibits the immune responses driven by T(H)1, T(H)2, and T(H)17 cells. IL-6 is involved in T(H)17 development, down-regulating Treg differentiation. Our hypothesis suggested that an imbalance between T(H)17 and Tregs enhances immune responses among renal transplant patients. MATERIALS AND METHODS: We studied 26 end-stage renal disease (ESRD) subjects and 10 patients awaiting a second renal transplant after previous graft dysfunction. We assessed the number of CD4(+)CD25(+)Foxp3(+) cells and serum levels of IL-17, the prototypic interleukin of T(H)17 cells. RESULTS: We observed a lower number of CD4(+)CD25(+)Foxp3(+) T cells among patients with previous graft dysfunction than those with ESRD (median 3.37 vs 8.63 cells/mm(3), P = .008). In contrast, IL-17 serum levels were augmented in graft dysfunction (median 4.45 pg/mL) compared with ESRD patients (1.39 pg/mL, P = .036), suggesting a proinflammatory state in patients awaiting a second renal transplant. CONCLUSION: The emerging alloresponse from a previous transplant favors the generation of T(H)17 instead of Treg cells. The enhanced activity of T(H)17 cells in retransplanted patients may down-regulate Treg cells, producing a proinflammatory environment that favors rejection of the next transplant.

Changes in the expression of the immunoglobulin-like transcript 3 (ILT3) and ILT4 receptors in renal allograft recipients: effect of donor and recipient aging.

INTRODUCTION: The aim of the present study was to investigate the number and phenotype of pre- and posttransplant peripheral blood dendritic cells (DCs) in kidney graft recipients to correlate with CD4(+)CD25(high) Treg and CD8(+)CD28(-) cells. Data were analyzed according to the age of the donor-recipient pairs. MATERIALS AND METHODS: A cohort of 49 cadaveric kidney transplant recipients was prospectively studied pretransplant and 6 months posttransplant by three-color flow cytometry with specific monoclonal antibodies. Patients were subgrouped according to age (elderly were considered above 60 years old and young below 55 years old) in the following donor-recipient pairs: aged/aged, young/aged, aged/young, young/young. RESULTS: At 6 months posttransplant, the proportion of cells tended to increase when the donor was young, regardless of the recipient. Importantly, there was a significant correlation between the numbers of immunoglobulin-like transcript 4(+) DCs and CD4(+)CD25(high) Treg cells before transplantation (r = .476, P = .004) and at 6 months (r = .408, P = .013). A significant association was also observed between ILT4(+) DCs and CD8(+)CD28(-) pretransplant (r = .540, P = .001) and at 12 months posttransplant (r = .609, P = .012). CONCLUSIONS: Renal grafts from young but not from aged donors seem to induce DC of a tolerogenic phenotype, both in aged and young recipients. These preliminary results suggested that donor age may have consequences in terms of tolerance induction.

Association between serum soluble CD30 and serum creatinine before and after renal transplantation.

OBJECTIVE: There is increasing evidence that circulating levels of soluble CD30 (sCD30) may represent a biomarker for outcome in kidney transplantation. The aim of this study was to measure the pre- and posttransplantation serum levels of sCD30 in cadaveric kidney transplant recipients and correlate them with serum creatinine. PATIENTS AND METHODS: Serum sCD30 was measured by a commercial enzyme-linked immunosorbent assay (ELISA) from prospective samples of 38 kidney allograft recipients serially transplanted at our center. Samples were collected at day 0 pretransplantation and at months 6, 12, 18, and 24 posttransplantation. We also studied sera from 29 patients with chronic kidney disease (CKD) at different stages of the K/DOQI guidelines, as a control group. RESULTS: Serum levels of sCD30 decreased significantly in samples posttransplantation compared with pretransplantation. The significant decrease after transplantation may be related to the improvement in renal function since we observed a significant correlation between serum levels of sCD30 and creatinine (sCr) at all times of the study. In addition, the patients with chronic renal failure showed a significant association between serum sCD30 and sCr (r = .454; P = .013). CONCLUSIONS: Our results did not suggest that the measurement of sCD30 may be used as a valuable biomarker in renal transplantation. Increased levels may be related to a decrease in its renal elimination.

Cytokine polymorphisms and risk of infection after kidney transplantation.

INTRODUCTION: Infection remains a significant cause of morbidity and mortality after solid organ transplantation. Genetic background has an influence on the incidence of infection. The aim of our study was to analyze the relationship between cytokine polymorphisms and infection in our kidney transplant recipients. METHODS: DNA from 255 kidney transplant recipients was isolated routinely. Polymerase chain reaction sequence-specific primer was performed using commercially available cytokine genotyping primer packs to determine polymorphisms of interleukin (IL)-10, transforming growth factor-beta, tumor necrosis factor-alpha, interferon-gamma, IL-6, IL-4, IL-2, IL-12, IL-4R alpha, IL-1RA, IL-1R, IL-1 beta, and IL-1 alpha. The appearance and number of infections within the first year after transplantation were identified retrospectively. RESULTS: One hundred twenty-two patients experienced at least one episode of infection in the first year after transplant. The frequency of the -511 IL-1beta CC genotype and the frequencies of the -1188 IL-12 CA and CC genotypes were significantly higher among the infected patients compared with the noninfected patients. We failed to observe significant differences in the genotype distribution of the other analyzed cytokines regarding the incidence of infection. After adjusting, recipient IL-1beta (-511 CC) genotype (relative risk [RR] 2.67, 95% confidence interval (CI) 1.30 to 5.49, P = .007) and recipient IL-12 (-1188 CA and CC) genotypes (RR 2.57, 95% CI 1.22 to 5.38, P = .012) predicted independently the risk of infection in the first year after kidney transplantation. CONCLUSION: Kidney transplant recipients with -511 IL-1beta CC genotype or with -1188 IL-12 CA and CC genotypes were at higher risk of developing infections in the first year after transplantation. Patients with genetic susceptibility to infection may benefit from less potent immunosuppressive therapy and more intense preventive measures.

Impact of HLA antibodies on transplant glomerulopathy.

The influence of humoral rejection on the development of chronic allograft nephropathy (CAN) is controversial, especially in relation to transplant glomerulopathy. The aim of our study was to analyse the influence of anti-HLA antibodies on the development of transplant glomerulopathy (cg0, cg1, cg2, and cg3; Banff'97). We selected all renal transplants patients from 1975 to 2003 who had a functioning graft for at least 6 months and a clinically indicated graft biopsy with CAN and chronic glomerular changes (case group). We studied the presence of anti-HLA antibodies (Ab) in the last serum taken while the graft was functioning and divided them into three groups according to the severity of glomerular lesions. We also selected 52 contemporary and comparable cases without transplant glomerulopathy (control group). A total of 77 case had transplant glomerulopathy: 39 cg1, 29 cg2, and 9 cg3. Pretransplant Ab titers and number of previous blood transfusions were higher among the subgroup with the most severe glomerulopathy. Patients who developed posttransplant anti-HLA Ab more frequently showed transplant glomerulopathy. Serum creatinine and proteinuria were higher among cases with chronic glomerulopathy, and more grafts were lost in that group. Thus, the presence of HLA-Ab is a key factor in the development of transplant glomerulopathy and chronic allograft rejection.