Functional Activity of the Complement System in Deceased Donors in Relation to Kidney Allograft Outcome.

Complement activation is considered one of the mediators of renal ischemia-reperfusion injury. Elevated levels of C5b-9, C3a, and C5a are detected in sera of deceased kidney donors. The goal of the study was to characterize the functional activity of complement pathways in donor sera and to assess their influence on transplant outcome. MATERIALS AND METHODS: Sixty-four deceased kidney donors (age 45 ± 16 years; 28 female, 36 male) and 27 healthy controls (age 42 ± 12 years; 14 female, 13 male) were enrolled in the study. The results of transplantation for the respective 122 kidney recipients were included in the analysis. The functional activities of classical (CP), lectin (LP), and alternative (AP) pathways were measured using Wielisa-kit (reference normal level = 100%). In most cases, decreased functional activity reflects the activation status of the pathway. RESULTS: The median (interquartile range) functional activities of the pathways in donor sera were CP 118 (89-150)%, LP 80 (20-127)%, and AP 74 (50-89)%, and did not differ from the control values CP 110 (102-115)%, LP 81 (26-106)%, AP 76 (61-88)%. The frequency of pathway activation observed in controls was CP 0%, LP 11%, and AP 0%. Deceased donors did not differ in activation of classical (11%) and lectin (13%) pathways, but presented a higher rate of alternative pathway activation (19%, P = .03). No significant influence of any pathway functional activity or its activation was proved to influence the transplant outcome. CONCLUSION: Complement activation via alternative pathway was observed in diseased donor sera. No predictive potential of donor complement functional activity on the transplant outcome could be proved.

B Cell Activating Factor (BAFF) in Long-term Kidney Transplant Recipients Is Not a Prognostic Marker for Allograft Dysfunction or Survival.

OBJECTIVE: B cell activating factor (BAFF) has been shown to play a role in B cell survival, maturation, and activation, and has been linked with renal transplant outcome. BAFF signaling has been associated with plasmablast survival, anti-HLA immunization, and loss of graft function. We aimed to analyze the interplay between BAFF, memory B cells, and plasmablasts in relation to allograft function in long-term kidney transplant (KTx) recipients and their anti-HLA sensitization. MATERIALS AND METHODS: This study included 70 long-term KTx recipients on standard immunosuppression 15 ± 6 years post transplantation (44 stable, 26 chronic allograft dysfunction, CAD) and 25 healthy volunteers. CD19+ B cells, memory B cells (CD19+CD27+), and plasmablasts (CD19+CD24-CD27++CD38++) were enumerated with flow cytometry. BAFF serum level and anti-HLA antibodies were assessed by Luminex bead arrays. RESULTS: We found no difference in BAFF levels between KTx recipients and controls (median, interquartile range: 1.67, 1.40-1.97 vs 1.78, 1.63-1.93 ng/mL, P = .478) and no correlation between BAFF level and cell counts. Recipients presented lower plasmablast count than controls (22.5, 8-57 vs 79, 48-166 cells/mL, P < .001). There was a positive correlation between estimated glomerular filtration rate and plasmablasts (rs = 0.30, P = .013) in recipients. Cell populations and BAFF were not related to the presence of anti-HLA antibodies. None of the parameters investigated was related to deterioration of allograft function during the 2-year follow-up. CONCLUSION: BAFF serum level is not related to anti-HLA sensitization, circulating memory B cells, plasmablast count, or allograft function. Circulating plasmablasts are associated with current allograft function but are not prognostic for future course.

Serum 25-Hydroxyvitamin D Level Is Not Related to Regulatory T Cells or Kidney Function in Renal Transplant Recipients.

BACKGROUND: Vitamin D and regulatory T cells (Tregs) are both involved in promoting peripheral tolerance and limiting chronic inflammatory diseases. Renal transplant recipients (RTRs) are likely to have low vitamin D levels, which may influence their immune status. AIM: The aim of our study was to assess the usefulness of serum 25-hydroxyvitamin D (25(OH)D) and Tregs in estimation of the protolerogenic milieu in RTRs within 1 year after kidney transplantation. METHODS: 26 RTRs (15M/11F, aged 49.1 ± 15.4 years) 3 to 13 months after kidney transplantation and 24 healthy volunteers were enrolled for the study. The serum level of 25(OH)D was measured with ELISA and peripheral blood immune cell populations (T lymphocytes, helper T lymphocytes, and Tregs) were assessed by flow cytometry. RESULTS: Severe 25(OH)D deficiency (<10 ng/mL) was found in one RTR (3%) and moderate deficiency (<20 ng/mL) in 12 (46%), while vitamin D sufficiency was found in 6 patients (23%). The RTRs did not differ from the control group in observed 25(OH)D levels. None of the cell populations were related to the level of 25(OH)D in the control group. In RTRs, there was a negative association between 25(OH)D and total T lymphocyte count (rs = -0.45, P = .023), but 25(OH)D was not related to any other cell population or kidney function. CONCLUSION: The results of our study suggest that serum 25(OH)D is not sufficiently reflective of vitamin D status to apply this measure in assessment of protolerogenic milieu in RTRs.

Lowering of Messenger Ribonucleic Acid Toll-Like Receptors 2-4,9 in Peripheral Blood Mononuclear Cells in Kidney Allograft Recipients, Relationships With Immunosuppressive Treatment, and Delayed Graft Function Occurrence.

BACKGROUND: Both tacrolimus (Tac) and cyclosporine (CsA) inhibit control peripheral blood mononuclear cells (PBMC) after stimulation of various Toll-like receptors (TLR) at supra-pharmacological concentrations. Earlier studies demonstrated that 24 hours after kidney transplantation (KT), the expression of the TLR4 messenger RNA (mRNA) in PBMC from patients with subsequent delayed graft function (DGF+) was lower than in patients without DGF (DGF-). An assessment was made of the interaction of immunosuppression with TLR mRNA in PBMC and to verify whether the reduced expression of TLR-2,3,4,9 mRNA in PBMC is permanent in DGF+. METHODS: We investigated mRNA expression of TLR in non-stimulated PBMC. All patients were transplanted more than 1 month before PBMC acquisition. Patients were divided into groups with respect to positive or negative history of delayed graft function (DGF+/-). RESULTS: The expression of TLR2, TLR3, and TLR9 in patients was lower than that in the control group. We found an association of Tac C0 with expression of TLR4 only and CsA dose per 1 kg body weight with TLR2 or up to 6 months after KT with TLR9. Mofetil mycophenolate (MMF)contributed to the change of TLR4 expression in the CsA group but not in the Tac group. TLR3 and TLR9 were nearly equally sensitive to both Tac and CsA, with a decrease of expression with respect to control. DGF+ was associated with variable degree of reduction of TLR2, TLR3, TLR4, and TLR 9 expression. CONCLUSIONS: We showed the importance of immunosuppression and delayed graft function as factors that modify the overall expression of mRNA-TLR PBMC for a period of time after KT. Patients with a history of DGF have chronically decreased expression of mRNA TLR2, TLR3, TLR4, and TLR9. This fact is associated with poorer graft function. Measuring the expression of the TLR in the upper range of therapeutic doses of calcineurin inhibitors and MMF gives the opportunity to assess the strength, effectiveness, and toxicity of immunosuppression.

A significant role for anti-human leukocyte antigen antibodies and antibody-mediated rejection in the biopsy-for-cause population.

The role of anti-human leukocyte antigen (HLA) antibodies and antibody-mediated rejection is well known, but our comprehension and the preventive measures we take seem to be insufficient. One of the major causes of premature renal transplant loss is recepients' immunologic hyperactivity to donors' antigens. Monitoring of humoral alloreactivity gives hope for early diagnosis and adequate therapy. The goal of our analysis was the assessment of the influence of anti-HLA antibodies on the function and survival of transplants. In our study we included 60 consecutive renal transplant recipients who had a renal transplant biopsy-for-cause performed due to insufficiency. Transplant biopsies were performed between the 7th day and 12th year (median, 2 years) after transplantation. Anti-HLA antibodies were present in 20 patients (33%). The patients were divided into 2 groups according the presence of anti-HLA antibodies. In a 12-month observation, 10/20 (50%) patients in the anti-HLA(+) group returned to dialysis in contrast with 7/40 (17.5%; P = .014) in the anti-HLA(-) group. Also, 8/10 (80%) of the anti-HLA(+) patients who lost the transplant had anti-HLA Abs class II and only 2/10 (20%) had anti-HLA Abs class I. Anti-HLA antibodies were specific to a donor (donor-specific antibodies [DSA]) in 8/10 (80%) of the patients who lost the transplant. Anti-HLA antibodies appeared de novo in 50% of patients who lost the transplant. Nonadherence was suspected in 50% of patients. Acute humoral rejection occurred in 1 patient. Also, 8/10 (90%) developed chronic active humoral rejection. Our study revealed that graft loss in the renal transplant biopsy-for-cause population with the presence of anti-HLA Abs during a 12-month observation reached 50%. Nonadherence in these patients was very high and amounted to 50%. Monitoring of renal transplant recipients and individualization of therapy considering humoral activity should prolong renal graft survival.

Non-HLA antibodies: angiotensin II type 1 receptor (anti-AT1R) and endothelin-1 type A receptor (anti-ETAR) are associated with renal allograft injury and graft loss.

INTRODUCTION: Non-HLA antibodies specific for angiotensin II type 1 receptor (anti-AT1R) and endothelin-1 type A receptor (anti-ETAR) of vascular cells activate signaling pathways leading to cell proliferation and vascular injury. The aim of this study was to evaluate the impact of non-HLA antibodies on kidney allograft morphology and function in patients who underwent a kidney biopsy due to renal function impairment. PATIENTS AND METHODS: The study included 65 consecutive renal transplant patients who were evaluated for the presence of non-HLA and anti-HLA antibodies at the time of transplant biopsy. Results of pre-transplant CDC cross-match were negative. A kidney allograft biopsy was performed between 6 days and 13 years (42 ± 49 months) after transplantation, and the diagnosis was made on the basis of the Banff criteria. The level >9 U/L of anti-AT1R and anti-ETAR antibodies was considered high. RESULTS: A high level of non-HLA antibodies (anti-AT1R and/or anti-ETAR) was found in 7 (10.7%) of 65 patients at the time of biopsy. Graft loss in the non-HLA-positive patients was significantly higher (71% in non-HLA-positive cases after 7.8 ± 2.6 months vs 11% after 6 months in non-HLA-negative cases [P = .00099]). In these non-HLA-positive patients, the mean anti-AT1R level was 15.3 ± 9.4 U/L and the mean anti-ETAR level was 13.8 ± 8.6 U/L. In only 2 of these patients were anti-HLA antibodies additionally detected: anti-class I in 1 and anti-class II in both patients. The mean serum creatinine level was 2.34 ± 0.6 mg/dL at the time of biopsy. Results of an early biopsy revealed acute vascular rejection (Banff grade IIB). Chronic allograft injury was found (grading cg1-3, cv1-2, ci1-2, ct1-2) in the remaining 6 patients. C4d was present in 3 of 7 patients. CONCLUSIONS: High levels of anti-AT1R and/or anti-ETAR antibodies were associated with morphological and functional allograft injury and graft loss in these study patients. Non-HLA antibodies can be helpful in assessing the risk of graft failure.

Significant infections after hand transplantation in a Polish population.

The study was conducted to assess serious infectious complications in five hand allograft recipients (four males, one female, age 40 ± 10 years), transplanted between 2006 and 2010. All donors and recipients were positive but one for cytomegalovirus (CMV) immunoglobulin G. All recipients received immunosuppressive therapy basiliximab, tacrolimus, mycophenolate mofetil and methylprednisolone. Until May 2013, there were four cases of severe infections requiring hospitalization. One patient developed CMV infection on the 28th postoperative day. Despite therapy with ganciclovir and prophylaxis with valganciclovir, reinfection episodes occurred both 4 weeks and 7 months later. The female recipient developed CMV infection 8 months after hand transplantation. After 3 weeks of ganciclovir treatment, the polymerase chain reaction results remained negative. We found that the CD4/CD8 T lymphocytes ratio differs in those two patients who had developed CMV disease in the past in comparison to the three remaining hand transplant recipients (mean 0.46 versus 1.7, respectively). Moreover, the ratio of patients who were CD4-8 negative to total T lymphocytes in CMV recovered patients was two-fold higher compared to the remaining recipients (10.0 versus 4.4, respectively). The female recipient was also hospitalized because of acute tonsillitis 25 months after hand transplantation, and successfully treated with amoxicillin clavulanate. The third recipient was hospitalized because of severe acute pain involving right lower limb, especially foot, 74 months after hand transplantation. After 48 hours, a painful vesicular rash occurred on the plantar as well as dorsal surface of right foot and herpes zoster was diagnosed. Immunosuppressive therapy after hand transplantation may be complicated by serious infections. CMV disease was associated with persistent alterations in T lymphocyte subsets.

Pathogenic role of antibodies against monomeric C-reactive protein in tubulointerstitial nephritis and uveitis syndrome.

Antibodies against monomeric C-reactive protein, which is a target antigen expressed both in kidney tubules and uveal cells, have been recently detected in patients with active tubulointerstitial nephritis and uveitis syndrome. We report the case of an 65-year-old woman with acute renal failure caused by biopsy-proven tubulointerstitial nephritis and the onset of uveitis 21 months later. The expression of monomeric C-reactive protein in kidney oligobiopsy was confirmed by immunohistochemical staining using mouse monoclonal antibody against human monomeric C-reactive protein. The levels of antibodies against monomeric C-reactive protein were 117% of the reference during the flare and 22% during the remission of the disease. The difference in the levels of antibodies against monomeric C-reactive protein during flare and remission, and above all positive biopsy staining, supports their pathogenic role in this disease.

The impact of de novo donor-specific anti-human leukocyte antigen antibodies on 5-year renal transplant outcome.

Numerous studies have shown that circulating donor-specific antibodies targeting human leukocyte antigen (HLA) are associated with accelerated renal transplant failure, but many patients with these antibodies have good graft function. The aim of our study was to investigate the long-term graft function and survival in patients with de novo post-transplant donor-specific anti-HLA antibodies (DSA). Our prospective study included 78 consecutive recipients with a negative crossmatch before transplantation. Recipient serum samples were assayed for DSA in week 2 and 1, 3, 6, 9, 12 months after transplantation using a complement-dependent lymphocytotoxic technique with donor lymphocytes. Additionally, patients with DSA and stable renal function in the first year were tested with a more sensitive flow-panel-reactive antibody. DSA were present in 34 (44%) of our patients during the first 12 months after transplantation. Biopsy-proved acute rejection occurred in 11 DSA-positive and 10 DSA-negative patients. Seven DSA-positive patients had antibody-mediated rejection and no DSA-negative ones developed humoral rejection. The serum creatinine level in DSA-positive patients was significantly higher (2.48 vs 1.43 mg/dL) in year 5. The 13 (38%) DSA-positive patients with good graft function in month 12 were stable during a 5-year follow-up: their serum creatinine was 1.46 ± 0.4 in year 1 and 1.56 ± 0.4 mg/dL in year 5 and nobody lost their allograft. One- and 5- year graft survivals were appropriately 85% and 59% in DSA-positive patients compared to 93% and 93% in DSA-negative patients. To sum up, post-transplant DSA had a significant influence on kidney function and graft survival but in 38% of patients the presence of DSA did not decrease a 5-year renal function. A good renal allograft function in the presence of DSA in the first year after transplantation and cessation of their production in the subsequent years may be a good prognostic marker for a long-term allograft function and survival.

Long-term follow-up of non-HLA and anti-HLA antibodies: incidence and importance in renal transplantation.

BACKGROUND: Detection of antibody-mediated injury is becoming increasingly important in post-transplant patient care. The role of donor-specific anti-human leukocyte antigen (HLA) antibodies in kidney transplant damage is known, whereas the significance of non-HLA antibodies remains an unresolved concern. The aim of the study was to determine the presence and influence on renal function of non-HLA and anti-HLA antibodies in stable patients at 5 years after kidney transplantation. METHODS: We evaluated the antibodies in 35 consecutive patients with stable renal function at 5 years after transplantation. RESULTS: Pretransplant screening for donor-specific antibodies by CDC cross-matches was negative in all patients. Anti-endothelial cell antibodies (AECA), anti-angiotensin II type 1 receptor antibodies (anti-AT1R), and anti-endothelin receptor antibodies (anti-ETAR) were assayed as non-HLA antibodies. Non-HLA antibodies were observed in 12 (34%) patients, including AECA (n = 5; 14%), anti- AT1R (n = 6; 17%), anti-ETAR (n = 4; 11%), and both anti-AT1R and anti-ETAR (n = 3). Among 13 (37%) patients with anti-HLA antibodies, 7 also had both non-HLA antibodies: AECA (n = 1), anti-AT1R (n = 3), and anti-ETAR (n = 3). The antibody-negative group (n = 13) showed significantly better renal function than the antibody-positive group (non-HLA and/or anti-HLA; n = 22). Biopsy-proven acute rejection had occurred in 2 of 13 (15%) antibody-negative versus 8 of 22 (36%) antibody-positive patients. These preliminary data revealed an high prevalence of autoantibody and alloantibody production among stable patients at 5 years after kidney transplantation. CONCLUSION: Simultaneous production of these antibodies and their association with reduced renal function suggests that active humoral immune responses are poorly controlled by immunosuppression.

Extracorporeal photopheresis as an antirejection prophylaxis in kidney transplant recipients: preliminary results.

Extracorporeal photopheresis (ECP) is considered a promising immunomodulatory therapy of acute allograft rejection in organ transplantation and graft-versus-host disease. Our aim was to investigate the biological responses of 10 patients who underwent kidney transplantation with ECP as prophylactic treatment. They received conventional immunosuppressive therapy plus ECP immediately after transplantation: 12 to 16 applications over the course of 2.5 months. ECP procedures were performed using an automated system for leukocyte separation and photoactivation with methoxsalen. All recipients were followed by estimated glomerular filtration rate (eGFR) and peripheral T, B, natural killer, T-regulatory (Treg) and dendritic cells (DC) counts and phenotypes. An acute rejection episode appeared in one control group recipient. The ECP group showed a positive trend to an higher GFR at months 3 (53±11 vs 47.1±9; P=.17) and 6 (67.5±10 vs 53.6±3; P=.03, Wilcoxon test). An increased percentage of Treg (CD3+ CD4+ CD25+) among the total CD3 cell count (4.9%±1% to 9.4%±15%) as well as inducible Treg (CD3+ CD8+ CD28-) was observed among CD3 cells (3.3%±3% to 11.8%±8%, P=.025) within 3 months of ECP treatment. A significant difference in the percentage of Treg was noted at month 3 (completed ECP) between the ECP and the control groups (9.4%±15% vs 3%±1%; P=.01). Addition of ECP to standard immunosuppression was associated with a significantly higher GFR at 6 months and with a significant increase in natural Treg among CD3 cells.