Nina Babel

Effect of anti-CD 20 antibody rituximab in patients with post-transplant lymphoproliferative disorder (PTLD)

Post‐transplant lymphoproliferative disorders (PTLD) are a life‐threatening complication following solid organ transplantation. Treatment with rituximab, a humanized anti‐CD20 monoclonal antibody, has proved to be a promising approach and shown a low toxicity profile. Between February 1999 and April 2002, we conducted a multicentre phase II trial investigating rituximab as single agent in 17 patients with PTLD. Transplanted organs were heart (n = 5), kidney (n = 4), lung (n = 4) and liver (n = 4). Patients were treated with four weekly doses of 375 mg/m2 of rituximab. The mean follow‐up time is 24.2 months. Histology was distributed in 10 diffuse large cell‐, 2 marginal zone‐, 1 Burkitt‐like lymphoma, 1 Hodgkin‐like PTLD and 3 polymorphic lymphoproliferations. Therapy was well tolerated and no severe adverse events were observed. The mean overall survival period is 37.0 months with 11 patients still living. In total, 9 patients (52.9%) achieved a complete remission, with a mean duration of 17.8 months. Partial remission was observed in 1 patient, minor remission in 2 patients, no change in 3 patients and 1 patient experienced progressive disease. Two patients relapsed, at intervals 3 and 5 months after obtaining complete remission. Rituximab proved to be well tolerated and effective in the treatment of PTLD.

Tumor necrosis factor–alpha, transforming growth factor–β1, and interleukin-10 gene polymorphisms: implication in protection or susceptibility to dengue hemorrhagic fever

Dengue virus infection has emerged as one of the most important arthropod-borne viral diseases. Some dengue infected individuals develop the severe, life-threatening form of the disease, dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). Host genetic factors may be relevant and may predispose some individuals to the severe illness. Human leukocyte antigen (HLA), FcγR, tumor necrosis factor (TNF)-α, and dendritic cell-specific intracellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN), among others genes have been associated with the pathogenesis of dengue. Little is known, however, about the predictive value of cytokine genotypes for the clinical outcome of dengue infection. In this study, the TNF-α, interleukin (IL)-6, interferon (IFN)-γ, IL-10 and transforming growth factor (TGF)-β1 gene single nucleotide polymorphisms (SNP) were studied by polymerase chain reaction-sequence-specific primer in a group of individuals with the antecedent of DHF during a secondary infection in the sequence dengue 1/dengue 2. A control group was also included. TNF-α (-308) A allele and IL-10 (-1082/-819/-592) ACC/ATA haplotype were significantly associated with DHF. TNF-α (-308) GG and TGF-β1 (c25) GG genotypes were associated with protection. Our results suggest that genetic predisposition to a high TNF-α production and a low IL-10 production seems to increase the susceptibility to DHF during a secondary dengue 2 infection, whereas TGF-β1 high producers might be protected for developing DHF.

BK virus-specific immunity kinetics: a predictor of recovery from polyomavirus BK-associated nephropathy.

Impaired BKV‐specific immunity is associated with development of BKV‐associated nephropathy. Suitable immunological parameters to identify patients at risk, however, are still debated. We monitored 18 kidney‐transplant recipients through the course of self‐limited BKV‐reactivation (n = 11) and BKV‐associated nephropathy (n = 7). BKV‐specific cellular immunity directed to nonstructural small and Large T‐antigen, and structural VP1–3 was analyzed in an interferon‐γ Elispot assay. BKV‐specific IgM and IgG were measured using an enzyme‐linked immunosorbent assay simultaneously. BKV‐specific cellular immunity directed to five BKV‐proteins increased significantly from diagnosis to resolution of BKV‐reactivation (p < 0.001). Patients with self‐limited BKV‐reactivation developed BKV‐specific T cells without therapeutic interventions, and cleared BKV‐reactivation within a median period of 1 month. Patients with BKV‐associated nephropathy, however, showed BKV‐specific T cells after a median period of 5 months after therapeutic interventions only, and cleared BKV‐reactivation after a median period of 8 months. Anti‐structural T cells were detected earlier than anti‐nonstructural T cells, which coincided with BKV‐clearance. Patients with BKV‐associated nephropathy showed the highest frequencies of BKV‐specific T cells at recovery, the highest increase in BKV‐specific IgG and persistence of increased IgM levels (p < 0.05). Our results suggest prognostic values of BKV‐specific immune monitoring to identify those patients at risk of BKV‐associated nephropathy and to aid in the management of therapeutic interventions.

Probiotic therapy in the prevention of pouchitis onset : Decreased interleukin-1β, interleukin-8, and interferon-γ gene expression

Background: Probiotic therapy has been shown to prevent the onset of pouchitis and to improve the quality of life in ulcerative colitis patients who required ileal pouch anal anastomosis. Pouchitis has been associated with elevated levels of proinflammatory cytokines and chemokines. Methods: In this retrospective analysis of archived endoscopic samples from responding patients enrolled in the above‐mentioned trial, we were interested in studying mucosal gene expression of the pleiotropic proinflammatory cytokines (interleukin‐1&bgr;, interleukin‐6), TH1 cytokines (interferon‐&ggr;, tumor necrosis factor‐&agr;, interleukin‐12), regulatory cytokines (interleukin‐10, transforming growth factor‐&bgr;), and the chemokine interleukin‐8. In addition to assessment of cytokine gene expression, the presence of polymorphonuclear cells in the mucosal tissue was evaluated. Results: Data show that patients who were treated with probiotics had significant lower mucosal mRNA expression levels of interleukin‐1&bgr;, interleukin‐8, and interferon‐&ggr; compared with placebo‐treated patients. Conclusions: In addition, a lower number of polymorphonuclear cells was present in the tissue of patients within the probiotic group compared with the number of polymorphonuclear cells in the tissue of patients receiving placebo and patients having an episode of pouchitis. Conclusions: These data suggest that probiotic treatment regulates the mucosal immune response by reducing mucosal levels of neutrophil‐chemoattractant IL‐8 and tissue influx of polymorphonuclear cells, and may further act by inhibition of T‐cell activation, by reinforcement of barrier function and by a tight control of the potent pro‐inflammatory cytokine IL‐1&bgr;.

Sustained BK viruria as an early marker for the development of BKV-associated nephropathy: analysis of 4128 urine and serum samples.

Background. BKV reactivation plays the causative role in the development of BKV-associated nephropathy (BKVAN). Because of the lack of effective therapy, early diagnosis of BKV reactivation is paramount for the prevention of BKVAN. Resting in uroepithelial cells, BKV is excreted first in urine before it can be detected in plasma. The present study analyzed predictive value of BK viruria for the development of BK viremia and its possible advantage for the early BKVAN prediction. Methods. Total of 4128 urine and serum samples obtained from renal transplant patients were analyzed for BKV positivity by real-time polymerase chain reaction in 433 patients in cross-sectional and in 233 patients in longitudinal manner, respectively. The prospective longitudinal analysis included seven measurements during the first posttransplant year. Results. A total of 7% and 19% patients were positive for BKV in serum and urine, respectively. Sustained BK viruria showed sensitivity of 100% and specificity of 94% for BK viremia and was associated with significantly higher level of BK load than the patients with transient viruria (P<0.01). Interestingly, BK viremia was preceded by BK viruria: the peak of viral load and number of positive patients appeared during the third and fifth posttransplant month for urine and serum, respectively. BKVAN diagnosed in 21.4% of patient with persistent BK viruria appeared 5 and 11 weeks after BKV reactivation in serum and urine, respectively, was detected. Conclusion. Sustained BK viruria is a reliable marker allowing an early identification of patients at high risk of BKVAN development and therefore assure precocious therapeutic interventions.

TCR Repertoire Analysis by Next Generation Sequencing Allows Complex Differential Diagnosis of T Cell–Related Pathology

Clonotype analysis is essential for complete characterization of antigen‐specific T cells. Moreover, knowledge on clonal identity allows tracking of antigen‐specific T cells in whole blood and tissue infiltrates and can provide information on antigenic specificity. Here, we developed a next generation sequencing (NGS)‐based platform for the highly quantitative clonotype characterization of T cells and determined requirements for the unbiased characterization of the input material (DNA, RNA, ex vivo derived or cell culture expanded T cells). Thereafter we performed T cell receptor (TCR) repertoire analysis of various specimens in clinical settings including cytomegalovirus (CMV), polyomavirus BK (BKV) reactivation and acute cellular allograft rejection. Our results revealed dynamic nature of virus‐specific T cell clonotypes; CMV reactivation was linked to appearance of new highly abundant antigen‐specific clonalities. Moreover, analysis of clonotype overlap between BKV‐, alloantigen‐specific T cell–, kidney allograft‐ and urine‐derived lymphocytes provided hints for the differential diagnosis of allograft dysfunction and enabled appropriate therapy adjustment. We believe that the established approach will provide insights into the regulation of virus‐specific/anti‐tumor immunity and has high diagnostic potential in the clinical routine.

BK polyomavirus infection and nephropathy: the virus–immune system interplay

Reactivation of latent BK polyomavirus (BKV) infection continues to be a major challenge in renal graft recipients. Progression of BKV infection to BKV-associated nephropathy (BKVAN) leads to graft loss in up to 60% of affected patients. Interestingly, although >80% of healthy adults are seropositive for BKV, BKVAN occurs almost exclusively in transplanted kidneys, which raises questions about its underlying pathogenetic mechanisms. Intragraft inflammation and an insufficient antiviral immune response seem to be the most important risk factors. Early studies revealed an association between the rate of recovery of BKV-specific cellular immunity (which shows high interindividual variation) and BK viral clearance, which determines the clinical course of BKV infection. In patients with prompt recovery of BKV-specific T cells, BKV infection can be controlled at the early reactivation stage and does not progress to BKVAN. By contrast, in patients with persistent BKV reactivation caused by insufficient BKV-specific immunity, continued viral replication and inflammation ultimately lead to graft injury and/or BKVAN. As the chronic course of BKV infection can be prevented in most patients by prompt restoration of BKV-specific immunity, frequent monitoring of BK viral load and targeted, timely modification or reduction of immunosuppression is strongly recommended for affected patients.

Salvage chemotherapy for refractory and relapsed posttransplant lymphoproliferative disorders (PTLD) after treatment with single-agent rituximab.

Background. Single-agent rituximab has demonstrated encouraging efficacy and tolerability in posttransplant lymphoproliferative disorders (PTLDs) failing to respond to immunosuppression reduction (IR). This retrospective analysis was undertaken to determine the efficacy and safety of salvage therapy in recipients of solid organ transplants with progression of PTLD after rituximab first-line therapy. Methods. Eleven patients who had received IR and single-agent rituximab were analyzed. Of these, 10 had received CHOP salvage chemotherapy. One patient with limited disease received tumor irradiation and further IR. Most patients (73%) had late PTLD (median onset of disease 145 months posttransplant), and most (83%) had monomorphic histology; 36% had EBV-association. Results. IR and irradiation therapy re-induced complete remission (CR) and allowed long-term disease control in a patient with polymorphic PTLD relapse. CHOP therapy achieved CR in five (50%) and partial remission (PR) in two (20%) patients. Four of five (80%) patients achieving CR remained in CR at a median follow-up of 44.2 months. Of the patients achieving PR, one is currently alive, and the second died from transplant rejection after converting to CR after consolidative chemotherapy. Patients with stable disease (two) and progressive disease (one) have died from PTLD. There was one possible CHOP-associated death (acute cardiac event) and two patients had to be switched to less-toxic monotherapies. Median overall survival was 46.5 months (95% confidence interval: 23.6–49.1 months). Conclusions. CHOP salvage therapy achieved a favorable overall response rate of 70% in this setting, indicating that PTLD generally remains chemotherapy-sensitive after progression following first-line rituximab.

Immunohistochemical Detection of Hypoxia-Inducible Factor-1α in Human Renal Allograft Biopsies

Although it generally is accepted that renal hypoxia may occur in various situations after renal transplantation, direct evidence for such hypoxia is lacking, and possible implications on graft pathophysiology remain obscure. Hypoxia-inducible factors (HIF) are regulated at the protein level by oxygen-dependent enzymes and, hence, allow for tissue hypoxia detection. With the use of high-amplification HIF-1alpha immunohistochemistry in renal biopsies, hypoxia is shown at specific time points after transplantation with clinicohistologic correlations. Immediately after engraftment, in primarily functioning grafts, abundant HIF-1alpha is present and correlates with cold ischemic time >15 h and/or graft age >50 yr (P < 0.04). In contrast, a low HIF-1alpha score correlates with primary nonfunction, likely reflecting loss of oxygen consumption for tubular transport. Protocol biopsies at 2 wk show widespread HIF-1alpha induction, irrespective of histology. Beyond 3 mo, both protocol biopsies and indicated biopsies are virtually void of HIF-1alpha, with the only exception being clinical/subclinical rejection. HIF-derived transcriptional adaptation to hypoxia may counterbalance, at least partly, the negative impact of cold preservation and warm reflow injury. Transient hypoxia at 2 wk may be induced by hyperfiltration, hypertrophy, calcineurin inhibitor-induced toxicity, or a combination of these. Lack of detectable HIF-1alpha at 3 mo and beyond suggests that at this time point, graft oxygen homeostasis occurs. The strong correlation between hypoxia and clinical/subclinical rejection in long-term grafts suggests that hypoxia is involved in such graft dysfunction, and HIF-1alpha immunohistochemistry could enhance the specific diagnosis of acute rejection.

High levels of CMV-IE-1-specific memory T cells are associated with less alloimmunity and improved renal allograft function.

BACKGROUND Cytomegalovirus (CMV) infection has been associated with allograft rejection in solid organ transplantation. However, the immunologic mechanisms behind this observation have not been elucidated. One proposed mechanism is direct cross-reactivity of antiviral T-cells with allogeneic MHC/peptide complexes, a process termed heterologous immunity. Another model favours indirect stimulation of alloimmunity by CMV-induced proinflammatory cytokines and upregulation of MHC class II and adhesion molecules. Recently, we found that protection from CMV disease was correlated with high levels of CMV-immediate early-1 (IE-1) specific IFN-gamma-producing T-cell responses in heart and lung transplant recipients. The aim of this study was to define the relation of CMV-specific T-cell responses to acute rejection, donor-reactive memory T cells, and allograft function after kidney transplantation. METHODS To address this issue, IFN-gamma-producing T-cell responses following ex-vivo stimulation with pools of overlapping peptides representing the CMV pp65 and IE-1 proteins, as well as donor-reactive IFN-gamma-producing T-cells were determined at multiple time points before (pre-Tx) and during the first 6 months posttransplant (post-Tx) in 36 kidney transplant recipients using an enzyme linked immunoabsorbent spot assay (ELISPOT). RESULTS CMV-specific T cells were not exclusively detectable in CMV seropositive patients, as 3/12 seronegative patients had significant pre- and post-Tx pp65/IE-1-specific T-cell responses. In patients with detectable anti-CMV antibody or T-cell responses, no difference in CMV-specific T-cell frequencies was found between patients with versus without acute rejection. However, early (week 1, r=0.457, p=0.037) and average IE-1-specific T-cell responses (r=-0.415, p=0.032) during 6 months post-Tx showed a significant inverse correlation with average post-Tx donor-reactive T-cell responses. Furthermore, average post-Tx IE-1-specific T-cell responses correlated significantly with 6 and 12 months glomerular filtration rate (GFR). In contrast, pp65-specific T-cell responses did not correlate with donor-reactive T cells or graft function. Only 2/36 patients developed CMV disease, both showing very weak IE-1-specific T-cell responses during the whole monitoring period. CONCLUSION No evidence for heterologous immunity could be found in patients with high levels of CMV-specific T cells. On the contrary, less alloreactivity and improved graft function were found in patients with strong IE-1-specific T-cell responses. These results emphasize the importance of immediate early antigens (IE) as targets for T-cell immunity to CMV. We hypothesize that IE-1-specific T cells might effectively suppress IE-1-induced indirect effects such as inflammation and upregulation of MHC class II and adhesion molecules.