Florian Emmerich

Long-term outcome of ABO-incompatible living donor kidney transplantation based on antigen-specific desensitization. An observational comparative analysis

BACKGROUNDnABO-incompatible living donor kidney transplantation based on specific conditioning has been successfully adopted by transplant centres worldwide. Excellent short-term results have been reported in small cohorts. However, long-term data and comparative analyses are still sparse. We report on the outcome of 40 consecutive ABO-incompatible living donor kidney transplant recipients and compare their clinical course to a control group of 43 ABO-compatible living donor transplant patients transplanted during the same time period.nnnMETHODSnThis is an observational single-centre analysis of 40 consecutive patients undergoing ABO-incompatible kidney grafting between April 2004 and April 2009, using a protocol of rituximab, antigen-specific immunoadsorption, intravenous immunoglobulin, basiliximab induction and oral triple immunosuppression with tacrolimus, mycophenolic acid and prednisone. Forty-three ABO-compatible kidney transplant recipients served as controls. The control group had also received basiliximab induction and an identical initial maintenance immunosuppression. The two groups were observed for an average of 39 and 19 months, respectively.nnnRESULTSnThere was a significantly higher incidence of lymphoceles requiring surgical revisions in the ABO-incompatible group. However, this surgical complication did not affect patient or graft survival. Mean serum creatinine, estimated glomerular filtration rate and proteinuria did not differ between the two groups. Furthermore, ABO-incompatible and ABO-compatible patients had the same incidence of humoral and cellular rejections. Despite a more aggressive induction therapy, no differences in infectious or malignant complications were observed.nnnCONCLUSIONSnABO-incompatible living donor kidney transplantation utilizing a combination of rituximab and antigen-specific immunoadsorption yielded results identical to ABO-compatible transplantation despite a significantly higher number of human leukocyte antigen mismatches.

Genome-wide Profiling in AML Patients Relapsing after Allogeneic Hematopoietic Cell Transplantation

Molecular pathogenesis of relapse after allogeneic hematopoietic cell transplantation is poorly understood. Data regarding relapse mechanisms after transplantation is scarcely available. We investigated genomic aberrations (GAs) in 21 patients undergoing related and unrelated HLA-matched transplantation in leukemic blasts before transplant and at relapse after transplantation. We found a higher number of GAs after transplantation, suggesting increased genomic instability during relapse. Two of 21 patients showed a large homozygous region spanning the whole HLA-locus on chromosome 6p in the relapse sample. In both patients sequence-based HLA typing of the blasts revealed a loss of the patient-specific allele at the mismatched locus leading to homozygosity for the HLA haplotype shared by the patient and the donor. In addition, GAs were found in critical regions such as 12p13, 13q12.2, and 17p13. Our results suggest that escape from immunologic surveillance may be a relevant mechanism of relapse after transplantation in patients with GAs on chromosome 6p. A combination of continuous immunologic pressure mediated by donor T cells and clonal evolution of myeloid leukemia may result in acquired GAs after transplantation.

An update on ABO-incompatible kidney transplantation

ABO‐incompatible kidney transplantation is nowadays a well‐established procedure to expand living donor transplantation to blood group incompatible donor/recipient constellations. In the last two decades, transplantation protocols evolved to more specific isohaemagglutinin elimination techniques and established competent antirejection protection protocols without the need of splenectomy. ABOi kidney transplantation associated accommodation despite isohaemagglutinin reappearance, C4d positivity of peritubular capillaries as well as the increased incidence of bleeding complications is currently under intense investigation. However, most recent data show excellent graft survival rates equivalent to ABO‐compatible kidney transplantation outcome.

The Role of HLA DQ2 and DQ8 in Dissecting Celiac-Like Disease in Common Variable Immunodeficiency

ObjectivesGastrointestinal manifestations are frequent in patients with common variable immunodeficiency (CVID), and some of the patients present with celiac-like features. Diagnosing celiac disease (CD) in CVID however is challenging, as autoantibody detection and histopathology of the small intestine cannot reliably discriminate between classic CD and a celiac-like disease in these individuals. For the development of classic gluten-sensitive CD a certain HLA haplotype involving the loci DQA1* and DQB1* and encoding two different HLA DQ heterodimers is the prerequisite. We aimed to determine the frequency of these haplotypes in CVID patients with suspected CD. Furthermore, we report on autoimmune manifestations and the lymphocyte phenotype in these patients.MethodsBy retrospective analysis data on gastrointestinal symptoms, diet, concurrent autoimmune diseases, and routine laboratory values were collected. CVID patients were classified according to their B-cell phenotype. Expression of HLA-DQA1* and HLA-DQB1* alleles were determined by genetic analysis.ResultsTwenty out of 250 CVID patients presented with a clinical phenotype resembling celiac disease. Four (20xa0%) out of these CVID patients carried the CD-associated HLA DQ2.5 or DQ8 heterodimer, while HLA DQ2.5 was present in 100xa0% of a CD control cohort. Gluten-free diet (GFD) resulted in a clinical and histological response in two out of four patients with HLA high-risk alleles for CD. The response could not be assessed in the remaining two patients, as these patients did not adhere sufficiently long to GFD. The percentage of autoimmune manifestations other than CD was high (50xa0%) in CVID patients presenting with a CD-like enteropathy, and most of these patients had an expansion of B-cells with low expression of CD21 (CD21low B-cells).ConclusionsIn CVID patients with suspected celiac disease typing of the HLA loci DQA1 and DQB1 can help to identify those that have a genetic susceptibility for CD. In CVID patients with a celiac-like phenotype but negative for CD-associated HLA-DQ markers, an autoimmune enteropathy (AIE) as part of an extended autoimmune dysregulation needs to be considered. This has important implications for further diagnostics and therapy of these patients.

One hundred ABO-incompatible kidney transplantations between 2004 and 2014: a single-centre experience

BACKGROUNDnABO-incompatible kidney transplantation (ABOi KTx) expands the living donor transplantation options. However, long-term outcome data, especially in comparison with ABO-compatible kidney transplantation (ABOc KTx), remain limited. Since the first ABOi KTx in Germany on 1 April 2004 at our centre, we have followed 100 ABOi KTx over up to 10 years.nnnMETHODSnOne hundred ABOi KTx and 248 ABOc KTx from 1 April 2004 until 28 October 2014 were analysed in this observational, single-centre study. Three ABOi KTx and 141 ABOc KTx were excluded because of cyclosporine A-based immunosuppression, and 1 ABOc KTx was lost to follow-up.nnnRESULTSnMedian estimated 10-year patient and graft survival in ABOi KTx was 99 and 94%, respectively, and surpassed ABOc-KTx patient and graft survival of 80 and 88%, respectively. The incidence rate of antibody-mediated rejections was 10 and 8%, and that of T-cell-mediated rejections was 17 and 20% in ABOi KTx and ABOc KTx, respectively. Infectious and malignant complications in ABOi KTx were not more common than in ABOc KTx. However, postoperative lymphoceles occurred more frequently in ABOi KTx. Subgroup analysis of ABOi-KTx patients revealed that patients with high-titre isohaemagglutinins before transplantation had equal long-term results compared with low-titre isohaemagglutinin patients.nnnCONCLUSIONnTaken together, long-term outcome of ABOi KTx is not inferior to ABOc KTx. Incidences of rejection episodes, infectious complications and malignancies are not increased, despite the more vigorous immunosuppression in ABOi KTx. Our data provide further evidence that ABOi KTx with living donation is a safe, successful and reasonable option to reduce the organ shortage.

B-cell signaling in persistent polyclonal B lymphocytosis (PPBL).

Persistent polyclonal B lymphocytosis (PPBL) is a benign hematological disorder characterized by a selective expansion of circulating polyclonal marginal zone (MZ)‐like B cells. Previous reports demonstrated that cases of PPBL showed poor activation, proliferation and survival of B cells in vitro, yet the underlying defect remains unknown. Here we report for the first time an attenuated activation of the canonical NF‐κB (nuclear factor of kappa light polypeptide gene enhancer in B cells) and mitogen‐activated protein kinase/extracellular signal‐regulated kinase pathway after CD40 stimulation. This defect was selective, as alternative NF‐κB signaling after CD40 stimulation and both B‐cell receptor‐ and Toll‐like receptor 9‐mediated activation remained unaffected. Reduced canonical NF‐κB activation resulted in decreased IκBα and CD40 expression in resting cells. In PPBL patients, expression of Bcl‐xL in MZ‐like B cells did not increase upon activation, consistent with the high apoptosis rates of PPBL‐derived B cells that were observed in vitro. The B‐cell phenotype of mice with selective knockouts of early components of the CD40 signaling pathway resembles PPBL, but sequencing corresponding genes in sorted MZ‐like B cells of PPBL patients did not reveal relevant genetic alterations. Nevertheless, the frequently observed mutations in early signaling components of the NF‐κB pathway in MZ lymphomas underline the relevance of our findings for the pathogenesis of PPBL.

Recurrence of persistent polyclonal B lymphocytosis (PPBL) after rituximab treatment

Dear Editor, Persistent polyclonal B lymphocytosis (PPBL) is a rare entity with a polyclonal increase of marginal zone-like B lymphocytes in the peripheral blood [1, 2], most prevalent in smoking middle-aged women [3]. Patients display elevated immunoglobulin M (IgM) and sometimes decreased IgG levels in the serum [4–6]. Splenomegaly and/or polyclonal lymphocytic infiltration of the bone marrow may be present [7, 8]. The disease has been associated with HLA-DR7 positivity and chromosomal abnormalities [6], but its etiology remains elusive. Herein, we report its recurrence after rituximab treatment, revealing interesting aspects of its putative pathophysiology. A 44-year-old Caucasian womanwith a history of Sudeck’s dystrophy and hysterectomy presented with epigastric pain. The patient was an active smoker. Awork-up revealed splenomegaly (3.6×16.5 cm) in the absence of enlarged lymph nodes. A differential blood count showed normal leukocyte count (9440/μl) with lymphocytosis (4863/μl, normal 1000– 2800/μl), and blood smear showed atypical, agranular large lymphocytes with bilobated nuclei (Fig. 1a). IgM was elevated (3.18 g/l, normal 0.4–2.3 g/l, Fig. 1b) without monoclonal bands in immunofixation, and IgG was reduced (4.09 g/l, normal 7–16 g/l). A bone marrow biopsy showed normal hematopoiesis and mild lymphocytosis without blasts or light chain restriction. Relative lymphocytosis in the peripheral blood was traced back to a B lymphocytosis (3457/μl, normal 100–500/μl, Fig. 1c) with no detectablemonoclonality in flow cytometry and IgH clonality analysis (Fig. 1d). The patient was HLA-DR7 negative. Albeit a watch-and-wait strategy is generally indicated in this situation, the patient was treated four times with 375 mg/ m rituximab resulting in the normalization of her spleen size (3.7×10.8 cm) and IgM level (2.1 g/l), as well as an eradication of her peripheral blood B lymphocytes (0/μl). Eighteen months after rituximab treatment, her B cell counts were back to normal (307/μl) and a mild polyclonal IgM elevation had returned (2.47 g/l). B cell numbers continued to rise over the next months (Fig. 1c), lymphocytes with bilobated nuclei reappeared, and B cell subpopulation analysis 2 years after the rituximab treatment showed an expansion of marginal zone-like B lymphocytes in the peripheral blood (52.1 % of B cells, normal <30.8 %) indicating recurrence of PPBL. However, her spleen size was still normal. FISH analysis revealed one or two 3q gains in 3.5 % of B cells, respectively (Fig. 1e, cutoffs for non-B cells 0.5–1.0 %). The recurrence of the PPBL phenotype after eradication of the recirculating B cell pool is remarkable and gives rise to various speculations on the etiology of PPBL. Given the high cellular burden in PPBL, an insufficient eradication and re-expansion of affected B cells in peripheral tissues is a possible explanation; however, the eradication from peripheral blood was complete. Alternatively, as CD20 expression is C. Wehr : L. Houet : S. Gutenberger :K. Warnatz (*) Center for Chronic Immunodeficiency (CCI), University Medical Center Freiburg, University of Freiburg, Breisacherstrasse 117, 79106 Freiburg, Germany e-mail: klaus.warnatz@uniklinik-freiburg.de

Activation of human macrophages by human corneal allogen in vitro

Purpose To study distinct aspects of human monocyte-derived macrophage (MDM) activation by human corneal tissue as a possible initial stage in human corneal allograft rejection. Methods Human monocytes were isolated from peripheral blood mononuclear cells (PBMC) and differentiated into MDM. Human corneas with or without endothelium were fragmented using a standardized protocol. MDM were stimulated with human corneal fragments, corneal fragment supernatant, lipopolysaccharide (LPS) or interferon-gamma (IFNγ), and expression profiles for 34 cytokines were determined in MDM-conditioned media using a Luminex bead-based multiplex assay. Data from clinical aqueous humour samples served for comparison and validation. To assess cell recruitment, immunogenicity of corneal endothelial cells (CEC), monocyte survival and differentiation, we applied transwell migration assays, cell viability assays and fluorescence-activated cell sorting, respectively. Results Corneal fragments induced MDM to release distinct cytokines into the medium. Media thus conditioned in vitro by stimulated MDM shared cytokine patterns, namely MCP-1, MIP-1α and MIP-1β, with human aqueous humor samples obtained in human corneal allograft rejection. The presence of CEC in tissue fragments used for MDM stimulation attenuated the upregulation of distinct pro-inflammatory chemokines, like MCP-3 and IL-8, reduced the monocyte survival time, and diminished monocyte-to-macrophage differentiation induced by conditioned media. Distinct anti-inflammatory cytokines, like IL-4 and IL-13, were upregulated in the presence of corneal endothelium. Cornea fragment-stimulated MDMs induced recruitment of monocytes from a PBMC pool in a transwell migration model, modulated immune cell viability and promoted further immune cell recruitment and differentiation. Conclusions Human macrophages respond to allogenic corneal tissue and generate an inflammatory milieu. This can drive further recruitment of immunocompetent cells and modulate cell survival and differentiation of the cells recruited. These observations are consistent with the hypothesis that macrophages play a significant role in the initiation of corneal transplant rejection. Our data also indicate that distinct aspects of early human corneal transplant rejection can be modelled in vitro.