Jens Goebel

Abnormalities in the alternative pathway of complement in children with hematopoietic stem cell transplant-associated thrombotic microangiopathy

Hematopoietic stem cell transplant (HSCT)-associated thrombotic microangiopathy (TMA) is a complication that occurs in 25% to 35% of HSCT recipients and shares histomorphologic similarities with hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP). The hallmark of all thrombotic microangiopathies is vascular endothelial cell injury of various origins, resulting in microangiopathic hemolytic anemia, platelet consumption, fibrin deposition in the microcirculation, and tissue damage. Although significant advances have been made in understanding the pathogenesis of other thrombotic microangiopathies, post-HSCT TMA remains poorly understood. We report an analysis of the complement alternative pathway, which has recently been linked to the pathogenesis of both the Shiga toxin mediated and the atypical forms of HUS, with a focus on genetic variations in the complement Factor H (CFH) gene cluster and CFH autoantibodies in six children with post-HSCT TMA. We identified a high prevalence of deletions in CFH-related genes 3 and 1 (delCFHR3-CFHR1) and CFH autoantibodies in these patients with HSCT-TMA. Conversely, CFH autoantibodies were not detected in 18 children undergoing HSCT who did not develop TMA. Our observations suggest that complement alternative pathway dysregulation may be involved in the pathogenesis of post-HSCT TMA. These findings shed light on a novel mechanism of endothelial injury in transplant-TMA and may therefore guide the development of targeted treatment interventions.

Eculizumab Therapy in Children with Severe Hematopoietic Stem Cell Transplantation–Associated Thrombotic Microangiopathy

We recently observed that dysregulation of the complement system may be involved in the pathogenesis of hematopoietic stem cell transplantation-associated thrombotic microangiopathy (HSCT-TMA). These findings suggest that the complement inhibitor eculizumab could be a therapeutic option for this severe HSCT complication with high mortality. However, the efficacy of eculizumab in children with HSCT-TMA and its dosing requirements are not known. We treated 6 children with severe HSCT-TMA using eculizumab and adjusted the dose to achieve a therapeutic level >99 μg/mL. HSCT-TMA resolved over time in 4 of 6 children after achieving therapeutic eculizumab levels and complete complement blockade, as measured by low total hemolytic complement activity (CH50). To achieve therapeutic drug levels and a clinical response, children with HSCT-TMA required higher doses or more frequent eculizumab infusions than currently recommended for children with atypical hemolytic uremic syndrome. Two critically ill patients failed to reach therapeutic eculizumab levels, even after dose escalation, and subsequently died. Our data indicate that eculizumab may be a therapeutic option for HSCT-TMA, but HSCT patients appear to require higher medication dosing than recommended for other conditions. We also observed that a CH50 level ≤ 4 complement activity enzyme units correlated with therapeutic eculizumab levels and clinical response, and therefore CH50 may be useful to guide eculizumab dosing in HSCT patients as drug level monitoring is not readily available.

Diagnostic and risk criteria for HSCT-associated thrombotic microangiopathy: a study in children and young adults

Transplant-associated thrombotic microangiopathy (TMA) leads to generalized endothelial dysfunction that can progress to multiorgan injury, and severe cases are associated with poor outcomes after hematopoietic stem cell transplantation (HSCT). Identifying patients at highest risk for severe disease is challenging. We prospectively evaluated 100 consecutive HSCT recipients to determine the incidence of moderate and severe TMA and factors associated with poor overall outcomes. Thirty-nine subjects (39%) met previously published criteria for TMA. Subjects with TMA had a significantly higher nonrelapse mortality (43.6% vs 7.8%, P < .0001) at 1 year post-HSCT compared with those without TMA. Elevated lactate dehydrogenase, proteinuria on routine urinalysis, and hypertension were the earliest markers of TMA. Proteinuria (>30 mg/dL) and evidence of terminal complement activation (elevated sC5b-9) in the blood at the time of TMA diagnosis were associated with very poor survival (<20% at 1 year), whereas all TMA subjects without proteinuria and a normal sC5b-9 serum concentration survived (P < .01). Based on these prospective observations, we conclude that severe TMA occurred in 18% of HSCT recipients in our cohort and propose an algorithm to identify the highest-risk patients who might benefit from prompt clinical interventions.

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.

A new paradigm: Diagnosis and management of HSCT-associated thrombotic microangiopathy as multi-system endothelial injury

Hematopoietic stem cell transplantation (HSCT)-associated thrombotic microangiopathy (TA-TMA) is now a well-recognized and potentially severe complication of HSCT that carries a high risk of death. In those who survive, TA-TMA may be associated with long-term morbidity and chronic organ injury. Recently, there have been new insights into the incidence, pathophysiology, and management of TA-TMA. Specifically, TA-TMA can manifest as a multi-system disease occurring after various triggers of small vessel endothelial injury, leading to subsequent tissue damage in different organs. While the kidney is most commonly affected, TA-TMA involving organs such as the lung, bowel, heart, and brain is now known to have specific clinical presentations. We now review the most up-to-date research on TA-TMA, focusing on the pathogenesis of endothelial injury, the diagnosis of TA-TMA affecting the kidney and other organs, and new clinical approaches to the management of this complication after HSCT.

Differing Effects of Rapamycin or Calcineurin Inhibitor on T‐Regulatory Cells in Pediatric Liver and Kidney Transplant Recipients

In a cross‐sectional study, we assessed effects of calcineurin inhibitor (CNI) or rapamycin on T‐regulatory (Treg) cells from children with stable liver (n = 53) or kidney (n = 9) allografts several years posttransplant. We analyzed Treg number, phenotype, suppressive function, and methylation at the Treg‐specific demethylation region (TSDR) using Tregs and peripheral blood mononuclear cells. Forty‐eight patients received CNI (39 as monotherapy) and 12 patients received rapamycin (9 as monotherapy). Treg numbers diminished over time on either regimen, but reached significance only with CNI (r =−0.424, p = 0.017). CNI levels inversely correlated with Treg number (r =−0.371, p = 0.026), and positively correlated with CD127+ expression by Tregs (r = 0.437, p = 0.023). Patients with CNI levels >3.6 ng/mL had weaker Treg function than those with levels <3.6 ng/mL, whereas rapamycin therapy positively correlated with Treg numbers (r = 0.628, p = 0.029) and their expression of CTLA4 (r = 0.726, p = 0.041). Overall, CTLA4 expression, TSDR demethylation and an absence of CD127 were important for Treg suppressive function. We conclude that rapamycin has beneficial effects on Treg biology, whereas long‐term and high dose CNI use may impair Treg number, function and phenotype, potentially acting as a barrier to attaining host hyporesponsiveness to an allograft.

Daclizumab (Zenapax®) inhibits early interleukin-2 receptor signal transduction events

Daclizumab, a humanized antibody against the interleukin-2 (IL-2) receptor (R) alpha-chain, is a promising new immunosuppressant in transplantation. As its exact mechanism of action has remained unclear, we examined its short-term effects on primary human T lymphocytes expressing the high-affinity IL-2R. Daclizumab exposure for 20 min neither affected T cell viability nor their surface expression of the IL-2R alpha-, beta-, or gamma-chains. However, after IL-2 stimulation (200 U/ml, 20 min), immunoblots of cell lysates demonstrated attenuation of the IL-2-induced tyrosine phosphorylation of 65-75 kDa proteins by Daclizumab, but not by isotype controls. Since this is the molecular weight of the IL-2R beta- and gamma-chains, which are both tyrosine-phosphorylated by IL-2, we next examined the effect of Daclizumab on their IL-2-induced tyrosine phosphorylation. In immunoblots of IL-2R beta- and gamma-chain-immunoprecipitates the tyrosine phosphorylation of both chains by IL-2, but not by IL-15, was attenuated in the presence of Daclizumab. Furthermore, co-immunoprecipitation experiments showed that Daclizumab inhibited the IL-2-induced association of these chains, a prerequisite for their mutual tyrosine phosphorylation. Lastly, we demonstrated that Daclizumab inhibits the receptor-downstream induction of the IL-2-activated DNA-binding protein STAT5 in gel shift assays. We conclude that Daclizumab directly and specifically interferes with IL-2 signaling at the receptor level by inhibiting the association and subsequent phosphorylation of the IL-2R beta- and gamma-chains induced by ligand binding. Under our experimental conditions, Daclizumab had no effects on cell viability, and it did not modulate the surface expression of the IL-2R alpha-, beta-, or gamma-chains.

Blood, and Not Urine, BK Viral Load Predicts Renal Outcome in Children with Hemorrhagic Cystitis following Hematopoietic Stem Cell Transplantation

BK virus is a significant cause of hemorrhagic cystitis after hematopoietic stem cell transplantation (HSCT). However, its role in nephropathy post-HSCT is less studied. We retrospectively evaluated clinical outcomes in pediatric HSCT patients with hemorrhagic cystitis. Although most of these patients had very high urine BK viral loads (viruria), patients with higher BK plasma loads (viremia) had significant renal dysfunction, a worse clinical course, and decreased survival. Patients with a peak plasma BK viral load of >10,000 copies/mL (high viremia) were more likely to need dialysis and aggressive treatment for hemorrhagic cystitis compared to patients with ≤ 10,000 copies/mL (low viremia). Conversely, most patients with low viremia had only transient elevations in creatinine, and less severe hemorrhagic cystitis that resolved with supportive therapy. Overall survival (OS) at 1 year post-HSCT was 89% in the low viremia group and 48% in the high viremia group. We conclude that the degree of BK viremia, and not viruria, may predict renal, urologic, and overall outcome in the post-HSCT population.

Prednisolone Plus Albuterol Versus Albuterol Alone in Mild to Moderate Bronchiolitis

To evaluate combination therapy of mild to moderate bronchiolitis with bronchiodilators and corticosteroids, we treated 51 young children with first-time wheezing and symptoms of respiratory tract infection with albuterol plus either prednisolone or placebo for 5 days. Disease severity was scored on days 0, 2, 3, and 6. On day 2, prednisolone resulted in significantly lower scores (2.7 ±1.4 vs. 4.0 ±1.5 in all patients evaluated, p<0.05) than placebo, whereas there was no detectable difference on day 6, suggesting that addition of prednisolone to albuterol transiently accelerates recovery from bronchiolitis. The clinical significance of this effect needs to be evaluated in further studies.

Ethical Considerations for Participation of Nondirected Living Donors in Kidney Exchange Programs

Kidneys from nondirected donors (NDDs) have historically been allocated directly to the deceased donor wait list (DDWL). Recently, however, NDDs have participated in kidney exchange (KE) procedures, including KE ‘chains’, which have received considerable media attention. This increasing application of KE chains with NDD participation has occurred with limited ethical analysis and without ethical guidelines. This article aims to provide a rigorous ethical evaluation of NDDs and chain KEs. NDDs and bridge donors (BDs) (i.e. living donors who link KE procedures within KE chains) raise several ethical concerns including coercion, privacy, confidentiality, exploitation and commercialization. In addition, although NDD participation in KE procedures may increase transplant numbers, it may also reduce NDD kidney allocation to the DDWL, and disadvantage vulnerable populations, particularly O blood group candidates. Open KE chains (also termed ‘never‐ending’ chains) result in a permanent diversion of NDD kidneys from the DDWL. The concept of limited KE chains is discussed as an ethically preferable means for protecting NDDs and BDs from coercion and minimizing ‘backing out’, whereas ‘honor systems’ are rejected because they are coercive and override autonomy. Recent occurrences of BDs backing out argue for adoption of ethically based protective measures for NDD participation in KE.