Conrad A. Baldamus

Relationship Between the Reactivity to Hepatitis B Virus Vaccination and the Frequency of MHC Class I, II and III Alleles in Haemodialysis Patients

To study the immunoreactivity genes in a heterogeneous human population needs a large number of individuals. Associations between HLA antigens and immunoresponse to viral or bacterial antigens have been studied with controversial results. As a homogeneous population, the MHC class I, II and III allele distribution was studied in 153 end‐stage renal disease patients (ESRD, average duration of renal replacement: 8.2+5.1 years) immunized with a recombinant hepatitis B vaccine in accordance to the standard vaccination schedule. Thirty‐four patients with an antibody titre of less than 10 U/l following the last booster injection were considered as non‐responders while 119 patients with antibody titre equal to or more than 10 U/l were considered as responders. The responder group was divided into two subgroups: low responders (antibody titre: 1000 U/l) and high responders (antibody titre: > 1000 U/ 1). Marked differences were observed between responders and non‐responders in the occurrence of carriers of different MHC class I, II and III alleles. Homozygotes for HLA—A1, HLA—B8, HLA—DR3 and HLA—DQ2 were found almost exclusively in the non‐responder group and significantly more heterozygotes for these alleles were found in the non‐responder group compared to the responders. Similar albeit less marked differences were found in the frequency of some MHC class III alleles (C4A*6, C4A*QO, Bf*F, BPS0.7). Within the responder group, carriers of HLA—A2, HLA—B7 and HLA—DR4 were found to be clustered in the low responder sub‐group whereas carriers of HLA—A1, HLA—B27, HLA—Cw2, C4A*6 and Bf*F were observed more frequently in the group of high responders. Similar differences were found with extended haplotypes as well. For example, the extended haplotypes HLA—Al, B8, BfS, C4AQO, C4B1, DR3, DQ2 and HLA—A1, B8, BfF, C4A6, C4B2, DR3, DQ2 were present in nine of 34 cases of non‐responders but only in one of 119 case of responders (P <0.000001). These observations indicate that the presence or absence of certain MHC alleles even in heterozygous form determine the responsiveness to hepatitis B vaccination in end‐stage renal disease patients, and among responders, the intensity of antibody response is also markedly influence by immunogenetic factors.

Long-term safety and tolerability of epoetin zeta, administered intravenously, for maintenance treatment of renal anemia.

IntroductionThe aim of this trial was to gather data on the long-term safety of a new erythropoietin preparation (epoetin zeta), focusing on the formation of anti-erythropoietin antibodies, when administered intravenously for maintenance of target hemoglobin concentration in anemic patients with end-stage renal failure receiving chronic hemodialysis. In addition, we aimed to provide information on the efficacy of epoetin zeta under open, noncontrolled conditions.MethodsPatients received epoetin zeta intravenously, 1–3 times/week for 56 weeks (overall patient group, n=745) or 108 weeks (Bulgarian subgroup, n=164). The aim of treatment was to maintain hemoglobin values between 10.5 and 12.5 g/dL with constant epoetin dosage. Primary (safety) endpoints were the occurrence of anti-erythropoietin antibodies and the evaluation of adverse events (AEs). Secondary (efficacy) endpoints included the mean weekly dose of epoetin per kg of body weight and mean hemoglobin concentrations.ResultsNo patients developed neutralizing anti-erythropoietin antibodies. The most commonly reported AEs were infections and infestations (34.1%); followed by injury, poisoning, and procedural complications (25.8%); and gastrointestinal disorders (21.9%); 37.3% of patients reported serious AEs. The hemoglobin values remained stable, with mean values after 56 weeks of 11.3–11.6 g/dL for the overall group and 11.1–11.6 g/dL for the Bulgarian subgroup. The dosage of epoetin zeta was stable throughout the course of the trial. No cases of lack of (or loss of ) efficacy were observed in the course of the trial.ConclusionsThe evaluation of the primary endpoints provided data supporting the intravenous administration of epoetin zeta in patients with chronic renal failure. Neutralizing antibodies against erythropoietin were not detected, and there were no reports of patients with increasing erythropoietin resistance. Our results suggest that intravenous administration of epoetin zeta is effective regarding its ability to maintain stabilized hemoglobin levels within the target range of 10.5–12.5 g/dL.

Th1/Th2 balance and CD45-positive T cell subsets in primary nephrotic syndrome

Abstract T cells are involved in the pathogenesis of nephrotic syndrome (NS). The aim of the study was to determine whether the activity of T-helper-1 (Th1) and T-helper-2 (Th2) cells and the distribution of the lymphocyte subsets, namely CD45RA+CD4+ (”naive” helper T cells, suppressor-inducer), CD45RA+CD8+ (”naive” suppressor T cells, suppressor-effector), CD45RO+CD4+ (”memory” helper T cells), are predictive for steroid sensitivity in children with primary NS. These parameters were assessed at the onset of disease, before initiation of steroid therapy. Two groups of NS children were retrospectively formed according to steroid sensitivity (SS) or resistance (SR). The activity of Th1 and Th2 cells was defined by the production of interleukin-2 (IL-2), interferon-γ, IL-4, and IL-10 in the supernatants of CD4+ T cell cultures activated with autologous monocytes presenting tetanus toxoid (TT). Peripheral lymphocyte subsets were determined using double- or triple-color flow cytometry. In SS children with NS we found a decreased proliferative response of CD4+ T cells to TT stimulation, cytokine synthesis indicating the predominance of Th2 activity, and an increased percentage of activated suppressor-inducer (CD45RA+ CD4+CD25+, 5.18±0.8, P<0.001) and suppressor-effector (CD45RA+CD8+CD25+, 2.05±0.6, P<0.01) cells, with the concomitant reduction of activated memory cells (CD45RO+CD4+CD25+, 0.2±0.1, P<0.001). In children with SRNS we found an increased proliferative response of CD4+ T cells to TT, a rise in activated memory (CD45RO+CD4+CD25+, 3.82±0.7, P<0.01) and suppressor-inducer peripheral T cells (CD45RA+ CD4+CD25+, 3.85±0.6, P<0.01), but a low percentage of activated suppressor-effector (CD45RA+CD8+ CD25+, 0.5±0.2, P<0.05) T cells. We conclude that prior to treatment the distribution of lymphocyte subpopulations in peripheral blood together with Th1 and Th2 cell activity provides a useful tool for evaluating the likelihood of steroid sensitivity in patients with primary NS.

Pharmacokinetics of omega-3-fatty acids during ingestion of fish oil preparations

An in vivo comparison of three dosages (3 g, 6 g, 12 g) of two different fish oil preparations in terms of plasma concentrations of their major active components eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) was performed. The plasma accumulation was measured during 28 days of ingestion and an equally long wash out period. Data were scrutinized for bioavailability in order to distinguish between the efficiency of the two preparations. Rapid increases in EPA and DHA plasma concentrations can be demonstrated at all dosages during a 28-day ingestion period. EPA accumulated more during ingestion of high than of low dosages of fish oil. DHA revealed almost identical increases and peak values in plasma concentrations in all subgroups. The present data demonstrate dose dependent increases of EPA concentrations whereas DHA plasma concentrations are comparable in all dosages investigated. Measurable EPA and DHA plasma concentration levels are inappropriate means to explain clinical effectiveness. These results were found in both commercially available fish oil preparations. Direct comparison of both preparations revealed no differences in bioavailability.

Genetic regulation of the impaired immune response to hepatitis-B vaccine associated with low TCR density in end stage renal disease patients: contribution of complement C4 and factor B alleles.

We have studied the relationship between T-cell receptor (TCR) density, genetic factors and the specific immune response in 153 end stage renal disease (ESRD) patients on haemodialysis immunised with HBsAg vaccine. One-hundred and nineteen patients raised a protective (> 10 U/ml) antibody response to hepatitis-B vaccination (responder, R), while 34 patients were found to be non-responders (NR). The density of the T-cell receptors was determined by flow cytometry. Proliferation of the T-cells induced by autologous monocytes presenting HBsAg was also measured and expressed as a stimulation index (SI). MHC class I, II and III alleles of the patients were also determined. The densities of TCR/CD3 receptors in NR patients were found to be significantly decreased as compared to the R patients (189 +/- 22 vs. 282 +/- 58 arbitrary units, P = 1.3 x 10(-7). TCR/CD3 receptor densities were found to be strongly associated (Spearman correlation coefficient: 0.84, P < 0.000001) with the SI values. Both parameters were found to be under dual genetic control: (a) very low density of the TCR/CD3 receptors and very low SI were found mainly in NR patients carrying HLA-A1, HLA-B8 and HLA-DR3 alleles; and (b) TCR/CD3 densities and function in R group were found to be significantly lower in carriers than in non-carriers of two MHC class III complement protein alleles: C4A*6, and Bf*F. Non-responsiveness to hepatitis-B vaccination was found to be associated with extremely increased neopterin levels. These findings indicate that both genetic and acquired factors contribute to the hepatitis-B vaccination failure in ESRD patients.

Differentia] transcriptional regulation of endothelin-1 by immunosuppressants FK506 and cyclosporin A

Abstract— Calcineurin antagonists FK506 and CsA, administered to treat organ allograft rejection, exert specific effects on renal vasoconstriction and nephrotoxicity, possibly due to endogenous vasoconstrictor release such as ET‐1. We investigated contribution of FK506 and CsA on regulation of prepro ET‐1 gene transcription in HUVEC. To conclude on transcriptional regulation, ET‐1 mRNA levels were quantified by Northern blot analysis upon stimulation with calcineurin antagonists, and newly transcribed luciferase gene, placed under the control of the rat ET‐1 promoter, was quantified by reporter gene assays, where luciferase activity reflects ET‐1 promoter activation. Calcium fluorometry was employed to examine calcium dependency of ET‐1 promoter‐dependent gene transcription. Northern blot analysis shows differential induction of prepro ET‐1 mRNA in favour of CsA over FK.506. Likewise, luciferase assays demonstrate stronger ET‐1 promoter‐dependent stimulation of the reporter gene by CsA than by FK506. Transcription of prepro ET‐1 gene upon stimulation with both calcineurin antagonists is regulated by intracellular calcium levels. Lack of extra‐ or intracellular calcium prevents ET‐1 promoter‐dependent gene transcription and ET‐1 mRNA induction. These observations demonstrate that calcineurin antagonists FK506 and CsA differ in quality to induce transcription of prepro ET‐1 in HUVEC via calcium‐dependent nuclear signalling events. To examine the contribution of ET‐1 in nephrotoxicity upon CsA and FK506 immunosuppression the availability of endothelin receptor antagonists or endothelin converting enzyme inhibitors is required.

Cyclosporin A induces prepro endothelin-1 gene transcription in human endothelial cells

Cyclosporin A employed in treatment of organ allograft rejection, is associated with hypertension possibly due to endothelin-1. We studied transcriptional regulation of endothelin-1 by cyclosporin A in human endothelial cells using cell transfection experiments and reporter gene assays. Human umbilical vein endothelial cells were established expressing a fusion gene of the coding sequence of the firefly luciferase gene, placed under the control of the rat endothelin-1 promoter. Luciferase assays demonstrate 2.8-fold stimulation of the reporter gene by cyclosporin A (P < 0.01), and Northern blot analysis shows induction of prepro endothelin-1 mRNA. Transcription is tightly repressed in the absence of the immunosuppressant, its regulation occurs Ca(2+)-dependent. Lack of extra- or intracellular Ca2+ prevents cyclosporin A-dependent endothelin-1 gene transcription and mRNA induction. These data demonstrate transcriptional regulation of endothelin-1 over a range of several orders of magnitude in human umbilical vein endothelial cells by cyclosporin A via Ca(2+)-dependent mechanisms. They support the critical role of endothelin- in cyclosporin A-associated hypertension.

Preferences of patients undergoing hemodialysis – results from a questionnaire-based study with 4,518 patients

Background Chronic kidney disease is an increasing health problem worldwide and in its final stage (stage V) can only be treated by renal replacement therapy, mostly hemodialysis. Hemodialysis has a major influence on the everyday life of patients and many patients report dissatisfaction with treatment. Little is known about which aspects of treatment are considered important by hemodialysis patients. The objective of this study was to rate the relative importance of different outcomes for hemodialysis patients and to analyze whether the relative importance differed among subgroups of patients. Patients and methods Within the framework of a yearly questionnaire which is distributed among patients receiving hemodialysis by the largest hemodialysis provider in Germany, we assessed the relative importance of 23 outcomes as rated on a discrete visual analog scale. Descriptive statistics were used to rank the outcomes. Subgroup analyses were performed using Mann–Whitney U or Kruskal–Wallis tests. Results Questionnaires of 4,518 hemodialysis patients were included in the analysis. The three most important outcomes were safety of treatment, health-related quality of life, and satisfaction with care. Further important outcomes were hospital stays, accompanying symptoms, hemodialysis duration, and the improvement or preservation of a good emotional state. Age, profession, and education had the strongest influence on relevant differences of preferences for outcomes; no relevant influence of sex or comorbidity was observed. Conclusion Outcomes concerning the delivery or provision of care and aspects influencing quality of life are rated by patients to be at least as important as clinical outcomes. Many of the outcomes judged to be important by the patients are not regularly considered in research, evaluation studies, or quality programs.

Prevalence of anti-erythropoietin antibodies in hemodialysis patients without clinical signs of pure red cell aplasia. Comparison between hypo- and normoresponsive patients treated with epoetins for renal anemia.

Background/Aims: The prevalence of anti-erythropoietin antibodies in renal patients without clinical evidence of pure red cell aplasia (PRCA) who respond poorly to epoetin is unknown. This study tested for anti-erythropoietin antibodies in hemodialysis patients who were either hypo- or normoresponsive to epoetin treatment. Methods: Epoetin hyporesponsiveness (hemoglobin ≤10.5 g/dl and epoetin ≧9,000 IU/week) and normoresponsiveness (hemoglobin >10.5 g/dl and epoetin <7,000 IU/week) were arbitrarily defined. Prevalence of anti-erythropoietin antibodies in hemodialysis patients without symptoms of PRCA was determined by screening sera of 536 patients from 35 German KfH dialysis units, using enzyme-linked immunosorbent assay (ELISA). Positive results were verified by radioimmunoprecipitation assay (RIP) and neutralizing activity was determined by bioassay. Results: Anti-erythropoietin antibodies were detected in 3 hyporesponsive and 3 normoresponsive patients using ELISA. One patient per group was verified as borderline by RIP testing; the other 4 were negative. The bioassay was negative for 1 patient; the other died unrelated to PRCA before testing. Follow-up with RIP testing after 15 months under continuous epoetin treatment was negative (4 patients, 2 deceased). Conclusion: This survey did not identify anti-erythropoietin antibodies in hemodialysis patient’s hyporesponsive to epoetin and does not support presumptive antibody screening as a routine work-up in these patients.

Ultrafiltration and Hemofiltration: Practical Applications

The modern era of hemofiltration began with its clinical application in 1976. Before that, Henderson and colleagues (1, 2), Quellhorst and associates (3–6), and Dorson and Markowitz (7, 8) had contributed to the physical principles (9, 10), reported first clinical results (6) and recycled purified ultrafiltrate (11). This work would have been inconceivable without the membrane technology developed by industrial firms, such as Amicon (9) and Sartorius (3). The dialysis community became curious about the new technology when in 1976 the first workshop on hemofiltration was held in Braunlage (Table 1) initiated by the German group of Quellhorst. At this meeting a variety of clinical benefits were reported (12) including improvement of anemia, neuropathy, lipid metabolism, hypertension control, hyperparathyroidism and symptomatology. This impressive list of advantages recommended hemofiltration as the panacea in end-stage renal disease (ESRD) treatment. Thereafter industry developed automatic balancing equipment (13) on a volumetric or gravimetric basis and different membranes for flat sheet or hollow fiber filters; simultaneously the complicated dynamics of fluid and solute transfer (9, 10) were elucidated.