Christof Weinstock

HLA Antibody Specification Using Single-Antigen Beads—A Technical Solution for the Prozone Effect

Background. Substantial progress in human leukocyte antigen antibody specification has been made by the introduction of Luminex single-antigen bead (SAB) assays. This progress was impaired when it turned out that this method is prone to a prozone effect leading to false-negative results in the case of high antibody titers. Testing serum and ethylenediaminetetraacetic acid (EDTA) plasma of one patient in parallel, we observed the prozone effect with the serum sample only. This led us to investigate complement component 1 (C1) as the cause of the prozone in SAB testing. We also found an easy way to overcome the prozone effect. Methods. Sera with a prozone effect were tested in the SAB assay, applying different methods of serum pretreatment to explore the parameters leading to the prozone. Results. The prozone was not observed when EDTA plasma or serum with EDTA added were tested. Further, addition of dithiothreitol, addition of C1 inhibitor, or heat inactivation of the sera abolished the prozone effect. Adding fresh nonimmune serum to heat-inactivated sera restored the prozone effect. Only beads showing a prozone were found to be covered with C1q. Conclusion. Our observations are consistent with the hypothesis that dissociation or destruction of complement C1 eliminates the prozone effect. Addition of EDTA to serum of highly immunized patients is the easiest way to avoid false-negative results in SAB testing caused by a prozone effect.

Effect of exhaustive exercise stress on the cytokine response.

Fifteen athletes were investigated 24 h before, 1 h after, and 20 h after an exhaustive exercise stress test (mean duration 68 min). Testing for cytokines was done in serum, urine, and the supernatants of whole blood cell cultures, which were stimulated with lipopolysaccharide (LPS), concanavalin A (Con A), or phythaemagglutinin (PHA). Elevated levels of interleukin 6 (IL-6) and soluble IL-2 receptor (sIL-2R) were found 1 h after the run in both serum and urine samples. TNF-alpha in serum was also increased, whereas IL-2 in urine was decreased after the exercise. All other testings in serum and urine (including IFN-gamma) gave borderline or negative results. In cell cultures, the LPS-induced release of the inflammatory cytokines TNF-alpha, IL-1, and IL-6 was suppressed 1 h after exercise. Also, the Con-A-induced and LPS-induced release of IFN-gamma, and the PHA-induced release of IL-2 were suppressed 1 h after exercise. In contrast, Con-A-induced release of IL-2 was mildly increased after the run. We conclude that exercise of the intensity and duration described here causes an activation of the immune system, which is immediately counter-regulated. Twenty hours after the exercise, most of the observed changes were back to pre-exercise levels, indicating only a short duration for this suppressive counter-regulation.

HSP expression in human leukocytes is modulated by endurance exercise.

PURPOSE Temperature increase, oxidative stress, and inflammatory reactions after endurance exercise were expected to stimulate the synthesis of heat shock proteins (HSP) in peripheral blood leukocytes. Furthermore, it was of interest whether regular endurance training influences HSP expression. METHODS The expression of HSP27, HSP60, HSP70, constitutive HSC70, and HSP90 in the cytoplasma and surface of lymphocytes, monocytes, and granulocytes of 12 trained athletes was analyzed by flow cytometry before and after (0, 3, and 24 h) a half marathon. Twelve untrained persons at rest were included as control. RESULTS After the race, there was a significantly greater percentage of leukocytes expressing cytoplasmic HSP27, HSP60, and HSP70 (P < 0.01), whereas HSC70 and HSP90 remained unchanged. The fluorescence intensity increased significantly in monocytes for HSP27 (0 and 3 h) and HSP70 (0, 3, and 24 h) and in granulocytes, only 24 h postexercise for HSP70. The percent values of trained athletes at rest were significantly lower compared with untrained persons (P < 0,01). CONCLUSIONS Strenuous exercise increased HSP expression in blood immediately after the run, indicating a protective function of HSP in leukocytes of athletes to maintain function after heavy exercise. The downregulation of HSP-positive cells in trained athletes at rest seems to be a result of adaptation mechanisms to regular endurance training.

Transplantation of a combination of CD133+ and CD34+ selected progenitor cells from alternative donors.

Positive selected haematopoietic stem cells are increasingly used for allogeneic transplantation with the CD34 antigen employed in most separation techniques. However, the recently described pentaspan molecule CD133 appears to be a marker of more primitive haematopoietic progenitors. Here we report our experience with a new CD133‐based selection method in 10 paediatric patients with matched unrelated (n = 2) or mismatched‐related donors (n = 8). These patients received a combination of stem cells (median = 29·3 × 106/kg), selected with either anti‐CD34 or anti‐CD133 coated microbeads. The proportion of CD133+ selected cells was gradually increased from patient to patient from 10% to 100%. Comparison of CD133+ and CD34+ separation procedures revealed similar purity and recovery of target populations but a lower depletion of T cells by CD133+ selection (3·7 log vs. 4·1 log, P < 0·001). Both separation procedures produced >90% CD34+/CD133+ double positive target cells. Engraftment occurred in all patients (sustained primary, n = 8; after reconditioning, n = 2). No primary acute graft versus host disease (GvHD) ≥ grade II or chronic GvHD was observed. The patients showed a rapid platelet recovery (median time to independence from substitution = 13·5 d), whereas T cell regeneration was variable. Five patients are alive with a median follow‐up of 10 months. Our data demonstrates the feasibility of CD133+ selection for transplantation from alternative donors and encourages further trials with total CD133+ separated grafts.

Effect of Vibrio parahaemolyticus haemolysin on human erythrocytes.

Haemolysin Kanagawa, a toxin from Vibrio parahaemolyticus, is known to trigger haemolysis. Flux studies indicated that haemolysin forms a cation channel. In the present study, channel properties were elucidated by patch clamp and functional significance of ion fluxes by fluorescence‐activated cell sorting (FACS) analysis. Treatment of human erythrocytes with 1 U ml−1 haemolysin within minutes induces a non‐selective cation permeability. Moreover, haemolysin activates clotrimazole‐sensitive K+ channels, pointing to stimulation of Ca2+‐sensitive Gardos channels. Haemolysin (1 U ml−1) leads within 5 min to slight cell shrinkage, which is reversed in Ca2+‐free saline. Erythrocytes treated with haemolysin (0.1 U ml−1) do not undergo significant haemolysis within the first 60 min. Replacement of extracellular Na+ with NMDG+ leads to slight cell shrinkage, which is potentiated by 0.1 U ml−1 haemolysin. According to annexin binding, treatment of erythrocytes with 0.1 U ml−1 haemolysin leads within 30 min to breakdown of phosphatidylserine asymmetry of the cell membrane, a typical feature of erythrocyte apoptosis. The annexin binding is significantly blunted at increased extracellular K+ concentrations and by K+ channel blocker clotrimazole. In conclusion, haemolysin Kanagawa induces cation permeability and activates endogenous Gardos K+ channels. Consequences include breakdown of phosphatidylserine asymmetry, which depends at least partially on cellular loss of K+.

Incomplete inhibition by eculizumab: mechanistic evidence for residual C5 activity during strong complement activation

Eculizumab inhibits the terminal, lytic pathway of complement by blocking the activation of the complement protein C5 and shows remarkable clinical benefits in certain complement-mediated diseases. However, several reports suggest that activation of C5 is not always completely suppressed in patients even under excess of eculizumab over C5, indicating that residual C5 activity may derogate the drugs therapeutic benefit under certain conditions. By using eculizumab and the tick-derived C5 inhibitor coversin, we determined conditions ex vivo in which C5 inhibition is incomplete. The degree of such residual lytic activity depended on the strength of the complement activator and the resulting surface density of the complement activation product C3b, which autoamplifies via the alternative pathway (AP) amplification loop. We show that at high C3b densities required for binding and activation of C5, both inhibitors reduce but do not abolish this interaction. The decrease of C5 binding to C3b clusters in the presence of C5 inhibitors correlated with the levels of residual hemolysis. However, by employing different C5 inhibitors simultaneously, residual hemolytic activity could be abolished. The importance of AP-produced C3b clusters for C5 activation in the presence of eculizumab was corroborated by the finding that residual hemolysis after forceful activation of the classical pathway could be reduced by blocking the AP. By providing insights into C5 activation and inhibition, our study delivers the rationale for the clinically observed phenomenon of residual terminal pathway activity under eculizumab treatment with important implications for anti-C5 therapy in general.

Successful use of eculizumab for treatment of an acute hemolytic reaction after ABO-incompatible red blood cell transfusion.

Transfusion of ABO major–incompatible red blood cells (RBCs) can activate the complement system and can cause severe and even lethal acute hemolytic reactions. The activation of the complement system with formation of C3a and C5a (anaphylatoxins) and the release of hemoglobin from the lysed RBCs are thought to mediate clinical signs like fever, hypotension, pain, and acute renal failure. Therapeutic inhibition of the complement cascade in case of ABO‐incompatible RBC transfusion would be desirable to ameliorate the signs and symptoms and to improve the outcome of the reaction.

A new blood group antigen is defined by anti-CD59, detected in a CD59-deficient patient

CD59 is a cell surface glycoprotein of approximately 20 kDa limiting the lytic activity of the terminal complement complex C5b‐9. Although CD59 is known as a red blood cell (RBC) antigen defined by monoclonal antibodies, it so far has not been identified as a blood group antigen, since the description of a human alloantibody was missing. In this study we show the presence of an anti‐CD59 in a patient affected by a homozygous CD59 deficiency.

Impaired Production of Cytokines in a Case of Human Leishmaniasis

A patient presented with the unique clinical picture of diffuse cutaneous and mucosal leishmaniasis caused by Leishmania tropica. Elevated serum levels of several cytokines including interleukin (IL) 2, interferon gamma (IFN-gamma), and tumor necrosis factor alpha were found. All cytokine levels returned to normal during therapy. No IL-10 or IL-4 levels were detectable. In whole blood cultures, induction of IFN-gamma by lipopolysaccharide (LPS) was completely negative, even after therapy. Concanavalin A (Con A)-induced release of IFN-gamma, like Con A-induced release of the other cytokines, was only initially impaired but returned to normal during therapy. Induction of the other cytokines by LPS was never impaired. The low expression of human leukocyte antigen DR on monocytes increased during IFN-gamma therapy but dropped when IFN-gamma treatment was ceased. We conclude that in this patient one or more of the routes of IFN-gamma production was impaired, thus resulting in insufficient IFN-gamma production in the infected lesions (although IFN-gamma was systemically present).

Molecular immunohaematology round table discussions at the AABB Annual Meeting, Denver 2013.

Well trained, experienced serologists in transfusion medicine laboratories have been familiar with blood group serology for decades. With the advent of molecular immunohaematology, there is a need to adopt and embrace the clinical and diagnostic applications in order for patients to benefit from the advances that this technology offers1–3. We organised an international forum to discuss molecular immunohaematology concepts that may be challenging even for some established professionals in the field of serology. The objectives of the session were two-fold. First, the session allowed networking among immunohaematology professionals who have an interest in the application of molecular immuno-haematology and blood group genetics while meeting with leaders in the field of molecular immunohaematology. By giving input and asking questions, the participants could define their knowledge relative to the experience of the group as a whole. Second, the discussions and the input from experienced professionals were recorded. This approach allowed documentation of current knowledge, as well as acceptance and concerns of the participants. It is useful to gathering information in this field, because the perception at the level of the participants is critical for shaping the adoption of molecular immunohaematology. We provide a summary report of the items discussed and issues raised by the participants. The results describe the status of molecular immunohaematology within this large group of experienced professionals in transfusion medicine laboratories and can guide targeted educational efforts.