Harry R. Koene

The Fc gamma RIIIA-158F allele is a risk factor for systemic lupus erythematosus

Objective. To study whether the Fc gamma RIIIA-158V/F polymorphism, which affects IgG binding affinity, is a risk factor for systemic lupus erythematosus (SLE), Methods. We genotyped a group of 70 Caucasian SLE patients for all known Fc gamma R polymorphisms. Of this group, 45 patients (64%) had nephritis, In 35 patients, this diagnosis was confirmed by renal biopsy, Results, In the total group of 70 SLE patients, the frequency of the Fc gamma RIIIA-158F allele was 0.74, versus 0.57 in healthy controls (P = 0.003), The genotype distribution of the Fc gamma RIIIA-158V/F polymorphism was also significantly different from that of the control population (P = 0.004). The distribution of the other Fc gamma R polymorphisms-Fc gamma RIIA-131R/H, Fc gamma RIIIB-NA(1/2), and Fc gamma RIIIA-48L/R/H-was similar in SLE patients and controls, Conclusion. In our group of SLE patients, only the distribution of the alleles of the Fc gamma RIII4-158V/F polymorphism nas significantly different from that in the control group. This might indicate that macrophage expression of the Fc gamma RIIIA-158F isoform is involved in the disturbed clearance of immune complexes in patients with SLE.

NA‐phenotype‐dependent differences in neutrophil FcγRIIIb expression cause differences in plasma levels of soluble FcγRIII

Soluble FcγRIII in plasma is primarily derived from neutrophils and is a measure of the total body neutrophil mass. We have developed a new, sensitive ‘sandwich’ ELISA to measure soluble FcγRIII in plasma and released FcγRIII in cell supernatants. Both sFcγRIIIa, derived from NK cells and sFcγRIIIb, derived from neutrophils are detected in the assay. However, plasma analysis of FcγRIIIB gene‐deficient donors suggested that sFcγRIIIa contributes only marginally to the total amount measured in healthy individuals. Furthermore, we observed that plasma of homozygous NA1‐positive donors contained lower amounts of sFcγRIII than plasma of homozygous NA2‐positive donors. Heterozygous donors were found to have intermediate levels of sFcγRIII in their plasma. Hemizygous FcγRIIIB gene‐deficient donors were found to have half the amount of sFcγRIII in their plasma compared to donors with two FcγRIIIB alleles. These NA phenotype‐dependent differences in plasma sFcγRIII could not be contributed to either an assay artefact or NA‐dependent differences in shedding of FcγRIIIb upon neutrophil activation. Calibration curves constructed with plasma of homozygous donors did not reveal NA‐dependent differences in antibody affinity. Measurement of released FcγRIIIb in supernatants of neutrophils stimulated with PMA, and inhibition of this signal with human IgG revealed no NA‐dependent differences. However, NA‐dependent differences in neutrophil FcγRIIIb expression were present, comparable to the differences found in plasma levels of sFcγRIII. Differences in the amounts of released FcγRIII in supernatants of NA‐typed apoptotic neutrophils were similar to initial differences in FcγRIIIb expression, again being lower in NA1‐positive than in heterozygous and NA2‐positive donors. In conclusion, NA‐dependent differences in plasma levels of soluble FcγRIII seem to be caused by differences in expression of the receptor on the neutrophil membrane.

Levels of soluble Fc gammaRIII correlate with disease severity in sepsis

Neutrophil activation is thought to play a crucial role in the pathogenesis of sepsis. During activation, neutrophils adhere to and migrate through the endothelium. Therefore, the amount of circulating neutrophils does not adequately reflect the total amount of neutrophils that are involved in the pathophysiologic process of this condition. In this study we test the hypothesis that the severity of sepsis is associated with the total body mass of neutrophils as reflected in the plasma concentration of soluble Fcγ receptor type III (sFcγRIII). Nineteen patients with sepsis (12 male, seven female, median age of 69 years, range 29–87 years) were included in this study. Ten healthy volunteers served as controls. Plasma sFcγRIII concentrations were measured by ELISA. Other parameters that were studied were leucocyte count, plasma concentrations of lactoferrin and soluble L‐selectin, and surface expression of CD11b and CD66b on circulating neutrophils. Disease activity was measured using the Acute Physiology and Chronic Health Evaluation (APACHE) II score. Soluble FcγRIII levels were elevated in sepsis patients whereas soluble L‐selectin levels were moderately decreased compared with healthy controls. Markers of cell activation were significantly increased in sepsis patients. Soluble FcγRIII correlated with disease severity as measured by the APACHE score (P < 0.05, r = 0.53), whereas the other parameters did not correlate with the APACHE score. In conclusion, this study demonstrates that soluble FcγRIII is a useful marker for disease severity in patients with sepsis.

The Metalloproteinase Inhibitor GI5402 Inhibits Endotoxin-Induced Soluble CD27 and CD16 Release in Healthy Humans

ABSTRACT Metalloproteinases have been implicated in the cleavage of a number of cell surface immune receptors. Oral administration of the metalloproteinase inhibitor GI5402 attenuated the release of soluble CD27 and CD16 into the circulation after intravenous endotoxin injection in healthy humans.

Platelet-mediated proteolytic down regulation of the anticoagulant activity of protein S in individuals with haematological malignancies

The natural anticoagulant protein S contains a so-called thrombin- sensitive region (TSR), which is susceptible to proteolytic cleavage. We have previously shown that a platelet-associated protease is able to cleave protein S under physiological plasma conditions in vitro . The aim of the present study was to investigate the relation between platelet-associated protein S cleaving activity and in vivo protein S cleavage, and to evaluate the impact of in vivo protein S cleavage on its anticoagulant activity. Protein S cleavage in healthy subjects and in thrombocytopenic and thrombocythaemic patients was evaluated by immunological techniques. Concentration of cleaved and intact protein S was correlated to levels of activated protein C (APC)-dependent and APC-independent protein S anticoagulant activity. In plasma from healthy volunteers 25% of protein S is cleaved in the TSR. While in plasma there was a clear positive correlation between levels of intact protein S and both APC-dependent and APC-independent protein S anticoagulant activities, these correlations were absent for cleaved protein S. Protein S cleavage was significantly increased in patients with essential thrombocythaemia (ET) and significantly reduced in patients with chemotherapy-induced thrombocytopenia. In ET patients on cytoreductive therapy, both platelet count and protein S cleavage returned to normal values. Accordingly, platelet transfusion restored cleavage of protein S to normal values in patients with chemotherapy-induced thrombocytopenia. In conclusion, proteases from platelets seem to contribute to the presence of cleaved protein S in the circulation and may enhance the coagulation response in vivo by down regulating the anticoagulant activity of protein S.

Phase 1/2 study of lenalidomide combined with low-dose cyclophosphamide and prednisone in lenalidomide-refractory multiple myeloma.

The prognosis of multiple myeloma (MM) patients who become refractory to lenalidomide and bortezomib is very poor, indicating the need for new therapeutic strategies for these patients. Next to the development of new drugs, the strategy of combining agents with synergistic activity may also result in clinical benefit for patients with advanced myeloma. We have previously shown in a retrospective analysis that lenalidomide combined with continuous low-dose cyclophosphamide and prednisone (REP) had remarkable activity in heavily pretreated, lenalidomide-refractory MM patients. To evaluate this combination prospectively, we initiated a phase 1/2 study to determine the optimal dose and to assess its efficacy and safety in lenalidomide-refractory MM patients. The maximum tolerated dose (MTD) was defined as 25 mg lenalidomide (days 1-21/28 days), combined with continuous cyclophosphamide (50 mg/d) and prednisone (20 mg/d). At the MTD (n = 67 patients), the overall response rate was 67%, and at least minimal response was achieved in 83% of the patients. Median progression-free survival and overall survival were 12.1 and 29.0 months, respectively. Similar results were achieved in the subset of patients with lenalidomide- and bortezomib-refractory disease as well as in patients with high-risk cytogenetic abnormalities, defined as t(4;14), t(14;16), del(17p), and/or ampl(1q) as assessed by fluorescence in situ hybridization. Neutropenia (22%) and thrombocytopenia (22%) were the most common grade 3-4 hematologic adverse events. Infections (21%) were the most common grade 3-5 nonhematologic adverse events. In conclusion, the addition of continuous low-dose oral cyclophosphamide to lenalidomide and prednisone offers a new therapeutic perspective for multidrug refractory MM patients. This trial was registered at www.clinicaltrials.gov as #NCT01352338.

Multi-center randomized open label phase II trial on three rituximab dosing schemes in immune thrombocytopenia patients.

The overall short-term treatment efficacy of rituximab (R) in immune thrombocytopenia (ITP) is reported to be approximately 58%.[1][1],[2][2] With four once-weekly 375 mg/m2 doses, responses of 31% after two years,[2][2] and 21% after five years[2][2] can be expected with a median clinical

Lenalidomide combined with low-dose cyclophosphamide and prednisone modulates Ikaros and Aiolos in lymphocytes, resulting in immunostimulatory effects in lenalidomide-refractory multiple myeloma patients

We recently showed that the outcome of multiple myeloma (MM) patients treated in the REPEAT study (evaluation of lenalidomide combined with low-dose cyclophosphamide and prednisone (REP) in lenalidomide-refractory MM) was markedly better than what has been described with cyclophosphamide-prednisone alone. The outcome with REP was not associated with plasma cell Cereblon expression levels, suggesting that the effect of REP treatment may involve mechanisms independent of plasma cell Cereblon-mediated direct anti-tumor activity. We therefore hypothesized that immunomodulatory effects contribute to the anti-MM activity of REP treatment, rather than plasma cell Cereblon-mediated effects. Consequently, we now characterized the effect of REP treatment on immune cell subsets in peripheral blood samples collected on day 1 and 14 of cycle 1, as well as on day 1 of cycle 2. We observed a significant mid-cycle decrease in the Cereblon substrate proteins Ikaros and Aiolos in diverse lymphocyte subsets, which was paralleled by an increase in T-cell activation. These effects were restored to baseline at day one of the second cycle, one week after lenalidomide interruption. In vitro, lenalidomide enhanced peripheral blood mononuclear cell-mediated killing of both lenalidomide-sensitive and lenalidomide-resistant MM cells in a co-culture system. These results indicate that the Cereblon-mediated immunomodulatory properties of lenalidomide are maintained in lenalidomide-refractory MM patients and may contribute to immune-mediated killing of MM cells. Therefore, combining lenalidomide with other drugs can have potent effects through immunomodulation, even in patients considered to be lenalidomide-refractory.

Cereblon loss and up-regulation of c-Myc are associated with lenalidomide resistance in multiple myeloma patients

Multiple myeloma (MM) patients who become refractory to anti-MM drugs have a very poor prognosis. Therefore, it is important to gain insight into the mechanisms of resistance to these drugs. Immunomodulatory drugs (IMiDs) have immune-stimulatory and anti-angiogenic properties as well as direct anti-

Short-term efficacy and safety of antithymocyte globulin treatment in elderly patients with acquired aplastic anaemia.

Blech, S., Ebner, T., Ludwig-Schwellinger, E., Stangier, J. & Roth, W. (2008) The metabolism and disposition of the oral direct thrombin inhibitor, dabigatran, in humans. Drug Metabolism and Disposition, 36, 386–399. Glund, S., Stangier, J., Schmohl, M., Gansser, D., Norris, S., van Ryn, J., Lang, B., Ramael, S., Moschetti, V., Gruenenfelder, F., Reilly, P. & Kreuzer, J. (2015) Safety, tolerability, and efficacy of idarucizumab for the reversal of the anticoagulant effect of dabigatran in healthy male volunteers: a randomised, placebo-controlled, double-blind phase 1 trial. Lancet, 386, 680–690. Levy, J.H., Ageno, W., Chan, N.C., Crowther, M., Verhamme, P. & Weitz, J.I.; Subcommittee on Control of Anticoagulation. (2016) When and how to use antidotes for the reversal of direct oral anticoagulants: guidance from the SSC of the ISTH. Journal of Thrombosis and Haemostasis, 14, 623–627. Peetermans, M., Pollack, C. Jr, Reilly, P., Liesenborghs, L., Jacquemin, M., Levy, J.H., Weitz, J.I. & Verhamme, P. (2016) Idarucizumab for dabigatran overdose. Clinical Toxicology, 54, 644– 646. Reilly, P.A., Lehr, T., Haertter, S., Connolly, S.J., Yusuf, S., Eikelboom, J.W., Ezekowitz, M.D., Nehmiz, G., Wang, S., Wallentin, L. & Investigators, R.E.-L.Y. (2014) The effect of dabigatran plasma concentrations and patient characteristics on the frequency of ischemic stroke and major bleeding in atrial fibrillation patients: the RE-LY Trial (Randomized Evaluation of Long-Term Anticoagulation Therapy). Journal of the American College of Cardiology, 63, 321–328. van Ryn, J., Stangier, J., Haertter, S., Liesenfeld, K.H., Wienen, W., Feuring, M. & Clemens, A. (2010) Dabigatran etexilate–a novel, reversible, oral direct thrombin inhibitor: interpretation of coagulation assays and reversal of anticoagulant activity. Thrombosis and Haemostasis, 103, 1116– 1127. Stangier, J., Rathgen, K., St€ahle, H., Gansser, D. & Roth, W. (2007) The pharmacokinetics, pharmacodynamics and tolerability of dabigatran etexilate, a new oral direct thrombin inhibitor, in healthy male subjects. British Journal of Clinical Pharmacology, 64, 292–303.