[Thrombotic thrombocytopenic purpura-a differential diagnostic challenge in an emergency]. / Thrombotisch-thrombozytopenische Purpura ­ eine differenzialdiagnostische Herausforderung im Notfall.

Thrombotic thrombocytopenic purpura (TTP), which comprises thrombocytopenia, elevated lactate dehydrogenase levels, and anemia in the combination of organ involvement, is a rare but life-threatening condition associated with an extremely high lethality in the acute phase if left untreated. Using the example of a 49-year-old woman admitted to the hospital with unexplained abdominal symptoms and subfebrile temperatures, recommendations for the emergency situation are presented. Increased awareness of the disease and targeted further diagnostics with determination of the PLASMIC score or ADAMTS13 activity may lead directly to diagnosis of TTP; delayed diagnosis is usually associated with secondary complications.

Kidney Dysfunction Is Associated with Thrombosis and Disease Severity in Myeloproliferative Neoplasms: Implications from the German Study Group for MPN Bioregistry.

Inflammation-induced thrombosis represents a severe complication in patients with myeloproliferative neoplasms (MPN) and in those with kidney dysfunction. Overlapping disease-specific attributes suggest common mechanisms involved in MPN pathogenesis, kidney dysfunction, and thrombosis. Data from 1420 patients with essential thrombocythemia (ET, 33.7%), polycythemia vera (PV, 38.5%), and myelofibrosis (MF, 27.9%) were extracted from the bioregistry of the German Study Group for MPN. The total cohort was subdivided according to the calculated estimated glomerular filtration rate (eGFR, (mL/min/1.73 m2)) into eGFR1 (≥90, 21%), eGFR2 (60-89, 56%), and eGFR3 (<60, 22%). A total of 29% of the patients had a history of thrombosis. A higher rate of thrombosis and longer MPN duration was observed in eGFR3 than in eGFR2 and eGFR1. Kidney dysfunction occurred earlier in ET than in PV or MF. Multiple logistic regression analysis identified arterial hypertension, MPN treatment, increased uric acid, and lactate dehydrogenase levels as risk factors for kidney dysfunction in MPN patients. Risk factors for thrombosis included arterial hypertension, non-excessive platelet counts, and antithrombotic therapy. The risk factors for kidney dysfunction and thrombosis varied between MPN subtypes. Physicians should be aware of the increased risk for kidney disease in MPN patients, which warrants closer monitoring and, possibly, early thromboprophylaxis.

Real-world data confirm the effectiveness of caplacizumab in acquired thrombotic thrombocytopenic purpura.

Acquired thrombotic thrombocytopenic purpura (aTTP) is a rare but life-threatening condition. In 2018, the nanobody caplacizumab was approved for the treatment of adults experiencing an acute episode of aTTP, in conjunction with plasma exchange (PEX) and immunosuppression for a minimum of 30 days after stopping daily PEX. We performed a retrospective, observational analysis on the use of caplacizumab in 60 patients from 29 medical centers in Germany during acute disease management. Caplacizumab led to a rapid normalization of the platelet count (median, 3 days; mean 3.78 days). One patient died after late treatment initiation due to aTTP-associated complications. In 2 patients with initial disease presentation and in 4 additional patients with laboratory signs of an exacerbation or relapse after the initial therapy, PEX-free treatment regimens could be established with overall favorable outcome. Caplacizumab is efficacious in the treatment of aTTP independent of timing and ancillary treatment modalities. Based on this real-world experience and published literature, we propose to administer caplacizumab immediately to all patients with an acute episode of aTTP. Treatment decisions regarding the use of PEX should be based on the severity of the clinical presentation and known risk factors. PEX might be dispensable in some patients.

ADAMTS13 and VWF activities guide individualized caplacizumab treatment in patients with aTTP.

Introduction of the nanobody caplacizumab was shown to be effective in the treatment of acquired thrombotic thrombocytopenic purpura (aTTP) in the acute setting. The official recommendations include plasma exchange (PEX), immunosuppression, and the use of caplacizumab for a minimum of 30 days after stopping daily PEX. This study was a retrospective, observational analysis of the use of caplacizumab in 60 patients from 29 medical centers in Germany. Immunosuppressive treatment led to a rapid normalization of ADAMTS13 activities (calculated median, 21 days). In 35 of 60 patients, ADAMTS13 activities started to normalize before day 30 after PEX; in 11 of 60 patients, the treatment was extended beyond day 30; and in 5 patients, it was extended even beyond day 58 due to persistent autoimmune activity. In 34 of 60 instances, caplacizumab was stopped before day 30 with a favorable outcome whenever ADAMTS13 activities were >10%. In contrast, 11 of 34 patients with ADAMTS13 activities <10% at the time of stopping caplacizumab treatment developed a nonfavorable outcome (disease exacerbation or relapse). In some cases, prolongation of the treatment interval to every other day was feasible and resulted in a sustained reduction of von Willebrand factor activity. ADAMTS13 activity measurements are central for a rapid diagnosis in the acute setting but also to tailor disease management. An ADAMTS13 activity-guided approach seems safe for identifying the individual time point when to stop caplacizumab to prevent overtreatment and undertreatment; this approach will result in significant cost savings without jeopardizing the well-being of patients. In addition, von Willebrand factor activity may serve as a biomarker for drug monitoring.

pH-Dependent Protonation of Surface Carboxylate Groups in PsbO Enables Local Buffering and Triggers Structural Changes.

Photosystem II (PSII) catalyzes the splitting of water, releasing protons and dioxygen. Its highly conserved subunit PsbO extends from the oxygen-evolving center (OEC) into the thylakoid lumen and stabilizes the catalytic Mn4 CaO5 cluster. The high degree of conservation of accessible negatively charged surface residues in PsbO suggests additional functions, as local pH buffer or by affecting the flow of protons. For this discussion, we provide an experimental basis, through the determination of pKa values of water-accessible aspartate and glutamate side-chain carboxylate groups by means of NMR. Their distribution is strikingly uneven, with high pKa values around 4.9 clustered on the luminal PsbO side and values below 3.5 on the side facing PSII. pH-dependent changes in backbone chemical shifts in the area of the lumen-exposed loops are observed, indicating conformational changes. In conclusion, we present a site-specific analysis of carboxylate group proton affinities in PsbO, providing a basis for further understanding of proton transport in photosynthesis.

Incidence of acquired thrombotic thrombocytopenic purpura in Germany: a hospital level study.

BACKGROUND: Acquired thrombotic thrombocytopenic Purpura (aTTP) is a life-threatening ultra-orphan disease with a reported annual incidence between 1.5 and 6.0 cases per million in Europe and mainly affecting otherwise young and healthy adults aged 40 years on average. The goal of this study was to assess the incidence of aTTP in Germany. METHODS: A systematic review was performed to determine the published evidence on the aTTP epidemiology in Germany. To obtain additional evidence on the proportion of aTTP cases within the national Thrombotic Microangiopathy (TMA) population a hospital-level study was performed, using a retrospective data collection approach. Diagnosis of aTTP was confirmed if ADAMTS13 level were < 10% and/or the medical records explicitly mentioned aTTP diagnosis. The aggregated hospital data were then projected to the national level using logistic regression techniques. RESULTS: The systematic literature search did not provide incidence estimates of aTTP in Germany. Eight centers (≈27% of the top 30 TMA hospitals) delivered data according to a predefined data collection form. On average (year 2014-2016) a total number of 172 aTTP episodes per year was projected (95% confidence interval [95%CI]: 132-212). The majority were newly diagnosed aTTP cases (n = 121; 95%CI: 105-129), and 51 were recurrent aTTP cases (95%CI: 27-84). The average annual projected incidence (year 2014-2016) of aTTP episodes was 2.10 per million inhabitants in Germany (95%CI: 1.60-2.58). CONCLUSIONS: The determined annual incidence of newly diagnosed aTTP cases and the overall annual incidence of aTTP episodes in Germany confirm the ultra-orphan character of aTTP. An external validation against international registries (France, UK and USA) shows that our findings are quite comparable with those international incidence rates.

Serologic response to meningococcal vaccination in patients with cold agglutinin disease (CAD) in the novel era of complement inhibition.

Cold agglutinin disease (CAD) is a rare, potentially life-threatening acquired autoimmune hemolytic anemia characterized by hemagglutination and hemolysis due to immunoglobulin-mediated (usually IgMκ) classic complement pathway activation. Complement inhibition (CI) represents a novel treatment option to control hemolysis. Due to CI patients (pts) are susceptible to encapsulated bacteria e.g. N. meningitidis. Therefore, meningococcal vaccination on CI is mandatory. In this study serologic response to the tetravalent conjugate vaccine Menveo® was analyzed in CAD pts on eculizumab treatment (DECADE trial) using rabbit serum as complement source (rSBA). Protective rSBA titers varied for meningococcal serogroups and over time reflecting an early decline to even non-protective rSBA titers. These data highlight the importance of serologic analyses under chronic CI. Currently, re-vaccination with a tetravalent meningococcal conjugate vaccine every 3 years is recommended on chronic CI. However, re-vaccination on CI might further rely on serologic analyses, implying even early booster vaccinations similar to adults with (functional) asplenia.

Structural and functional characterization of a maltose/maltodextrin ABC transporter comprising a single solute binding domain (MalE) fused to the transmembrane subunit MalF.

Canonical ATP-binding cassette import systems rely on extracellular substrate binding proteins (SBP) for function. In gram-negative bacteria, SBPs are usually freely diffusible in the periplasm and, where studied, exist in excess over their cognate transporters. However, in vitro studies with the maltose transporter of Escherichia coli (MalFGK2) have demonstrated that mechanistically one copy of its SBP (MalE) per transport complex is sufficient for activity. To address whether such a condition is physiologically relevant, we have characterized a homolog of the E. coli system from the gram-negative bacterium Bdellovibrio bacteriovorus which has a single copy of a maltose binding domain fused to the MalF subunit. Both transporters share substrate specificity for maltose and linear maltodextrins. Specific ATPase and transport activities of the B. bacteriovorus transporter were comparable to those of the E. coli system assayed at a 1:1 M ratio of MalE to the transport complex. While MalEEc was able to additionally increase ATPase activity of MalFGK2Bb, the isolated MalE domain of B. bacteriovorus failed to stimulate the E. coli system. Strikingly, interactions of the MalE domain with the transmembrane subunits during the transport cycle as studied by site-specific cross-linking were found to differ from those observed for E. coli MalE-FGK2.

Improving the accuracy and resolution of neutron crystallographic data by three-dimensional profile fitting of Bragg peaks in reciprocal space.

Neutron crystallography is a powerful technique for directly visualizing the locations of H atoms in biological macromolecules. This information has provided key new insights into enzyme mechanisms, ligand binding and hydration. However, despite the importance of this information, the application of neutron crystallography in biology has been limited by the relatively low flux of available neutron beams and the large incoherent neutron scattering from hydrogen, both of which contribute to weak diffraction data with relatively low signal-to-background ratios. A method has been developed to fit weak data based on three-dimensional profile fitting of Bragg peaks in reciprocal space by an Ikeda-Carpenter function with a bivariate Gaussian. When applied to data collected from three different proteins, three-dimensional profile fitting yields intensities with higher correlation coefficients (CC1/2) at high resolutions, decreased Rfree factors, extended resolutions and improved nuclear density maps. Importantly, additional features are revealed in nuclear density maps that may provide additional scientific information. These results suggest that three-dimensional profile fitting will help to extend the capabilities of neutron macromolecular crystallography.

Eculizumab in cold agglutinin disease (DECADE): an open-label, prospective, bicentric, nonrandomized phase 2 trial.

Cold agglutinin disease (CAD) is a complement-dependent disorder, with extravascular and intravascular hemolysis resulting from initial or terminal complement activation, respectively. We tested the efficacy and safety of eculizumab, an inhibitor of the terminal complement pathway. Treatment-requiring patients received 600 mg eculizumab weekly for 4 weeks, followed 1 week later by 900 mg every other week through week 26. The primary end point was the difference in the lactate dehydrogenase level between the first and the last day of therapy. Twelve patients with chronic CAD and 1 patient with an acute cold agglutinin syndrome were included. The median lactate dehydrogenase level decreased from 572 U/L (interquartile range [IQR], 534-685) to 334 U/L (IQR, 243-567; P = .0215), paralleled by an increase in hemoglobin from 9.35 g/dL (IQR, 8.80-10.80) to 10.15 g/dL (IQR, 9.00-11.35; P = .0391; Wilcoxon signed-rank test). Three patients maintained and 8 patients acquired transfusion independence, and 1 patient each showed a reduced or increased transfusion requirement, respectively (P = .0215; exact McNemar's test). Patients with cold agglutinins with a thermal amplitude of 37°C tended to have less pronounced lactate dehydrogenase responses than patients with cold agglutinins with narrower thermal amplitudes. In the latter, responses were observed at lower serum levels of eculizumab than they were in the former. In contrast to hemolysis, cold-induced circulatory symptoms remained unaffected. In conclusion, eculizumab significantly reduced hemolysis and transfusion requirement in patients with CAD. Suppression of hemolysis caused by cold agglutinins with a wide thermal amplitude may require higher eculizumab doses than used here. The trial is registered with EudraCT (#2009-016966-97) and www.clinicaltrials.gov (#NCT01303952).

Leptomeningeal Myelomatosis: A Rare but Devastating Manifestation of Multiple Myeloma Diagnosed Using Cytology, Flow Cytometry, and Fluorescent in situ Hybridization.

INTRODUCTION: Involvement of the central nervous system in patients with multiple myeloma is a rare event. We evaluated the diagnostic workup and prognosis of patients with leptomeningeal myelomatosis (LMM). METHODS: Between April 2005 and April 2016, we identified 16 cases with LMM. The involvement was diagnosed by magnetic resonance imaging (MRI), cerebrospinal fluid (CSF) cytology as well as flow cytometry. Fluorescence in situ hybridization (FISH) was used in 8/16 cases. In 1 case, genome-wide screening for genetic alterations using single-nucleotide polymorphism (SNP) array analysis was performed. RESULTS: The median time from initial diagnosis until the occurrence of LMM was 434 days. At diagnosis, the median age was 60 years. The median cell count was 21/µL (range 1-1,333/µL). All CSF samples showed malignant pleocytosis, confirmed by flow cytometry in 12/16 patients. FISH revealed high-risk features in the majority of samples. Treatment for LMM consisted of intrathecal chemotherapy and radiation therapy. Genome-wide screening assays revealed different subclones. The outcome was dismal with a median overall survival after the diagnosis of LMM of 82 days. CONCLUSION: By combining several technical procedures, it is possible to identify most patients with LMM. Management of affected patients is challenging and the survival short after a diagnosis of LMM.

The Differential Diagnosis and Treatment of Thrombotic Microangiopathies.

BACKGROUND: Thrombotic microangiopathies are rare, life-threatening diseaseswhose care involves physicians from multiple specialties. The past five years haveseen major advances in our understanding of the pathophysiology, classification,and treatment of these conditions. Their timely diagnosis and prompt treatment cansave lives. METHODS: This review is based on pertinent articles published up to 17 December2017 that were retrieved by a selective search of the National Library of Medicine'sPubMed database employing the terms "thrombotic microangiopathy," "thromboticthrombocytopenic purpura," "hemolytic-uremic syndrome," "drug-induced TMA," and"EHEC-HUS." RESULTS: The classic types of thrombotic microangiopathy are thrombotic thrombo -cytopenic purpura (TTP) and typical hemolytic-uremic syndrome (HUS), also knownas enterohemorrhagic Escherichia coli-associated HUS (EHEC-HUS). There are anumber of further types from which these must be differentiated. The key test,beyond a basic hematological evaluation including a peripheral blood smear, ismeasurement of the blood level of the protease that splits von Willebrand factor,which is designated ADAMTS13 (a disintegrin and metalloprotease with thrombo -spondin type 1 motif, member 13). The quantitative determination of ADAMTS13, ofADAMTS13 activity, and of the ADAMTS13 inhibitor serves to differentiate TTP fromother types of thrombotic microangiopathy. As TTP requires urgent treatment,plasmapheresis should be begun as soon as TTP is suspected on the basis of afinding of hemolysis with schistocytes and thrombocytopenia. The treatment shouldbe altered as indicated once the laboratory findings become available. CONCLUSION: Rapid differential diagnosis is needed in order to determine the specifictype of thrombotic microangiopathy that is present, because only patients with TTPand only a very small percentage of those with atypical hemolytic-uremic syndrome(aHUS) can benefit from plasmapheresis. The establishment of a nationwideregistry in Germany with an attached biobank might help reveal yet unknowngenetic predispositions.

Small heat shock protein speciation: novel non-canonical 44 kDa HspB5-related protein species in rat and human tissues.

When analyzing small stress proteins of rat and human tissues by electrophoretic methods followed by western blotting, and using the anti-HspB1/anti-HspB5 antibody clone 8A7, we unexpectedly found a protein with a molecular mass of ~44 kDa. On two-dimensional gels, this protein resolved into four distinct species. Electrophoretic and immunological evidence suggests that this 44 kDa protein is a derivative of HspB5, most likely a covalently linked HspB5 dimer. This HspB5-like 44 kDa protein (HspB5L-P44) is particularly abundant in rat heart, brain, and renal cortex and glomeruli. HspB5L-P44 was also found in human brains, including those from patients with Alexander disease, a condition distinguished by cerebral accumulation of HspB5. Gray matter of such a patient contained an elevated amount of HspB5L-P44. A spatial model of structurally ordered dimeric HspB5 α-crystallin domains reveals the exposed and adjacent position of the two peptide segments homologous to the HspB1-derived 8A7 antigen determinant peptide (epitope). This explains the observed extraordinary high avidity of the 8A7 antibody towards HspB5L-P44, as opposed to commonly used HspB5-specific antibodies which recognize other epitopes. This scenario also explains the remarkable fact that no previous study reported the existence of HspB5L-P44 species. Exposure of rat endothelial cells to UV light, an oxidative stress condition, temporarily increased HspB5L-P44, suggesting physiological regulation of the dimerization. The existence of HspB5L-P44 supports the protein speciation discourse and fits to the concept of the protein code, according to which the expression of a given gene is reflected only by the complete set of the derived protein species.

Selective Utilization of Benzimidazolyl-Norcobamides as Cofactors by the Tetrachloroethene Reductive Dehalogenase of Sulfurospirillum multivorans.

The organohalide-respiring bacterium Sulfurospirillum multivorans produces a unique cobamide, namely, norpseudo-B12, which serves as cofactor of the tetrachloroethene (PCE) reductive dehalogenase (PceA). As previously reported, a replacement of the adeninyl moiety, the lower base of the cofactor, by exogenously applied 5,6-dimethylbenzimidazole led to inactive PceA. To explore the general effect of benzimidazoles on the PCE metabolism, the susceptibility of the organism for guided biosynthesis of various singly substituted benzimidazolyl-norcobamides was investigated, and their use as cofactor by PceA was analyzed. Exogenously applied 5-methylbenzimidazole (5-MeBza), 5-hydroxybenzimidazole (5-OHBza), and 5-methoxybenzimidazole (5-OMeBza) were found to be efficiently incorporated as lower bases into norcobamides (NCbas). Structural analysis of the NCbas by nuclear magnetic resonance spectroscopy uncovered a regioselectivity in the utilization of these precursors for NCba biosynthesis. When 5-MeBza was added, a mixture of 5-MeBza-norcobamide and 6-MeBza-norcobamide was formed, and the PceA enzyme activity was affected. In the presence of 5-OHBza, almost exclusively 6-OHBza-norcobamide was produced, while in the presence of 5-OMeBza, predominantly 5-OMeBza-norcobamide was detected. Both NCbas were incorporated into PceA, and no negative effect on the PceA activity was observed. In crystal structures of PceA, both NCbas were bound in the base-off mode with the 6-OHBza and 5-OMeBza lower bases accommodated by the same solvent-exposed hydrophilic pocket that harbors the adenine as the lower base of authentic norpseudo-B12 In this study, a selective production of different norcobamide isomers containing singly substituted benzimidazoles as lower bases is shown, and unique structural insights into their utilization as cofactors by a cobamide-containing enzyme are provided.IMPORTANCE Guided biosynthesis of norcobamides containing singly substituted benzimidazoles as lower bases by the organohalide-respiring epsilonproteobacterium Sulfurospirillum multivorans is reported. An unprecedented specificity in the formation of norcobamide isomers containing hydroxylated or methoxylated benzimidazoles was observed that implicated a strict regioselectivity of the norcobamide biosynthesis in the organism. In contrast to 5,6-dimethylbenzimidazolyl-norcobamide, the incorporation of singly substituted benzimidazolyl-norcobamides as a cofactor into the tetrachloroethene reductive dehalogenase was not impaired. The enzyme was found to be functional with different isomers and not limited to the use of adeninyl-norcobamide. Structural analysis of the enzyme equipped with either adeninyl- or benzimidazolyl-norcobamide cofactors visualized for the first time structurally different cobamides bound in base-off conformation to the cofactor-binding site of a cobamide-containing enzyme.

Protein crystallization and initial neutron diffraction studies of the photosystem II subunit PsbO.

The PsbO protein of photosystem II stabilizes the active-site manganese cluster and is thought to act as a proton antenna. To enable neutron diffraction studies, crystals of the ß-barrel core of PsbO were grown in capillaries. The crystals were optimized by screening additives in a counter-diffusion setup in which the protein and reservoir solutions were separated by a 1% agarose plug. Crystals were cross-linked with glutaraldehyde. Initial neutron diffraction data were collected from a 0.25 mm3 crystal at room temperature using the MaNDi single-crystal diffractometer at the Spallation Neutron Source, Oak Ridge National Laboratory.

Thrombotic Microangiopathy after Allogeneic Stem Cell Transplantation: A Comparison of Eculizumab Therapy and Conventional Therapy.

We report the results of a single-center analysis of a cohort of 39 patients treated between 1997 and 2016 for transplantion-associated thrombotic microangiopathy. We evaluated 2 subgroups of patients: 24 patients treated between 1997 and 2014 who received conventional therapy and 15 patients treated with the complement-inhibiting monoclonal antibody eculizumab between 2014 and 2016. The conventional therapy group was treated predominantly with defibrotide alone or in combination with plasmapheresis or rituximab. Despite an initial response rate of 61%, only 4 patients (16%) were long-term survivors, 2 of whom had a low-risk thrombotic microangiopathy without multiorgan damage. Progression of thrombotic micorangiopathy and bacterial/fungal infections contributed equally to treatment failure. The overall response rate in the eculizumab group was significantly higher, at 93%. In addition, we were able to stop eculizumab treatment in 5 patients (33%), all of whom had high-risk thrombotic microangiopathy, due to sustained recovery. Despite the very good response in the eculizumab-treated group, we did not observe a significant improved overall survival, due primarily to a high rate of infection-related mortality (70%). Therefore, further studies are needed to identify the optimal therapeutic management approach for transplantation-associated thrombotic microangiopathy to improve its dismal outcome.

Structural insights into the light-driven auto-assembly process of the water-oxidizing Mn4CaO5-cluster in photosystem II.

In plants, algae and cyanobacteria, Photosystem II (PSII) catalyzes the light-driven splitting of water at a protein-bound Mn4CaO5-cluster, the water-oxidizing complex (WOC). In the photosynthetic organisms, the light-driven formation of the WOC from dissolved metal ions is a key process because it is essential in both initial activation and continuous repair of PSII. Structural information is required for understanding of this chaperone-free metal-cluster assembly. For the first time, we obtained a structure of PSII from Thermosynechococcus elongatus without the Mn4CaO5-cluster. Surprisingly, cluster-removal leaves the positions of all coordinating amino acid residues and most nearby water molecules largely unaffected, resulting in a pre-organized ligand shell for kinetically competent and error-free photo-assembly of the Mn4CaO5-cluster. First experiments initiating (i) partial disassembly and (ii) partial re-assembly after complete depletion of the Mn4CaO5-cluster agree with a specific bi-manganese cluster, likely a di-µ-oxo bridged pair of Mn(III) ions, as an assembly intermediate.