[Pancytopenia and hemolysis--diagnosis, differential diagnosis and therapy of pernicious anemia]

Pernicious anemia and Vitamin B12 deficiency have a wide range of symptoms and are a common finding in the elderly. A 73 year old female is admitted to the hospital because of dyspnea, fatigue and loss of appetite and weight. While previous medical history and physical examination are inconspicuous, laboratory findings show severe pancytopenia with macrocytosis, low reticulocyte count and marked signs of hemolysis. A very low serum level of vitamin B12 and chronic atrophic type A gastritis upon endoscopy with presence of parietal cell antibodies in the serum lead to the diagnosis of pernicious anemia. Complete restitution is achieved by parenteral vitamin B12 substitution. Nowadays, severe pernicious anemia is only rarely seen. The differential diagnosis of pancytopenia (with macrocytic anemia) combined with hemolysis and the essential hints to the diagnosis of pernicious anemia are discussed, and thereby practical aspects including therapy actualized.

Biocompatibility assessment of the first generation PediaFlow pediatric ventricular assist device

The PediaFlow pediatric ventricular assist device is a miniature magnetically levitated mixed flow pump under development for circulatory support of newborns and infants (3-15 kg) with a targeted flow range of 0.3-1.5 L/min. The first generation design of the PediaFlow (PF1) was manufactured with a weight of approximately 100 g, priming volume less than 2 mL, length of 51 mm, outer diameter of 28 mm, and with 5-mm blood ports. PF1 was evaluated in an in vitro flow loop for 6 h and implanted in ovines for three chronic experiments of 6, 17, and 10 days. In the in vitro test, normalized index of hemolysis was 0.0087 ± 0.0024 g/100L. Hemodynamic performance and blood biocompatibility of PF1 were characterized in vivo by measurements of plasma free hemoglobin, plasma fibrinogen, total plasma protein, and with novel flow cytometric assays to quantify circulating activated ovine platelets. The mean plasma free hemoglobin values for the three chronic studies were 4.6 ± 2.7, 13.3 ± 7.9, and 8.8 ± 3.3 mg/dL, respectively. Platelet activation was low for portions of several studies but consistently rose along with observed animal and pump complications. The PF1 prototype generated promising results in terms of low hemolysis and platelet activation in the absence of complications. Hemodynamic results validated the magnetic bearing design and provided the platform for design iterations to meet the objective of providing circulatory support for young children with exceptional biocompatibility.

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Elevated transaminases in asymptomatic patients can be detected in more than 5 % of the investigations. If there are no obvious reasons, the finding should be confirmed within the next 3 months. Frequent causes are non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcohol, hepatitis B or C, hemochromatosis and drugs or toxins. Rarer causes are autoimmune hepatitis, M. Wilson and α1-antitrypsine deficiency. There are also non-hepatic causes such as celiac disease or hemolysis and myopathies in the case of an exclusive increase of ASAT. I recommend a two-step investigational procedure; the more frequent causes are examined first before the rare causes are studied. The value of the proposed investigations is discussed.

Oxidative stress precedes peak systemic inflammatory response in pediatric patients undergoing cardiopulmonary bypass operation

Oxidative stress seems to contribute to cardiopulmonary bypass (CPB)-related postoperative complications. Pediatric patients are particularly prone to these complications. With this in mind, we measured oxidative stress markers in blood plasma of 20 children undergoing elective heart surgery before, during, and up to 48 h after cessation of CPB, along with inflammatory parameters and full analysis of iron status. Ascorbate levels were decreased by approximately 50% (P < 0.001) at the time of aorta cross-clamp removal (or pump switch-off in 4 patients with partial CPB), and associated with corresponding increases in dehydroascorbate (P < 0.001, r = -0.80) and malondialdehyde (P < 0.01, r = -0.59). In contrast to the immediate oxidative response, peak levels of IL-6 and IL-8 were not observed until 3-12 h after CPB cessation. The early loss of ascorbate correlated with duration of CPB (P < 0.002, r = 0.72), plasma hemoglobin after cross-clamp removal (P < 0.001, r = 0.70), and IL-6 and IL-8 levels at 24 and 48 h after CPB (P < 0.01), but not with postoperative lactate levels, strongly suggesting that hemolysis, and not inflammation or ischemia, was the main cause of early oxidative stress. The correlation of ventilation time with early changes in ascorbate (P < 0.02, r = 0.55), plasma hemoglobin (P < 0.01, r = 0.60), and malondialdehyde (P < 0.02, r = 0.54) suggests that hemolysis-induced oxidative stress may be an underlying cause of CPB-associated pulmonary dysfunction. Optimization of surgical procedures or therapeutic intervention that minimize hemolysis (e.g., off-pump surgery) or the resultant oxidative stress (e.g., antioxidant treatment) should be considered as possible strategies to lower the rate of postoperative complications in pediatric CPB.

Percutaneous left-ventricular support with the Impella-2.5-assist device in acute cardiogenic shock: results of the Impella-EUROSHOCK-registry

BACKGROUND Acute cardiogenic shock after myocardial infarction is associated with high in-hospital mortality attributable to persisting low-cardiac output. The Impella-EUROSHOCK-registry evaluates the safety and efficacy of the Impella-2.5-percutaneous left-ventricular assist device in patients with cardiogenic shock after acute myocardial infarction. METHODS AND RESULTS This multicenter registry retrospectively included 120 patients (63.6±12.2 years; 81.7% male) with cardiogenic shock from acute myocardial infarction receiving temporary circulatory support with the Impella-2.5-percutaneous left-ventricular assist device. The primary end point evaluated mortality at 30 days. The secondary end point analyzed the change of plasma lactate after the institution of hemodynamic support, and the rate of early major adverse cardiac and cerebrovascular events as well as long-term survival. Thirty-day mortality was 64.2% in the study population. After Impella-2.5-percutaneous left-ventricular assist device implantation, lactate levels decreased from 5.8±5.0 mmol/L to 4.7±5.4 mmol/L (P=0.28) and 2.5±2.6 mmol/L (P=0.023) at 24 and 48 hours, respectively. Early major adverse cardiac and cerebrovascular events were reported in 18 (15%) patients. Major bleeding at the vascular access site, hemolysis, and pericardial tamponade occurred in 34 (28.6%), 9 (7.5%), and 2 (1.7%) patients, respectively. The parameters of age >65 and lactate level >3.8 mmol/L at admission were identified as predictors of 30-day mortality. After 317±526 days of follow-up, survival was 28.3%. CONCLUSIONS In patients with acute cardiogenic shock from acute myocardial infarction, Impella 2.5-treatment is feasible and results in a reduction of lactate levels, suggesting improved organ perfusion. However, 30-day mortality remains high in these patients. This likely reflects the last-resort character of Impella-2.5-application in selected patients with a poor hemodynamic profile and a greater imminent risk of death. Carefully conducted randomized controlled trials are necessary to evaluate the efficacy of Impella-2.5-support in this high-risk patient group.

Designed cell penetrating peptide dendrimers efficiently internalize cargo into cells

Redesigning linear cell penetrating peptides (CPPs) into a multi-branched topology with short dipeptide branches gave cell penetrating peptide dendrimers (CPPDs) with higher cell penetration, lower toxicity and hemolysis and higher serum stability than linear CPPs. Their use is demonstrated by delivering a cytotoxic peptide and paclitaxel into cells.

On the Dark Side of Therapies with Immunoglobulin Concentrates: The Adverse Events

Therapy by human immunoglobulin G (IgG) concentrates is a success story ongoing for decades with an ever increasing demand for this plasma product. The success of IgG concentrates on a clinical level is documented by the slowly increasing number of registered indication and the more rapid increase of the off-label uses, a topic dealt with in another contribution to this special issue of Frontiers in Immunology. A part of the success is the adverse event (AE) profile of IgG concentrates which is, even at life-long need for therapy, excellent. Transmission of pathogens in the last decade could be entirely controlled through the antecedent introduction by authorities of a regulatory network and installing quality standards by the plasma fractionation industry. The cornerstone of the regulatory network is current good manufacturing practice. Non-infectious AEs occur rarely and mainly are mild to moderate. However, in recent times, the increase in frequency of hemolytic and thrombotic AEs raised worrying questions on the possible background for these AEs. Below, we review elements of non-infectious AEs, and particularly focus on hemolysis and thrombosis. We discuss how the introduction of plasma fractionation by ion-exchange chromatography and polishing by immunoaffinity chromatographic steps might alter repertoire of specificities and influence AE profiles and efficacy of IgG concentrates.

Genetic variations in complement factors in patients with congenital thrombotic thrombocytopenic purpura with renal insufficiency.

The congenital form of thrombotic thrombocytopenic purpura (TTP) is caused by genetic mutations in ADAMTS13. Some, but not all, congenital TTP patients manifest renal insufficiency in addition to microangiopathic hemolysis and thrombocytopenia. We included 32 congenital TTP patients in the present study, which was designed to assess whether congenital TTP patients with renal insufficiency have predisposing mutations in complement regulatory genes, as found in many patients with atypical hemolytic uremic syndrome (aHUS). In 13 patients with severe renal insufficiency, six candidate complement or complement regulatory genes were sequenced and 11 missense mutations were identified. One of these missense mutations, C3:p.K155Q mutation, is a rare mutation located in the macroglobulin-like 2 domain of C3, where other mutations predisposing for aHUS cluster. Several of the common missense mutations identified in our study have been reported to increase disease-risk for aHUS, but were not more common in patients with as compared to those without renal insufficiency. Taken together, our results show that the majority of the congenital TTP patients with renal insufficiency studied do not carry rare genetic mutations in complement or complement regulatory genes.

Phosphocreatine interacts with phospholipids, affects membrane properties and exerts membrane-protective effects

A broad spectrum of beneficial effects has been ascribed to creatine (Cr), phosphocreatine (PCr) and their cyclic analogues cyclo-(cCr) and phospho-cyclocreatine (PcCr). Cr is widely used as nutritional supplement in sports and increasingly also as adjuvant treatment for pathologies such as myopathies and a plethora of neurodegenerative diseases. Additionally, Cr and its cyclic analogues have been proposed for anti-cancer treatment. The mechanisms involved in these pleiotropic effects are still controversial and far from being understood. The reversible conversion of Cr and ATP into PCr and ADP by creatine kinase, generating highly diffusible PCr energy reserves, is certainly an important element. However, some protective effects of Cr and analogues cannot be satisfactorily explained solely by effects on the cellular energy state. Here we used mainly liposome model systems to provide evidence for interaction of PCr and PcCr with different zwitterionic phospholipids by applying four independent, complementary biochemical and biophysical assays: (i) chemical binding assay, (ii) surface plasmon resonance spectroscopy (SPR), (iii) solid-state (31)P-NMR, and (iv) differential scanning calorimetry (DSC). SPR revealed low affinity PCr/phospholipid interaction that additionally induced changes in liposome shape as indicated by NMR and SPR. Additionally, DSC revealed evidence for membrane packing effects by PCr, as seen by altered lipid phase transition. Finally, PCr efficiently protected against membrane permeabilization in two different model systems: liposome-permeabilization by the membrane-active peptide melittin, and erythrocyte hemolysis by the oxidative drug doxorubicin, hypoosmotic stress or the mild detergent saponin. These findings suggest a new molecular basis for non-energy related functions of PCr and its cyclic analogue. PCr/phospholipid interaction and alteration of membrane structure may not only protect cellular membranes against various insults, but could have more general implications for many physiological membrane-related functions that are relevant for health and disease.

Phenotypic and genotypic identification of streptococci and related bacteria isolated from bovine intramammary infections.

BACKGROUND Streptococcus spp. and other Gram-positive, catalase-negative cocci (PNC) form a large group of microorganisms which can be found in the milk of cows with intramammary infection. The most frequently observed PNC mastitis pathogens (major pathogens) are Streptococcus uberis, Strep. dysgalactiae, and Strep. agalactiae. The remaining PNC include a few minor pathogens and a large nonpathogenic group. Improved methods are needed for the accurate identification and differentiation of PNC. A total of 151 PNC were collected from cows with intramammary infection and conclusively identified by 16S rRNA sequencing as reference method. Nine phenotypic microbiological tests (alpha-hemolysis, CAMP reaction, esculin hydrolysis, growth on kanamycin esculin azide agar and on sodium chloride agar, inulin fermentation, hippurate hydrolysis, leucine aminopeptidase and pyrrolidonyl peptidase activity), multiplex PCR for the three major pathogens (target genes for Strep. uberis, Strep. dysgalactiae and Strep. agalactiae: pauA, 16S rRNA, and sklA3, respectively), and mass spectroscopy using the matrix-assisted laser desorption ionization-time of flight (MALDI-TOF MS) were evaluated for the diagnosis and discrimination of the three clinically most relevant PNC. RESULTS The probability that a strain of Strep. uberis, Strep. dysgalactiae and Strep. agalactiae was correctly identified by combining the results of the 9 phenotypic tests was 92%, 90%, and 100%, respectively. Applying the multiplex PCR, all strains of the three major pathogens were correctly identified and no false positive results occurred. Correct identification was observed for all strains of Strep. uberis and Strep. agalactiae using MALDI-TOF MS. In the case of Strep. dysgalactiae, some variability was observed at the subspecies level, but all strains were allocated to one single cluster. CONCLUSIONS The results of the present study show that reliable identification of the clinically most relevant PNC (Strep. uberis, Strep. agalactiae and Strep. dysgalactiae) can be obtained by use of a combination of colony morphology, hemolysis type and catalase reaction, and a multiplex PCR with specific primers restricted to these 3 pathogens. The MALDI-TOF MS is a fast method that shows promising results, although identification of Strep. dysgalactiae at the subspecies level is not yet satisfactory.

Convergent synthesis and cellular uptake of multivalent cell penetrating peptides derived from Tat, Antp, pVEC, TP10 and SAP

Cell penetrating peptides (CPP) are peptides of 10 to 30 residues derived from natural translocating proteins. Multivalency is known to enhance cellular uptake for the Tat peptide and closely related polycationic sequences. To test whether multivalency effects on cellular uptake might also occur with other CPP types, we prepared multivalent versions of the strongly cationic Tat, the amphipathic sequences Antp, pVEC and TP10, and the polyproline helix SAP by convergent thioether ligation of the linear CPP onto multivalent scaffolds, and evaluated their uptake in HeLa and CHO cells, intracellular localization, cytotoxicity and hemolysis. While multivalency did not increase the cellular uptake of pVEC or SAP, multivalency effects on uptake comparable to Tat were observed with TP10 and Antp, which are attributable to their polycationic nature. The efficient synthetic protocol for these divalent CPP and their localization in the cytoplasm suggest that CPP might be useful for application in cargo delivery into cells.

Anesthesia in patients with glucose-6-phosphate dehydrogenase deficiency: case report and perioperative anesthesiologic management

Glucose-6-phosphate dehydrogenase (G6PD) deficiency, a frequent congenital human enzyme defect, is the most frequent cause of hemolytic anemia triggered by drugs or infectious diseases. Drugs which induce acute hemolysis in patients with G6PD deficiency are often used in anesthesia and perioperative pain therapy. Considering the fact that patients from geographic regions with a high prevalence of the disease are often treated in European hospitals, special attention should be paid to this problem. We report a case of a 30-year-old female patient with favism and review the disease and anesthesia-related implications.

A naturally occurring prfA truncation in a Listeria monocytogenes field strain contributes to reduced replication and cell-to-cell spread

Listeria (L.) monocytogenes is an environmental bacterium that may become an intracellular pathogen upon ingestion to cause gastroenteritis, septicaemia, abortions, and/or fatal infections of the central nervous system. We here describe a L. monocytogenes field strain (JF5171) isolated from a bovine placenta in the context of abortion, which exhibited attenuation in bovine brain-slice cultures. The whole genome of strain JF5171 was sequenced, and the invasion, replication, and intercellular spread of JF5171 were further analyzed by quantification of colony forming units and immunofluorescence studies. Phospholipase and hemolysis activity of JF5171 were also quantified along with transcription levels of actA, hly and prfA. The data obtained were compared to those of the widely used L. monocytogenes reference strain, EGD-e. JF5171 exhibited reduced replication and lower levels of phospholipase and hemolysis activity. Invasion and cell-to-cell spread was strongly decreased compared to EGD-e, and actin polymerization was absent. A frame shift deletion was identified in the JF5171 coding region of the major regulator for virulence, prfA. This resulted in a truncated C-terminus sequence (WEN* vs. WGKLN*). In addition, a point mutation resulted in a lysine to arginine substitution at amino acid position 197. Complementation with prfA from EGD-e and with (EGD-e) prfA-K197N increased the replication and spread efficiency of JF5171. In contrast, complementation with the truncated version of prfA had no effect. Taken together, these results suggest that the truncated C-terminus of prfA considerably contributes to the strongly attenuated phenotype observed in vitro.

An iron stable isotope comparison between human erythrocytes and plasma

We present precise iron stable isotope ratios measured by multicollector-ICP mass spectrometry (MC-ICP-MS) of human red blood cells (erythrocytes) and blood plasma from 12 healthy male adults taken during a clinical study. The accurate determination of stable isotope ratios in plasma first required substantial method development work, as minor iron amounts in plasma had to be separated from a large organic matrix prior to mass-spectrometric analysis to avoid spectroscopic interferences and shifts in the mass spectrometer's mass-bias. The 56Fe/54Fe ratio in erythrocytes, expressed as permil difference from the “IRMM-014” iron reference standard (δ56/54Fe), ranges from −3.1‰ to −2.2‰, a range typical for male Caucasian adults. The individual subject erythrocyte iron isotope composition can be regarded as uniform over the 21 days investigated, as variations (±0.059 to ±0.15‰) are mostly within the analytical precision of reference materials. In plasma, δ56/54Fe values measured in two different laboratories range from −3.0‰ to −2.0‰, and are on average 0.24‰ higher than those in erythrocytes. However, this difference is barely resolvable within one standard deviation of the differences (0.22‰). Taking into account the possible contamination due to hemolysis (iron concentrations are only 0.4 to 2 ppm in plasma compared to approx. 480 ppm in erythrocytes), we model the pure plasma δ56/54Fe to be on average 0.4‰ higher than that in erythrocytes. Hence, the plasma iron isotope signature lies between that of the liver and that of erythrocytes. This difference can be explained by redox processes involved during cycling of iron between transferrin and ferritin.

Current insights into thrombotic microangiopathies: Thrombotic thrombocytopenic purpura and pregnancy.

The complex relation between thrombotic thrombocytopenic purpura (TTP) and pregnancy is concisely reviewed. Pregnancy is a very strong trigger for acute disease manifestation in patients with hereditary TTP caused by double heterozygous or homozygous mutations of ADAMTS13 (ADisintegrin And Metalloprotease with ThromboSpondin type 1 domains, no. 13). In several affected women disease onset during their first pregnancy leads to the diagnosis of hereditary TTP. Without plasma treatment mother and especially fetus are at high risk of dying. The relapse risk during a next pregnancy is almost 100% but regular plasma transfusion starting in early pregnancy will prevent acute TTP flare-up and may result in successful pregnancy outcome. Pregnancy may also constitute a mild risk factor for the onset of acute acquired TTP caused by autoantibody-mediated severe ADAMTS13 deficiency. Women having survived acute acquired TTP may not be at very high risk of TTP relapse during an ensuing next pregnancy but seem to have an elevated risk of preeclampsia. Monitoring of ADAMTS13 activity and inhibitor titre during pregnancy may help to guide management and to avoid disease recurrence. Finally, TTP needs to be distinguished from the much more frequent hypertensive pregnancy complications, preeclampsia and especially HELLP (Hemolysis, Elevated Liver Enzymes, Low Platelet count) syndrome.