Hb Mizuho Case Report; Early Genomic Testing Facilitates a Life Changing Diagnosis.

Unstable variant hemoglobinopathies are an uncommon cause of hemolysis in the pediatric patient and may cause a delay in diagnosis if there is not a high index of suspicion. Hemoglobin (Hb) Mizuho is a rare unstable hemoglobinopathy caused by a pathogenic variant of the HBB gene with a severe phenotype. Here we report on the first known case of Hb Mizuho in Australia, presenting with features of acute and chronic hemolysis. The morphological features on blood film review, in conjunction with biochemical findings and other clinical features, did not immediately suggest an alternative diagnosis and a Next Generation Sequencing gene analysis approach was taken to investigate genes associated with red blood cell disorders and atypical uremic syndrome. The HBB Mizuho variant was detected and established the diagnosis. This report highlights the challenge of diagnosing Hb Mizuho on conventional testing and the need for early genomic testing to clarify a diagnosis.

Searching for Virulence Factors among Staphylococcus lugdunensis Isolates from Orthopedic Infections: Correlation of ß-hemolysin, hemolysin III, and slush Genes with Hemolytic Activity and Synergistic Hemolytic Activity.

Staphylococcus lugdunensis is an emerging high-virulent pathogen. Here, the presence and expression of virulence genes (icaA, fbl, vwbl, fbpA, slush A, B and C, and genes of the putative ß-hemolysin and hemolysin III) and the ability to induce synergistic hemolytic activity and hemolysis after 24, 48 and 72 h were investigated in a collection of twenty-two S. lugdunensis clinical isolates. The collection of isolates, mainly from implant orthopedic infections, had previously been grouped by ribotyping/dendrogram analysis and studied for biofilm matrices, biomasses and antibiotic resistances. Two isolates, constituting a unique small ribogroup sharing the same cluster, exhibited an amplicon size of the slush operon (S. lugdunensis synergistic hemolysin) which was shorter than the expected 977 bp. This outcome can predict the genetic lineage of the S. lugdunensis strains. One isolate (cra1342) presented two deletions: one of 90 bp in slush A and the other of 91 bp in slush B. Another isolate (N860314) showed a single 193 bp deletion, which encompassed part of the slush B terminal sequence and most of slush C. The isolate N860314 was devoid of hemolytic activity after 24 h, and the first consideration was that the deleted region deals with the coding of the active enzymatic site of the slush hemolysin. On the other hand, cra1342 and N860314 isolates with different slush deletions and with hemolytic activity after 24 and 48 h, respectively, could have replaced the hemolytic phenotype through other processes.

Utilizing Extraepitopic Amino Acid Substitutions to Define Changes in the Accessibility of Conformational Epitopes of the Bacillus cereus HlyII C-Terminal Domain.

Hemolysin II (HlyII)-one of the pathogenic factors of Bacillus cereus, a pore-forming ß-barrel toxin-possesses a C-terminal extension of 94 amino acid residues, designated as the C-terminal domain of HlyII (HlyIICTD), which plays an important role in the functioning of the toxin. Our previous work described a monoclonal antibody (HlyIIC-20), capable of strain-specific inhibition of hemolysis caused by HlyII, and demonstrated the dependence of the efficiency of hemolysis on the presence of proline at position 324 in HlyII outside the conformational antigenic determinant. In this work, we studied 16 mutant forms of HlyIICTD. Each of the mutations, obtained via multiple site-directed mutagenesis leading to the replacement of amino acid residues lying on the surface of the 3D structure of HlyIICTD, led to a decrease in the interaction of HlyIIC-20 with the mutant form of the protein. Changes in epitope structure confirm the high conformational mobility of HlyIICTD required for the functioning of HlyII. Comparison of the effect of the introduced mutations on the effectiveness of interactions between HlyIICTD and HlyIIC-20 and a control antibody recognizing a non-overlapping epitope enabled the identification of the amino acid residues N339 and K340, included in the conformational antigenic determinant recognized by HlyIIC-20.

Neuregulin-1 attenuates hemolysis- and ischemia induced-cerebrovascular inflammation associated with sickle cell disease.

OBJECTIVES: Individuals with sickle cell disease (SCD) are at severely heightened risk for cerebrovascular injury and acute cerebrovascular events, including ischemic and hemorrhagic stroke, potentially leading to impaired development and life-long physical and cognitive disabilities. Cerebrovascular injury specific to SCD includes inflammation caused by underlying conditions of chronic hemolysis and reduced cerebrovascular perfusion. The objectives of this study were to investigate whether expression of neuregulin-1ß (NRG-1), an endogenous neuroprotective polypeptide, is increased in SCD or experimental conditions mimicking the hemolysis and ischemic conditions of SCD, and to determine if treatment with exogenous NRG-1 reduces markers of cerebrovascular inflammation. MATERIALS AND METHODS: Plasma and brain-specific NRG-1 levels were measured in transgenic SCD mice. Endogenous NRG-1 levels and response to experimental conditions of excess heme and ischemia were measured in cultured human brain microvascular cells and astrocytes. Pre-treatment with NRG-1 was used to determine NRG-1's ability to ameliorate resultant cerebrovascular inflammation. RESULTS: Plasma and brain-specific NRG-1 were elevated in transgenic SCD mice compared to healthy controls. Neuregulin-1 expression was significantly increased in cultured human microvascular cells and astrocytes exposed to excess heme and ischemia. Pre-treatment with NRG-1 reduced inflammatory chemokine (CXCL-1 and CXCL-10) and adhesion molecule (ICAM-1 and VCAM-1) expression and increased pro-angiogenic factors (VEGF-A) in microvascular cells and astrocytes exposed to excess heme and ischemia. CONCLUSIONS: Elevated NRG-1 in SCD is likely a protective endogenous response to ongoing cerebrovascular insults caused by chronic hemolysis and reduced cerebrovascular perfusion. Administration of NRG-1 to reduce cerebrovascular inflammation may be therapeutically beneficial in SCD and warrants continued investigation.

Hematologic and systemic metabolic alterations due to Mediterranean class II G6PD deficiency in mice.

Deficiency of glucose-6-phosphate dehydrogenase (G6PD) is the single most common enzymopathy, present in approximately 400 million humans (approximately 5%). Its prevalence is hypothesized to be due to conferring resistance to malaria. However, G6PD deficiency also results in hemolytic sequelae from oxidant stress. Moreover, G6PD deficiency is associated with kidney disease, diabetes, pulmonary hypertension, immunological defects, and neurodegenerative diseases. To date, the only available mouse models have decreased levels of WT stable G6PD caused by promoter mutations. However, human G6PD mutations are missense mutations that result in decreased enzymatic stability. As such, this results in very low activity in red blood cells (RBCs) that cannot synthesize new protein. To generate a more accurate model, the human sequence for a severe form of G6PD deficiency, Med(-), was knocked into the murine G6PD locus. As predicted, G6PD levels were extremely low in RBCs, and deficient mice had increased hemolytic sequelae to oxidant stress. Nonerythroid organs had metabolic changes consistent with mild G6PD deficiency, consistent with what has been observed in humans. Juxtaposition of G6PD-deficient and WT mice revealed altered lipid metabolism in multiple organ systems. Together, these findings both establish a mouse model of G6PD deficiency that more accurately reflects human G6PD deficiency and advance our basic understanding of altered metabolism in this setting.

Multiple-ancestry genome-wide association study identifies 27 loci associated with measures of hemolysis following blood storage.

BackgroundThe evolutionary pressure of endemic malaria and other erythrocytic pathogens has shaped variation in genes encoding erythrocyte structural and functional proteins, influencing responses to hemolytic stress during transfusion and disease.MethodsWe sought to identify such genetic variants in blood donors by conducting a genome-wide association study (GWAS) of 12,353 volunteer donors, including 1,406 African Americans, 1,306 Asians, and 945 Hispanics, whose stored erythrocytes were characterized by quantitative assays of in vitro osmotic, oxidative, and cold-storage hemolysis.ResultsGWAS revealed 27 significant loci (P < 5 × 10-8), many in candidate genes known to modulate erythrocyte structure, metabolism, and ion channels, including SPTA1, ALDH2, ANK1, HK1, MAPKAPK5, AQP1, PIEZO1, and SLC4A1/band 3. GWAS of oxidative hemolysis identified variants in genes encoding antioxidant enzymes, including GLRX, GPX4, G6PD, and SEC14L4 (Golgi-transport protein). Genome-wide significant loci were also tested for association with the severity of steady-state (baseline) in vivo hemolytic anemia in patients with sickle cell disease, with confirmation of identified SNPs in HBA2, G6PD, PIEZO1, AQP1, and SEC14L4.ConclusionsMany of the identified variants, such as those in G6PD, have previously been shown to impair erythrocyte recovery after transfusion, associate with anemia, or cause rare Mendelian human hemolytic diseases. Candidate SNPs in these genes, especially in polygenic combinations, may affect RBC recovery after transfusion and modulate disease severity in hemolytic diseases, such as sickle cell disease and malaria.

Commercially Available Blocking Oligonucleotides Effectively Suppress Unwanted Hemolysis-Related miRNAs in a Large Whole-Blood RNA Cohort.

When sequencing small RNA libraries derived from whole blood, the most abundant microRNAs (miRs) detected are often miR-486-5p, miR-451a, and miR-92a-3p. These highly expressed erythropoietic miRs are released into the sample from red blood cell hemolysis. Next-generation sequencing of these unwanted miRs leads to a waste in sequencing cost and diminished detection of lowly expressed miRNAs, including many potential miRNA biomarkers. Previous work has developed a method to reduce targeted miRNAs using oligonucleotides that bind their target miRNA and prevent its ligation during library construction, although the extent to which oligonucleotides can be multiplexed and their effect on larger cohorts has not been thoroughly explored. We present a method for suppressing detection of three highly abundant heme miRs in a single multiplexed blocking oligonucleotide reaction. In a small paired-sample pilot (n = 8) and a large cohort of samples (n = 901), multiplexed oligos reduced detection of their target miRNAs by approximately 70%, allowing for an approximately 10-fold increase in reads mapping to nonheme miRs and increased detection of very lowly expressed miRs, with minimal off-target effects. By removing all three highly expressed erythropoietic miRNAs from next-generational sequencing libraries, this commercially available multiplexed blocking oligonucleotide method allows for greater detection of lowly expressed biomarkers, improving the efficacy, cost-efficiency, and sensitivity of biomarker studies and diagnostic tests.

Identification of genetic determinants of hemolytic activity of Riemerella anatipestifer using random transposon mutagenesis.

Riemerella anatipestifer causes epizootic infectious disease in poultry resulting in serious economic losses especially to the duck industry. In our previous study, R. anatipestifer was found to lyse duck erythrocytes in vitro. In the present study, a random Tn4351 mutagenesis library of hemolytic R. anatipestifer strain SX containing 4000 mutants was constructed to investigate the genetic basis of hemolytic activity. Thirty mutants with reduced hemolytic activity and one with increased hemolytic activity were screened and insertions in 24 genes were identified. Of these genes, four were predicted to encode outer membrane proteins, one encoded a cytoplasmic membrane protein, 11 encoded cytoplasmic proteins, and eight encoded proteins with unknown locations. Based on current annotations of the R. anatipestifer genomes, of the 24 genes, 7 (29.17%) were involved in iron utilization. The hemolytic activities of the complemented strains M2 (pRES-Riean_0790) and M18 (pRES-Riean_0653) were restored, indicating that both Riean_0653 and Riean_0790 are involved in the hemolytic activity of strain SX. However, the recombinant proteins rRiean_0317, rRiean_0790, rRiean_0653, rRiean_1027, rRiean_1143, and rRiean_1561 had no hemolytic activity, suggesting that none were hemolysins.

Hb Sun Prairie: A rare cause of chronic hemolysis in an Indian patient.

Haemolytic anaemia is a commonly encountered condition in clinical haematology practise. Dissecting the aetiology of haemolytic anaemia is of paramount importance for appropriate management. We describe a 29-years-old lady of Indian origin, who presented with fatigue and recurrent jaundice for 2 years. Examination revealed pallor, mild icterus, and splenomegaly. Blood tests showed anaemia, reticulocytosis, indirecthyperbilirubinemia, and high serum lactate dehydrogenase, consistent with haemolytic anaemia. Peripheral smear showed severely microcytic hypochromic red cells and polychromasia. Heinz bodies and inclusion bodies were seen with supravital staining. Haemoglobin high pressure liquid chromatography showed low HbA2 and normal HbF. Work-up for iron deficiency was negative. Polymerase chain reaction of the genomic DNA failed to identify common deletions in the HBA genes. Sangers sequencing of HBA2 gene revealed a homozygous missense mutation NM_000517.6: c.391G > C (p.Ala131Pro) leading to a highly unstable hemoglobin, Hb Sun Prairie. Mother was heterozygous for the same mutation, and father was unavailable for genetic testing. We highlight the role of sangers sequencing in unravelling the underlying aetiology of haemolytic anaemia. Pathophysiology and existing literature of Hb Sun Prairie has been discussed.

The Role of α1-Microglobulin (A1M) in Erythropoiesis and Erythrocyte Homeostasis-Therapeutic Opportunities in Hemolytic Conditions.

α1-microglobulin (A1M) is a small protein present in vertebrates including humans. It has several physiologically relevant properties, including binding of heme and radicals as well as enzymatic reduction, that are used in the protection of cells and tissue. Research has revealed that A1M can ameliorate heme and ROS-induced injuries in cell cultures, organs, explants and animal models. Recently, it was shown that A1M could reduce hemolysis in vitro, observed with several different types of insults and sources of RBCs. In addition, in a recently published study, it was observed that mice lacking A1M (A1M-KO) developed a macrocytic anemia phenotype. Altogether, this suggests that A1M may have a role in RBC development, stability and turnover. This opens up the possibility of utilizing A1M for therapeutic purposes in pathological conditions involving erythropoietic and hemolytic abnormalities. Here, we provide an overview of A1M and its potential therapeutic effect in the context of the following erythropoietic and hemolytic conditions: Diamond-Blackfan anemia (DBA), 5q-minus myelodysplastic syndrome (5q-MDS), blood transfusions (including storage), intraventricular hemorrhage (IVH), preeclampsia (PE) and atherosclerosis.

Calcium dynamically alters erythrocyte mechanical response to shear.

Red blood cells (RBC) are constantly exposed to varying mechanical forces while traversing the cardiovascular system. Upon exposure to mechanical stimuli (e.g., shear stress), calcium enters the cell and prompts potassium-efflux. Efflux of potassium is accompanied by a loss of intracellular fluid; thus, the volume of RBC decreases proportionately (i.e., 'Gárdos effect'). The mechanical properties of the cell are subsequently impacted due to complex interactions between cytosolic viscosity (dependent on cell hydration), the surface-area-to-volume ratio, and other molecular processes. The dynamic effects of calcium on RBC mechanics are yet to be elucidated, although accumulating evidence suggests a vital role. The present study thus examined the effects of calcium on contemporary biomechanical properties of RBC in conjunction with high-precision geometrical analyses with exposure to shear. Mechanical stimulation of RBC was performed using a co-axial Couette shearing system to deform the cell membrane; intracellular signaling events were observed via fluorescent imaging. Calcium was introduced into RBC using ionophore A23187. Increased intracellular calcium significantly impaired RBC deformability; these impairments were mediated by a calcium-induced reduction of cell volume through the Gárdos channel. Extracellular calcium in the absence of the ionophore only had an effect under shear, not at stasis. Under low shear, the presence of extracellular calcium induced progressive lysis of a sub-population of RBC; all remaining RBC exhibited exceptional capacity to deform, implying preferential removal of potentially aged cells. Collectively, we provide evidence of the mechanism by which calcium acutely regulates RBC mechanical properties.

Phosphotransferase System Uptake and Metabolism of the ß-Glucoside Salicin Impact Group A Streptococcal Bloodstream Survival and Soft Tissue Infection.

Streptococcus pyogenes (group A Streptococcus [GAS]), a major human-specific pathogen, relies on efficient nutrient acquisition for successful infection within its host. The phosphotransferase system (PTS) couples the import of carbohydrates with their phosphorylation prior to metabolism and has been linked to GAS pathogenesis. In a screen of an insertional mutant library of all 14 annotated PTS permease (EIIC) genes in MGAS5005, the annotated ß-glucoside PTS transporter (bglP) was found to be crucial for GAS growth and survival in human blood and was validated in another M1T1 GAS strain, 5448. In 5448, bglP was shown to be in an operon with a putative phospho-ß-glucosidase (bglB) downstream and a predicted antiterminator (licT) upstream. Using defined nonpolar mutants of the ß-glucoside permease (bglP) and ß-glucosidase enzyme (bglB) in 5448, we showed that bglB, not bglP, was important for growth in blood. Furthermore, transcription of the licT-blgPB operon was found to be repressed by glucose and induced by the ß-glucoside salicin as the sole carbon source. Investigation of the individual bglP and bglB mutants determined that they influence in vitro growth in the ß-glucoside salicin; however, only bglP was necessary for growth in other non-ß-glucoside PTS sugars, such as fructose and mannose. Additionally, loss of BglP and BglB suggests that they are important for the regulation of virulence-related genes that control biofilm formation, streptolysin S (SLS)-mediated hemolysis, and localized ulcerative lesion progression during subcutaneous infections in mice. Thus, our results indicate that the ß-glucoside PTS transports salicin and its metabolism can differentially influence GAS pathophysiology during soft tissue infection.

Intravascular hemolysis triggers ADP-mediated generation of platelet-rich thrombi in precapillary pulmonary arterioles.

Patients with hereditary or acquired hemolytic anemias have a high risk of developing in situ thrombosis of the pulmonary vasculature. While pulmonary thrombosis is a major morbidity associated with hemolytic disorders, the etiological mechanism underlying hemolysis-induced pulmonary thrombosis remains largely unknown. Here, we use intravital lung microscopy in mice to assess the pathogenesis of pulmonary thrombosis following deionized water-induced acute intravascular hemolysis. Acute hemolysis triggered the development of αIIbß3-dependent platelet-rich thrombi in precapillary pulmonary arterioles, which led to the transient impairment of pulmonary blood flow. The hemolysis-induced pulmonary thrombosis was phenocopied with intravascular ADP- but not thrombin-triggered pulmonary thrombosis. Consistent with a mechanism involving ADP release from hemolyzing erythrocytes, the inhibition of platelet P2Y12 purinergic receptor signaling attenuated pulmonary thrombosis and rescued blood flow in the pulmonary arterioles of mice following intravascular hemolysis. These findings are the first in vivo studies to our knowledge to suggest that acute intravascular hemolysis promotes ADP-dependent platelet activation, leading to thrombosis in the precapillary pulmonary arterioles, and that thrombin generation most likely does not play a significant role in the pathogenesis of acute hemolysis-triggered pulmonary thrombosis.

Differential expression of hemolysin genes in weakly and strongly hemolytic Brachyspira hyodysenteriae strains.

BACKGROUND: Swine dysentery (SD) is a diarrheal disease in fattening pigs that is caused by the strongly hemolytic species Brachyspira (B.) hyodysenteriae, B. hampsonii and B. suanatina. As weakly hemolytic Brachyspira spp. are considered less virulent or even non-pathogenic, the hemolysin is regarded as an important factor in the pathogenesis of SD. Four hemolysin genes (tlyA, tlyB, tlyC, and hlyA) and four putative hemolysin genes (hemolysin, hemolysin activation protein, hemolysin III, and hemolysin channel protein) have been reported, but their role in strong hemolysis is not entirely clear. Our study aimed to assess the transcriptional activity of eight (putative) hemolysin genes in a strongly hemolytic (B204) and a weakly hemolytic (G423) B. hyodysenteriae strain during non-hemolytic and hemolytic growth stages. RESULTS: Strongly and weakly hemolytic B. hyodysenteriae strains caused hemolysis on blood agar at different growth stages, namely during log phase (B204) and stationary/death phase (G423). During the lag, early log, late log (stationary phase in G423) and death phase (time points 1-4) strains differed in their hemolysin gene transcription patterns. At time point 1, transcription of the putative hemolysin gene was higher in B204 than in G423. At time point 2, tlyA and tlyC were upregulated in B204 during hemolysis. TlyB and hlyA were upregulated in both strains at all time points, but higher transcription rates were observed in the weakly hemolytic strain G423. The transcription activity of the hemolysin channel protein gene was quite similar in both strains, whereas the hemolysin activation protein gene was upregulated in the non-hemolytic stage of B204 at time point 4. Sequence analysis revealed deletions, insertions and single nucleotide polymorphisms in the G423 hlyA promoter, although without altering the transcription activity of this gene. CONCLUSION: Our data indicate a combined activity of TlyA and TlyC as the most probable underlying mechanism of strong hemolysis in B. hyodysenteriae. Further studies should verify if the expression of tlyA is upregulated by the putative hemolysin gene. Depending on their immunogenic potential TlyA and TlyC may serve as possible vaccine candidates, especially since vaccines for an effective control of swine dysentery are currently not available.

Negative charge of the AC-to-Hly linking segment modulates calcium-dependent membrane activities of Bordetella adenylate cyclase toxin.

Two distinct conformers of the adenylate cyclase toxin (CyaA) appear to accomplish its two parallel activities within target cell membrane. The translocating conformer would deliver the N-terminal adenylyl cyclase (AC) enzyme domain across plasma membrane into cytosol of cells, while the pore precursor conformer would assemble into oligomeric cation-selective pores and permeabilize cellular membrane. Both toxin activities then involve a membrane-interacting 'AC-to-Hly-linking segment' (residues 400 to 500). Here, we report the NMR structure of the corresponding CyaA411-490 polypeptide in dodecylphosphocholine micelles and show that it consists of two α-helices linked by an unrestrained loop. The N-terminal α-helix (Gly418 to His439) remained solvent accessible, while the C-terminal α-helix (His457 to Phe485) was fully enclosed within detergent micelles. CyaA411-490 weakly bound Ca2+ ions (apparent KD 2.6 mM) and permeabilized negatively charged lipid vesicles. At high concentrations (10 µM) the CyaA411-490 polypeptide formed stable conductance units in artificial lipid bilayers with applied voltage, suggesting its possible transmembrane orientation in the membrane-inserted toxin. Mutagenesis revealed that two clusters of negatively charged residues within the 'AC-to-Hly-linking segment' (Glu419 to Glu432 and Asp445 to Glu448) regulate the balance between the AC domain translocating and pore-forming capacities of CyaA in function of calcium concentration.

Differences in phenotypic and genetic characteristics of Trueperella pyogenes detected in slaughtered cattle and pigs with septicemia.

We investigated the hemolytic properties, biochemical properties, and possession of virulence factor genes of Trueperella pyogenes isolated from cattle and pigs with septicemia. The porcine strains showed significantly stronger hemolyticity than the bovine strains. In addition, T. pyogenes from cattle and pigs also differed in biochemical properties. Virulence factor genes (nanP, cbpA, fimC, and fimE) were more prevalent in bovine strains, whereas other virulence factor genes (nanH and fimG) were more prevalent in porcine strains. T. pyogenes isolated from pig and cattle with septis cases in Japanese meat inspection showed variability in biochemical and genetic properties. Differences were observed between porcine and bovine strain in term of the hemolytic strength and possession of genes for factors promoting adhesions which are considered pathogenic.

Fish borne Edwardsiella tarda eha involved in the bacterial biofilm formation, hemolytic activity, adhesion capability and pathogenicity.

Edwardsiella tarda (E. tarda) is distributed widely in a variety of hosts including humans, other mammals and fish, and it is worthwhile to notice that E. tarda -caused fish infections lead to the most important bacterial disease in fish. Considering Eha acting as a transcriptional regulator in E. tarda strain ET13 have been reported previously, to better understand its pathogenesis due to this, a type of cell of epithelial cell line (Caco-2) infection model for the pathogen was established in the laboratory. We focused on studying various parameters such as lactate dehydrogenase release (to measure cytotoxicity) and cell adhesions, both of which are related to the bacterial pathogenesis. Furthermore biofilm formation, hemolytic activity, and adhesion to Caco-2 cells were decreased in an E.tarda mutant strain with deletion in-frame isogenic gene eha (∆eha) compared to the wild-type and the complementary strain eha+ (an engineered construct of ∆eha expressing eha); Meanwhile, we found that hemolytic activity and biofilm formation were significantly enhanced in the strain eha+. Moreover, the ∆eha strain had attenuated pathogenicity in the zebrafish infection model. The data also demonstrated that the series of genes fimA, esrB, gadB, mukF, katB, and katG are regulated by eha based on a quantitative reverse transcription polymerase chain reaction tests and analysis. Thus our research data indicated that eha has an impact on hemolytic activity, biofilm formation, adhesion, and pathogenicity of pathogenic strain ET13 and plays an essential role in manifesting the virulence factors.