To B or not to B cells-mediate a healthy start to life.

Maternal immune responses during pregnancy are critical in programming the future health of a newborn. The maternal immune system is required to accommodate fetal immune tolerance as well as to provide a protective defence against infections for the immunocompromised mother and her baby during gestation and lactation. Natural immunity and antibody production by maternal B cells play a significant role in providing such immunoprotection. However, aberrations in the B cell compartment as a consequence of maternal autoimmunity can pose serious risks to both the mother and her baby. Despite their potential implication in shaping pregnancy outcomes, the role of B cells in human pregnancy has been poorly studied. This review focuses on the role of B cells and the implications of B cell depletion therapy in pregnancy. It highlights the evidence of an association between aberrant B cell compartment and obstetric conditions. It also alludes to the potential mechanisms that amplify these B cell aberrances and thereby contribute to exacerbation of some maternal autoimmune conditions and poor neonatal outcomes. Clinical and experimental evidence suggests strongly that maternal autoantibodies contribute directly to the pathologies of obstetric and neonatal conditions that have significant implications for the lifelong health of a newborn. The evidence for clinical benefit and safety of B cell depletion therapies in pregnancy is reviewed, and an argument is mounted for further clinical evaluation of B cell-targeted therapies in high-risk pregnancy, with an emphasis on improving neonatal outcomes and prevention of neonatal conditions such as congenital heart block and fetal/neonatal alloimmune thrombocytopenia.

A haploid diamondback moth (Plutella xylostella L.) genome assembly resolves 31 chromosomes and identifies a diamide resistance mutation.

The diamondback moth, Plutella xylostella (L.), is a highly mobile brassica crop pest with worldwide distribution and can rapidly evolve resistance to insecticides, including group 28 diamides. Reference genomes assembled using Illumina sequencing technology have provided valuable resources to advance our knowledge regarding the biology, origin and movement of diamondback moth, and more recently with its sister species, Plutella australiana. Here we apply a trio binning approach to sequence and annotate a chromosome level reference genome of P. xylostella using PacBio Sequel and Dovetail Hi-C sequencing technology and identify a point mutation that causes resistance to commercial diamides. A P. xylostella population collected from brassica crops in the Lockyer Valley, Australia (LV-R), was reselected for chlorantraniliprole resistance then a single male was crossed to a P. australiana female and a hybrid pupa sequenced. A chromosome level 328 Mb P. xylostella genome was assembled with 98.1% assigned to 30 autosomes and the Z chromosome. The genome was highly complete with 98.4% of BUSCO Insecta genes identified and RNAseq informed protein prediction annotated 19,002 coding genes. The LV-R strain survived recommended field application doses of chlorantraniliprole, flubendiamide and cyclaniliprole. Some hybrids also survived these doses, indicating significant departure from recessivity, which has not been previously documented for diamides. Diamide chemicals modulate insect Ryanodine Receptors (RyR), disrupting calcium homeostasis, and we identified an amino acid substitution (I4790K) recently reported to cause diamide resistance in a strain from Japan. This chromosome level assembly provides a new resource for insect comparative genomics and highlights the emergence of diamide resistance in Australia. Resistance management plans need to account for the fact that resistance is not completely recessive.

Mean platelet diameter measurements to classify inherited thrombocytopenias.

INTRODUCTION: Mean platelet volume (MPV) assists the differential diagnosis of inherited thrombocytopenia (IT) but lacks standardisation and varies between automated analysers. Classification of IT based on mean platelet diameter (MPD) has been proposed by an international collaborative study but has not been validated. METHODS: To assess the applicability of MPD to classify forms of IT, digital images of blood films from patients with established genetic causes for IT were generated, and the MPD measured (ZEISS Axio-scanner and Image J software) by a blinded reviewer. Comparison was made to the proposed classification system. RESULTS: Mean platelet volume was measured in thrombocytopenia with different genetic aetiologies, bilallelic BSS (bBSS) (n = 1), monoallelic BSS (mBSS) (n = 2), MYH9-related disorders (MYH9-RD) (n = 11), GFI1B-related thrombocytopenia (RT) (n = 15), FLI1-RT (n = 2), TUBB1-RT (n = 3), ITGA2B/ITGB3-RT (n = 1), RUNX1-RT (n = 2) and controls (n = 54). bBSS and 82% of MYH9-RD samples had MPD >4 µm which correlated with "IT with giant platelets." Only 55% of samples expected in the "large platelet group" had MPD meeting the classification cut-off (MPD >3.2 µm). FLI1-RT MPD were significantly larger than expected whilst ITGA2B/ITGB3-RT MPD were smaller than proposed. MPD in FPD/AML were "normal." CONCLUSION: Platelet MPD measurements are a useful guide to classify IT, but the time taken to record measurements may limit clinical applicability.

Receptor homodimerization plays a critical role in a novel dominant negative P2RY12 variant identified in a family with severe bleeding.

Essentials Three dominant variants for the autosomal recessive bleeding disorder type-8 have been described. To date, there has been no phenotype/genotype correlation explaining their dominant transmission. Proline plays an important role in P2Y12R ligand binding and signaling defects. P2Y12R homodimer formation is critical for the receptor function and signaling. SUMMARY: Background Although inherited platelet disorders are still underdiagnosed worldwide, advances in molecular techniques are improving disease diagnosis and patient management. Objective To identify and characterize the mechanism underlying the bleeding phenotype in a Caucasian family with an autosomal dominant P2RY12 variant. Methods Full blood counts, platelet aggregometry, flow cytometry and western blotting were performed before next-generation sequencing (NGS). Detailed molecular analysis of the identified variant of the P2Y12 receptor (P2Y12R) was subsequently performed in mammalian cells overexpressing receptor constructs. Results All three referred individuals had markedly impaired ADP-induced platelet aggregation with primary wave only, despite normal total and surface P2Y12R expression. By NGS, a single P2RY12:c.G794C substitution (p.R265P) was identified in all affected individuals, and this was confirmed by Sanger sequencing. Mammalian cell experiments with the R265P-P2Y12R variant showed normal receptor surface expression versus wild-type (WT) P2Y12R. Agonist-stimulated R265P-P2Y12R function (both signaling and surface receptor loss) was reduced versus WT P2Y12R. Critically, R265P-P2Y12R acted in a dominant negative manner, with agonist-stimulated WT P2Y12R activity being reduced by variant coexpression, suggesting dramatic loss of WT homodimers. Importantly, platelet P2RY12 cDNA cloning and sequencing in two affected individuals also revealed three-fold mutant mRNA overexpression, decreasing even further the likelihood of WT homodimer formation. R265 located within extracellular loop 3 (EL3) is one of four residues that are important for receptor functional integrity, maintaining the binding pocket conformation and allowing rotation following ligand binding. Conclusion This novel dominant negative variant confirms the important role of R265 in EL3 in the functional integrity of P2Y12R, and suggests that pathologic heterodimer formation may underlie this family bleeding phenotype.

Reduced fibrinolysis and increased fibrin generation can be detected in hypercoagulable patients using the overall hemostatic potential assay.

BACKGROUND: Routinely available coagulation assays are not capable of detecting clinically defined hypercoagulable states. A number of global coagulation assays have been developed with the potential to evaluate hypercoagulability, which predisposes to the common clinical events of arterial and venous thromboembolism (VTE). OBJECTIVES: We hypothesized that the overall hemostatic potential (OHP) assay would show abnormal fibrin generation and lysis in patients with clinically defined hypercoagulable states. METHODS: We used the OHP assay as described by Blombäck and colleagues [1,2] in 161 clinically hypercoagulable patients with arterial or VTE, pregnancy complications or autoimmune disease. Eighty patients had associated antiphospholipid antibodies (APLA). Ninety-eight normal plasma donors were tested for comparison. RESULTS: We derived three new assay parameters for correlation with hypercoagulable states: the maximum optical density, maximum slope, and delay in onset of fibrin generation. We found significantly different assay results for all patients' parameters examined when compared with controls, indicating both increased fibrin generation and reduced fibrinolysis in hypercoagulable patients. The findings were similar whether samples were collected in association with an acute thrombotic event or not. Estimated assay sensitivity for detection of a clinically defined hypercoagulable state was 96%. CONCLUSIONS: The OHP assay is a simple, inexpensive global test that is useful for assessing patients with hypercoagulable states including APLA. OHP results are significantly abnormal in hypercoagulable groups compared with controls, indicating that both increased fibrin generation and reduced fibrinolysis contribute to hypercoagulable states. The assay may ultimately assist in tailoring clinical management to patients' individual requirements.

Use of a functional assay to diagnose protein S deficiency; inappropriate testing yields equivocal results.

Inherited deficiency of protein S (PS) is a rare but accepted risk factor for venous thromboembolism. There is accumulating evidence that inherited PS deficiency may be associated with a variety of adverse obstetric events. Acquired PS deficiency may be caused by a variety of clinical states including normal pregnancy. We conducted a retrospective audit of the results of screening for PS deficiency through our reference laboratory. The majority of patients in this audit with significantly reduced (<50%) free functional PS levels had a major confounding factor likely to cause acquired PS deficiency, most frequently pregnancy. Recommendations for PS testing for the diagnosis of hereditary PS deficiency include deferring testing until at least 40 days post-partum. It appears that these recommendations are not being adhered to leading to difficulty in the interpretation of results.

Thrombocytopenia and CD34 expression is decoupled from α-granule deficiency with mutation of the first growth factor-independent 1B zinc finger.

Essentials The phenotypes of different growth factor-independent 1B (GFI1B) variants are not established. GFI1B variants produce heterogeneous clinical phenotypes dependent on the site of mutation. Mutation of the first non-DNA-binding zinc-finger causes a mild platelet and clinical phenotype. GFI1B regulates the CD34 promoter; platelet CD34 expression is an indicator of GFI1B mutation. SUMMARY: Background Mutation of the growth factor-independent 1B (GFI1B) fifth DNA-binding zinc-finger domain causes macrothrombocytopenia and α-granule deficiency leading to clinical bleeding. The phenotypes associated with GFI1B variants disrupting non-DNA-binding zinc-fingers remain uncharacterized. Objectives To determine the functional and phenotypic consequences of GFI1B variants disrupting non-DNA-binding zinc-finger domains. Methods The GFI1B C168F variant and a novel GFI1B c.2520 + 1_2520 + 8delGTGGGCAC splice variant were identified in four unrelated families. Phenotypic features, DNA-binding properties and transcriptional effects were determined and compared with those in individuals with a GFI1B H294 fs mutation of the fifth DNA-binding zinc-finger. Patient-specific induced pluripotent stem cell (iPSC)-derived megakaryocytes were generated to facilitate disease modeling. Results The DNA-binding GFI1B variant C168F, which is predicted to disrupt the first non-DNA-binding zinc-finger domain, is associated with macrothrombocytopenia without α-granule deficiency or bleeding symptoms. A GFI1B splice variant, c.2520 + 1_2520 + 8delGTGGGCAC, which generates a short GFI1B isoform that lacks non-DNA-binding zinc-fingers 1 and 2, is associated with increased platelet CD34 expression only, without quantitative or morphologic platelet abnormalities. GFI1B represses the CD34 promoter, and this repression is attenuated by different GFI1B zinc-finger mutations, suggesting that deregulation of CD34 expression occurs at a direct transcriptional level. Patient-specific iPSC-derived megakaryocytes phenocopy these observations. Conclusions Disruption of GFI1B non-DNA-binding zinc-finger 1 is associated with mild to moderate thrombocytopenia without α-granule deficiency or bleeding symptomatology, indicating that the site of GFI1B mutation has important phenotypic implications. Platelet CD34 expression appears to be a common feature of perturbed GFI1B function, and may have diagnostic utility.

Monoclonal anti-idiotype antibody mimicking the pesticide binding site of cutinase: potential for broad specificity organophosphate recognition.

An anti-idiotype monoclonal antibody (Mab) able to bind the organophosphate pesticides, chlorfenvinphos (CFV), ethyl paraoxon, tetrachlorfenvinphos and demeton-s-methyl, has been produced using as immunogen a Mab which binds to the active site of cutinase. The principle of using an anti-idiotype antibody as the mimic of a site on a protein able to bind a group of ligands has, therefore, been demonstrated, and may have implications for future research on broad specificity immunoanalysis of groups of compounds.

Biophysical characteristics of anti-Gal(alpha)1-3Gal IgM binding to cell surfaces: implications for xenotransplantation.

BACKGROUND: Natural antibodies directed against cell surface carbohydrates are thought to be vital to host defense and to initiate the rejection of xenografts and ABO-incompatible allografts. The biophysical properties underlying the association and dissociation of these antibodies from cell surfaces is incompletely understood. We investigated those properties for the binding of Galalpha1-3Gal antibodies to porcine endothelial cell surfaces, because such interactions might be relevant to the clinical application of xenotransplantation. RESULTS AND CONCLUSIONS: The initial rate of binding of anti-Galalpha1-3Gal antibodies to endothelial cells was found to depend on antibody concentration, antibody diffusion, and antigen concentration. The presence of an intact glycocalyx had a greater impact on antibody binding than mobility of antigen in cell membranes. Disruption of glycocalyx increased the amount of antibody bound at equilibrium by more than 50%. Although the binding of anti-Galalpha1-3Gal antibodies to cell surfaces could be inhibited by soluble Galalpha1-3Gal, once bound, some anti-Galalpha1-3Gal could not be dissociated by competitive inhibitors of binding or by denaturation of the bound Ig with chaotropic reagents, but could be dissociated by reduction of disulfide bonds, suggesting that attachment to cell surfaces was, at least in part, by means other than specific reaction with the epitope.

A frameshift mutation at Gly975 in the transmembrane domain of GPIIb prevents GPIIb-IIIa expression--analysis of two novel mutations in a kindred with type I glanzmann thrombasthenia.

Two Hispanic siblings presenting with lifelong mucocutaneous bleeding were diagnosed clinically with Glanzmann thrombasthenia on the basis of a normal platelet count, prolonged bleeding time and absent platelet aggregation in response to multiple agonists. Quantitative analysis of the probands' platelets by flow cytometry showed a complete absence of GPIIb-IIIa, consistent with Type I thrombasthenia. Genetic analysis showed the probands to be compound heterozygotes for two novel mutations of GPIIb: a C1414>G mutation in exon 14, resulting in a premature termination codon replacing residue Tyr440, and the insertion of a G at position 3016 in exon 29, leading to a frameshift affecting the C-terminal half of the transmembrane domain and the cytoplasmic tail. The frameshifted sequence alters residues from Gly975 onwards and is predicted to significantly alter the hydropathy and charge profiles of the GPIIb transmembrane domain. The Type I phenotype associated with this mutation suggests that GPIIb residues 975-1008 contain critical structural motifs for heterodimer assembly, membrane retention, export from the ER and surface expression.

Folate-targeted non-viral DNA vectors for cancer gene therapy.

The vitamin folic acid exhibits high affinity for the endocytosed, membrane-bound folate receptor. Conjugation of folic acid via its gamma-carboxyl group to various macromolecules results in binding to, and endocytosis into, cells expressing the folate receptor. The folate receptor is overexpressed on a wide range of cancers, therefore folic acid has been investigated as a targeting ligand for the specific delivery of therapeutic compounds to cancer cells. This review will introduce folate-targeting of macromolecules to cancer cells in vitro and in vivo, and discuss the accumulation of such compounds in non-target tissues in vivo. Folate-targeting of non-viral DNA vectors in vitro and in vivo will be discussed in detail, with particular emphasis on the recent advances in this field.

Binding of the von Willebrand factor A1 domain to histone.

Activation of the von Willebrand Factor (vWF) A1 domain is a critical factor in regulating the interaction of vWF with its platelet membrane receptor, the glycoprotein (GP) Ib-IX-V complex. This activation controls vWF-dependent platelet adhesion at high shear. The vWF-GP Ib-IX-V interaction is induced in vivo by exposure of platelet-rich plasma to high shear force, or by association of vWF with one or more unidentified components of the subendothelial matrix. In vitro, soluble vWF is activated to bind to platelets by nonphysiological modulators, such as the bacterial glycopeptide, ristocetin, or the snake venom protein, botrocetin, or by removal of negatively-charged sialic acid residues. Analysis of vWF modulators and the very marked charge asymmetry of amino acid sequences within the A1 domain has led to an electrostatic model for vWF modulation. Endothelial membrane/matrix and detergent-soluble fractions of human placenta were screened for the ability to bind vWF by electrophoresis of extracts on SDS-polyacrylamide gels, electrotransferring to nitrocellulose and probing with fluid-phase 125I-labeled vWF or a 39/34-kDa vWF fragment (Leu-480-Gly-718) that encompasses the A1 domain. In the course of these studies, it was found that both vWF and the 39/34-kDa vWF fragment bound strongly to histone. Purified soluble histone also bound vWF since, like ristocetin, it induced vWF flocculation. Histone binding to vWF did not activate or inhibit vWF binding to platelets. While the vWF-histone interaction has no conceivable physiological role, it suggests that binding to the A1 domain of vWF alone is insufficient to modulate vWF adhesive activity. This implies that specific interactions of the vWF A1 domain with either ristocetin or botrocetin are required for GP Ib-IX-V recognition to occur.

Ophthalmic sequelae of infantile hemangiomas of the eyelids and orbit.

The major findings in a study of 51 infants and children with infantile hemangioma of the eyelid were as follows: Visual complications occurred in 27 patients. The most common complications were amblyopia (in 22) and strabismus (in 17). Amblyopia of 6/60 (20/200) or less was probably caused by stimulus-deprivation, but amblyopia in the range of 6/30 (20/100) to 6/12 (20/40) was likely caused by anisometropia or strabismus. Eyelid occlusion of six months or more invariably resulted in amblyopia of 6/60 (20/200) or less. Occlusion for even one month carried a risk of amblyopia. Each child must be considered individually for therapy, which must be started as early as possible. Patients should receive careful follow-up from the beginning to prevent severe amblyopia. For difficult cases, we need more efficacious, safe methods to induce regression of these tumors.

Characterization of mocarhagin, a cobra venom metalloproteinase from Naja mocambique mocambique, and related proteins from other Elapidae venoms.

Mocarhagin, a cobra venom metalloproteinase from Naja mocambique mocambique, has previously been shown to cleave selectively two mucin-like substrates on platelets and neutrophils within anionic amino acid sequences containing sulfated tyrosines. We now show that purified mocarhagin has haemagglutinin activity, and a similar profile for inhibition of mocarhagin-dependent haemagglutination and proteolysis suggests that the lectin-like domain may account for its substrate specificity. In addition, immunologically and functionally related proteins were detected in other Elapidae venoms.

Reducing intergroup bias: elements of intergroup cooperation.

The authors examined the potentially separable contributions of 2 elements of intergroup cooperation, interaction and common fate, and the processes through which they can operate. The manipulation of interaction reduced bias in evaluative ratings, which supports the idea that these components are separable, whereas the manipulation of common fate when the groups were interacting was associated with lower bias in nonverbal facial reactions in response to contributions by in-group and out-group members. Whereas interaction activated several processes that can lead to reduced bias, including decategorization, consistent with the common in-group identity model (S. L. Gaertner, J. F. Dovidio, P. A. Anastasio, B. A. Bachman, & M. C. Rust, 1993) as well as M. Hewstone and R. J. Brown's (1986) group differentiation model, the primary set of mediators involved participants' representations of the memberships as 2 subgroups within a superordinate entity.

Folic acid targeting of protein conjugates into ascites tumour cells from ovarian cancer patients.

Despite a wealth of in vitro data describing the use of folic acid for drug and DNA delivery into ovarian cancer cell lines, there have been no reports describing the targeting of such compounds to freshly isolated tumour cells. We have carried out a study to determine the usefulness of folic acid as a targeting ligand for ovarian cancer by measuring the uptake of folic acid-BSA-FITC in tumour cells isolated from the ascitic fluid of ovarian cancer patients. In 7 out of 7 patients we have found folic acid mediated uptake of the fluorescently labelled albumin, with the accumulation (average cell fluorescence) and differential uptake (ratio between receptor mediated and fluid phase uptake) varying between patients. Accumulation of folic acid-albumin FITC occurs in ascites tumour cells expressing the epithelial cell marker EMA, with a significant proportion of EMA negative cells also accumulating the conjugate. There is no correlation between cell cycle and uptake of folic acid-BSA-FITC. These results suggest that folic acid-targeting of therapeutics is a promising approach for the treatment of ovarian cancer.

An analysis, from photographs, of the results of four approaches to elongating the columella after repair of bilateral cleft lip.

A system for subjective and objective clinical analysis of the lower third of the nose before and after lengthening the columella was devised, and used the study of photographs of 77 patients who had had lengthening done by one of 4 approaches. It was found that each method was equally effective in lengthening the columella and in restoring a normal relationship between the structures in the tip of triangle (the basal view of the nose). However, there were certain peculiar advantages and disadvantages of each surgical technique, and these are discussed.