Viewpoint: The value of non-criteria antiphospholipid antibodies.

In 2006, at a meeting in Sydney, Australia, consensus was reached by an international group of specialists to establish a number of serological criteria that identify patients with a history of thrombosis or pregnancy complications as having antiphospholipid syndrome (APS). These criteria were originally formulated for research purposes and to compare clinical trials in different centres. However, these same criteria are now generally used and accepted for the diagnosis and treatment of patients. The practice of using these criteria for direct patient care requires that these criteria are based on sound scientific evidence. Indeed, for all the autoantibodies that are officially included in the serological criteria, it has been shown that they induce thrombosis and fetal loss when infused into mice. There are also a number of additional autoantibodies that have been identified in these patients but for these antibodies there was not enough evidence to meet the official APS criteria in 2006. Seventeen years have now passed since the consensus meeting, therefore, this review examines whether additional studies performed with these 'non-criteria' autoantibodies have provided sufficient results to suggest the inclusion of these autoantibodies in the official serological criteria of APS.

Viewpoint: Provoked thrombosis in antiphospholipid syndrome.

Unprovoked thrombosis (thrombosis occurring without an established environmental factor favouring the episode) is a classic feature of APS. In the general population, provoked venous thromboembolism (VTE) is clearly defined and has clinical and therapeutic differences compared with unprovoked VTE. Whether provoked VTE in the context of APS may lead to a limited treatment duration is not well established. Therefore, careful clinical and laboratory evaluation is needed to identify patients eligible for a limited duration of anticoagulation treatment after provoked VTE. Given the uncertainties of available data, the risks and benefits of treatment decisions should be clearly explained. Decisions should be shared by both the patient and physician. Cardiovascular risk factors are common in patients with APS with arterial thrombosis. There are insufficient data suggesting that cardiovascular risk factor control would allow the cessation of anticoagulation. In most instances, arterial thrombosis will require prolonged anticoagulants. A careful analysis of clinical characteristics and laboratory evaluation, particularly the aPL antibody profile, is needed to make decisions on a case-by-case basis.

Antibodies to Domain I ß2-Glycoprotein 1 in Patients with Antiphospholipid Syndrome and Systemic Lupus Erythematosus.

The role of antiphospholipid antibodies (aPL), which are not included in the Sydney diagnostic criteria, in antiphospholipid syndrome (APS) and systemic lupus erythematosus (SLE) is poorly understood. The aim of this study was to determine the clinical significance of IgG antibodies for domain 1 of ß2-glycoprotein 1 (ß2-GP1), IgG anti-ß2-GP1DI, in patients with APS with and without SLE. The study included 187 patients with APS with or without SLE, 49 patients formed the comparison group, and 100 apparently healthy individuals formed the control group. IgG/IgM antibodies to cardiolipin (aCL) and IgG/IgM anti-ß2-GP1 were determined by enzyme immunoassay (ELISA) in patients with or without APS, and IgG anti-ß2-GP1DI was determined by chemiluminescence assay (CLA) in all patients and controls. IgG anti-ß2-GP1DI was detected in 37 (71%) of 52 patients with primary APS (PAPS), in 6 (50%) of 12 patients with probable APS, in 42 (71%) of 59 patients with SLE + APS, in 17 (26%) of 64 patients with SLE, in 1 (2%) of the comparison group, and in none of the control group. IgG anti-ß2-GP1DI was significantly associated with PAPS and SLE + APS compared with the patients with SLE (p = 0.0002 and 0.0001, respectively). The association of IgG anti-ß2-GP1DI with clinical manifestations of APS (thrombosis (p = 0.001) and obstetric pathology (p = 0.04)) was detected. There was a significant association of IgG anti-ß2-GP1DI with arterial thrombosis (p = 0.002) and with late gestational obstetric pathology (p = 0.01). High specificity of IgG anti-ß2-GP1DI depending on the diagnosis and clinical manifestations of APS despite low sensitivity was noted: specificity was 84% for thrombosis, 94% for obstetric pathology, and 89% for APS. Isolated IgG anti-ß2-GP1DI positivity was reported in 2% of 50 aPL-negative patients and was not associated with APS manifestations. The frequency of IgG anti-ß2-GP1DI detection was higher in the patients with APS compared to the patients with SLE, comparison group, and control (p < 0.05). Positive IgG anti-ß2-GP1DI values were significantly associated with thrombotic complications and with obstetric pathology (p = 0.002 and p = 0.01, respectively). Specificity of IgG anti-ß2-GP1DI for APS and its clinical manifestations (thrombosis and obstetric pathology) was higher than sensitivity (89, 94, and 84%, respectively).

Acidic pH-Induced Conformational Changes in Chikungunya Virus Fusion Protein E1: a Spring-Twisted Region in the Domain I-III Linker Acts as a Hinge Point for Swiveling Motion of Domains.

Chikungunya virus (CHIKV), a mosquito-transmitted alphavirus, enters a cell through endocytosis, followed by viral and cell membrane fusion. The fusion protein, E1, undergoes an acid pH-induced pre- to postfusion conformation change during membrane fusion. As part of the conformation change, E1 dissociates from the receptor-binding protein, E2, and swivels its domains I and II over domain III to form an extended intermediate and then eventually to form a postfusion hairpin homotrimer. In this study, we tested if the domain I-III linker acts as a "hinge" for the swiveling motion of E1 domains. We found a conserved spring-twisted structure in the linker, stabilized by a salt bridge between a conserved arginine-aspartic acid pair, as a "hinge point" for domain swiveling. Molecular dynamics (MD) simulation of the CHIKV E1 or E2-E1 structure predicted that the spring-twisted region untwists at pH 5.5. Corroborating the prediction, introduction of a "cystine staple" at the hinge point, replacing the conserved arginine-aspartic acid pair with cysteine residues, resulted in loss of fusion activity of E1. MD simulation also predicted domain I-III swiveling at acidic pH. We tested if breaking the His 331-Lys 16 H bond between domains I and III, seen only in the prefusion conformation, is important for domain swiveling. When domains I and III are "stapled" by introducing a disulfide bond in between, E1 showed loss of fusion activity, implying that domain I and III dissociation is a critical acid pH-induced step in membrane fusion. However, replacement of His 331 with an acidic residue did not affect the pH threshold for fusion, suggesting His 331 is not an acid-sensing residue.IMPORTANCEAedes mosquito-transmitted viruses such as the Zika, dengue, and chikungunya viruses have spread globally. CHIKV, similar to many other enveloped viruses, enters cells in sequential steps: step 1 involves receptor binding followed by endocytosis, and step 2 involves viral-cell membrane fusion in the endocytic vesicle. The viral envelope surface protein, E1, performs membrane fusion. E1 is triggered to undergo conformational changes by acidic pH of the maturing endosome. Different domains of E1 rearrange during the pre- to postfusion conformation change. Using in silico analysis of the E1 structure and different biochemical experiments, we explained a structural mechanism of key conformational changes in E1 triggered by acidic pH. We noted two important structural changes in E1 at acidic pH. In the first, a spring-twisted region in a loop connecting two domains (I and III) untwists, bringing a swiveling motion of domains on each other. In the second, breaking of interactions between domains I and III and domain separation are required for membrane fusion. This knowledge will help devise new therapeutic strategies to block conformation changes in E1 and thus viral entry.

HCV-2a NS5A downregulates viral translation predominantly through domain I.

Hepatitis C virus (HCV) non-structural protein NS5A is a multifunctional protein with critical roles in viral replication and assembly. We previously showed that HCV-1b NS5A downregulates viral translation only in the presence of the poly(U/UC) tract in 3'UTR. As NS5A of different HCV genotypes may have different functions or carry out the same functions through genotype-specific mechanisms, we investigated the effect of HCV-2a NS5A on viral translation. We found that HCV-2a NS5A downregulates RNA translation of both HCV-2a and -1b, whereas the effect of HCV-1b NS5A is limited to HCV-1b only. In addition, individual regions of 3'UTR are not required for HCV-2a NS5A to downregulate viral RNA translation. We also found that HCV-2a NS5A inhibits capped mRNA translation. Mapping experiments showed that the translation downregulation by HCV-2a NS5A is predominantly mediated by domain I. Furthermore, we found that the integrity of serine-146 residue plays an important role in translation downregulation by NS5A. Our results increased our understanding on genotype-specific functions of HCV NS5A.

Domain I of hepatitis C virus NS5A associates with ACBD3 in a genotype-dependent manner.

Previously, it was found that the hepatitis C virus NS5A interacted with ACBD3 in a genotype-dependent manner. However, the region in NS5A responsible for association with ACBD3 is not clear. Domain I of NS5A was identified as critical for ACBD3 binding. By comparing the differences of amino acids in domain I from different genotypes of NS5A, it was found that key amino acids potentially corresponded to the affinity of the NS5A-ACBD3 interaction. The findings not only revealed that domain I of NS5A associates with ACBD3 but they also shed mechanistic light on how NS5A is associated with ACBD3 in a genotype-dependent manner.

Domain I of ß2GPI is capable of blocking serum IgA antiphospholipid antibodies binding in vitro: an effect enhanced by PEGylation.

OBJECTIVES: This study aims to inhibit antiphospholipid syndrome (APS) serum derived IgA anti-beta-2-glycoprotein I (aß2GPI) binding using Domain I (DI). METHODS: Serum from 13 APS patients was tested for IgA aß2GPI and Anti-Domain I. Whole IgA was purified by peptide M affinity chromatography from positive serum samples. Serum was tested for IgA aß2GPI binding in the presence and absence of either DI or of two biochemically modified variants containing either 20 kDa of poly(ethylene glycol) (PEG) or 40 kDa of PEG. RESULTS: Significant inhibition with DI was possible with average inhibition of 23% (N = 13). Further inhibitions using 20 kDa PEG-DI and 40 kDa PEG-DI variants showed significant inhibition (p = 0.0001) with both the 40 kDa PEG-DI and 20 kDa PEG-DI variants showing increased inhibition compared with DI alone (p = 0.0001 and p = 0.001, n = 10). CONCLUSIONS: Inhibition of IgA aß2GPI by DI is possible and can be enhanced by biochemical modification in a subset of patients.

Natural selection and genetic diversity of domain I of Plasmodium falciparum apical membrane antigen-1 on Bioko Island.

BACKGROUND: Plasmodium falciparum apical membrane antigen-1 (PfAMA-1) is a promising candidate antigen for a blood-stage malaria vaccine. However, antigenic variation and diversity of PfAMA-1 are still major problems to design a universal malaria vaccine based on this antigen, especially against domain I (DI). Detail understanding of the PfAMA-1 gene polymorphism can provide useful information on this potential vaccine component. Here, general characteristics of genetic structure and the effect of natural selection of DIs among Bioko P. falciparum isolates were analysed. METHODS: 214 blood samples were collected from Bioko Island patients with P. falciparum malaria between 2011 and 2017. A fragment spanning DI of PfAMA-1 was amplified by nested polymerase chain reaction and sequenced. Polymorphic characteristics and the effect of natural selection were analysed using MEGA 5.0, DnaSP 6.0 and Popart programs. Genetic diversity in 576 global PfAMA-1 DIs were also analysed. Protein function prediction of new amino acid mutation sites was performed using PolyPhen-2 program. RESULTS: 131 different haplotypes of PfAMA-1 were identified in 214 Bioko Island P. falciparum isolates. Most amino acid changes identified on Bioko Island were found in C1L. 32 amino acid changes identified in PfAMA-1 sequences from Bioko Island were found in predicted RBC-binding sites, B cell epitopes or IUR regions. Overall patterns of amino acid changes of Bioko PfAMA-1 DIs were similar to those in global PfAMA-1 isolates. Differential amino acid substitution frequencies were observed for samples from different geographical regions. Eight new amino acid changes of Bioko island isolates were also identified and their three-dimensional protein structural consequences were predicted. Evidence for natural selection and recombination event were observed in global isolates. CONCLUSIONS: Patterns of nucleotide diversity and amino acid polymorphisms of Bioko Island isolates were similar to those of global PfAMA-1 DIs. Balancing natural selection across DIs might play a major role in generating genetic diversity in global isolates. Most amino acid changes in DIs occurred in predicted B-cell epitopes. Novel sites mapped on a three dimensional structure of PfAMA-1 showed that these regions were located at the corner. These results may provide significant value in the design of a malaria vaccine based on this antigen.

Recombinant Human Serum Albumin Containing 3 Copies of Domain I, Has Significant in Vitro Antioxidative Capacity Compared to the Wild-Type.

Human serum albumin (HSA), the most abundant protein in serum, functions as carrier of drugs and contributes to maintaining serum colloid osmotic pressure. We report herein on the preparation of a genetic recombinant HSA, in which domains II and III were changed to domain I (triple domain I; TDI). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) results indicated that the purity of the TDI was equivalent to that of the wild type (WT). Both far- and near-UV circular dichroism (CD) spectra of the TDI showed that its structural characteristics were similar to the WT. Ligand binding capacity was examined by an ultrafiltration method using 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF) and ketoprofen as markers for site I and site II, respectively. The binding capacity of TDI for both ligands was lower than that for the wild type. TDI significantly suppressed the oxidation of dihydrorhodamine 123 (DRD) by H2O2 compared to the WT. Our current results suggest that TDI has great potential for further development as HSA a product having antioxidative functions.

Arginine 112 is involved in HCV translation modulation by NS5A domain I.

HCV NS5A has three domains, which have multiple roles in the viral life cycle. We previously found that NS5A is able to down-regulate HCV RNA translation through a mechanism requiring the polyU/UC region within the viral 3'UTR to which NS5A binds. In this study, we further investigated the role of domain I in modulating viral translation. Using a series of deletion and substitution mutants, we identified a number of positively charged residues that played a role in this modulatory effect, most prominently R112. The R112A mutation negated the ability of domain I and full-length NS5A to modulate viral translation. Additionally, the R112A mutation impeded domain I binding to the polyU/UC RNA, suggesting a mechanism for this down-regulatory effect. Finally, the R112A mutation rendered HCV replication deficient. These results collectively point to a crucial role for the R112 residue of NS5A in the modulation of HCV life cycle by NS5A.

Proof-of-concept study demonstrating the pathogenicity of affinity-purified IgG antibodies directed to domain I of ß2-glycoprotein I in a mouse model of anti-phospholipid antibody-induced thrombosis.

OBJECTIVE: IgG aPL against domain I of ß2-glycoprotein I (ß2GPI) [anti-DI (aDI)] is associated with the pathogenesis of APS, an autoimmune disease defined by thrombosis and pregnancy morbidity. To date, however, no study has demonstrated direct pathogenicity of IgG aDI in vivo. In this proof-of-concept study, we designed a novel system to affinity purify polyclonal aDI aPL in order to assess its prothrombotic ability in a well-characterized mouse microcirculation model for APS. METHODS: Two polyclonal IgG fractions were isolated from serum of a patient with APS, both with high aPL activity but differing in aDI activity (aDI-rich and aDI-poor). These IgG fractions were tested for their pathogenic ability in an in vivo mouse model of thrombosis. Male CD1 mice were injected intraperitoneally with either aDI-rich or aDI-poor IgG; as a control, IgG isolated from healthy serum was used. A pinch injury was applied to the right femoral vein and thrombus dynamics and tissue factor activity in isolated tissue were evaluated. RESULTS: Both aDI-rich and aDI-poor IgG retained aCL and anti-ß2GPI activity, while only aDI-rich IgG displayed high aDI activity, as defined by our in-house cut-offs for positivity in each assay. aDI-rich IgG induced significantly larger thrombi in vivo compared with aDI-poor IgG (P < 0.0001). Similarly, aDI-rich IgG significantly enhanced the procoagulant activity of carotid artery endothelium and peritoneal macrophages isolated from experimental animals (P < 0.01). CONCLUSION: These data directly demonstrate that the ability to cause thrombosis in vivo is concentrated in the aDI fraction of aPL.

Positron emission tomography/computed tomography and biomarkers for early treatment response evaluation in metastatic colon cancer.

BACKGROUND: Treatment options for metastatic colon cancer (mCC) are widening. We prospectively evaluated serial 2-deoxy-2-[18F]fluoro-d-glucose positron-emission tomography/computed tomography (PET/CT) and measurements of tissue inhibitor of metalloproteinases-1 (TIMP-1), carcinoembryonic antigen (CEA), and liberated domain I of urokinase plasminogen activator receptor (uPAR(I)) for early assessment of treatment response in mCC patients. METHODS: Thirty-three mCC patients scheduled for first-line chemotherapy with capecitabine and oxaliplatin (CAPOX) and bevacizumab participated; 27 were evaluated by PET/CT before treatment, after one and four treatment series. Morphological and metabolic response was independently assessed according to Response Evaluation Criteria in Solid Tumors and European Organization for Research and Treatment of Cancer PET criteria. Plasma TIMP-1, plasma uPAR(I), and serum CEA were determined. RESULTS: Metabolic response after one treatment course predicted the ability of CAPOX and bevacizumab to induce morphological response after four treatment series with a sensitivity of 80%, specificity of 69%, and odds ratio of 13.9 (95% confidence interval [CI] 1.9; 182). Early metabolically stable or progressive disease was associated with shorter progression-free survival (hazard ratio [HR] = 3.2 [CI 1.3; 7.8]). Biomarker levels at early evaluation were associated with shorter OS (TIMP-1 per unit increase on a log-2-transformed ng/mL scale: HR = 2.6 [CI 1.4; 4.9]; uPAR(I) per 25 fmol/mL increase: HR = 1.5 [CI 1.1; 2.1]). CONCLUSION: This monocentric study demonstrated predictive value of early metabolic PET response and prognostic value of TIMP-1 and uPAR(I) levels in mCC treated with CAPOX and bevacizumab. Results support investigation of PET/CT, TIMP-1, and uPAR(I) guided early treatment adaptation in mCC.

Tolerogenic dendritic cells specific for ß2-glycoprotein-I Domain-I, attenuate experimental antiphospholipid syndrome.

Tolerogenic dendritic cells (tDCs) have the potential to control the outcome of autoimmunity by modulating the immune response. The aim of this study was to uncover the tolerance efficacy attributed to beta-2-glycoprotein-I (ß2GPI) tDCs or ß2GPI domain-I (D-I) and domain-V (D-V)-tDCs in mice with antiphospholipid syndrome (APS). tDCs were pulsed with ß2GPI or D-I or D-V derivatives. Our results revealed that ß2GPI related tDCs phenotype includes CD80(high), CD86(high) CD40(high) MHC class II(high). The miRNA profiling encompass miRNA 23b(high), miRNA 142-3p(low) and miRNA 221(low). In addition the ß2GPI related tDCs showed reduced secretion of IL-1ß, IL-12 and IL-23. D-I tDCs treatment was more efficient than ß2GPI tDCs in inducing of tolerance in APS mice, manifested by lowered titers of anti- ß2GPI antibodies (Abs) and reduced percentage of fetal loss. Tolerance induction was accompanied by poor T cell response to ß2GPI, high numbers of CD4 + CD25 + FOXP3 + T-regulatory cells (Treg), reduced levels of IFNγ, IL-17 and increased expression of IL-10 and TGFß. Tolerance was successfully transferred by Treg cells from the tolerized mice to ß2GPI immunized mice. We conclude that predominantly D-I-tDCs and ß2GPI tDCs have the potential to attenuate experimental APS by induction of Treg cells, reduction of anti- ß2GPI Abs titers and increased expression of anti-inflammatory cytokines. We suggest that ß2-GPI-D-I-tDCs may offer a novel approach for developing therapy for APS patients.

Domain I: the hidden face of antiphospholipid syndrome.

IgG antiphospholipid antibodies (aPL) against ß2-glycoprotein I (ß2GPI) can target any of its five domains; however, aPL against the N-terminal domain (anti-DI, aDI) are considered the most clinically relevant in the antiphospholipid syndrome (APS). Circulating levels of aDI are elevated in patients with APS compared with disease and healthy controls, and crucially aDI are prothrombotic in in vivo and in vitro models. In addition, human recombinant DI has been shown to abrogate aPL-induced thrombosis in vivo. Therefore, although the potential of utilizing DI for management of APS is not yet fully defined, there is promise that DI could prove valuable both as a diagnostic and therapeutic tool.

Laboratory Testing for Non-criteria Antiphospholipid Antibodies: Antibodies Toward the Domain I of Beta2-Glycoprotein I (aDI).

Anti-ß2GPI antibodies (aß2GPI) are one of the laboratory criteria for antiphospholipid syndrome (APS), along with lupus anticoagulant (LA) and anticardiolipin antibodies (aCL). A subset of the aß2GPI are the antibodies directed toward the domain I of the ß2GPI (aDI). The aDI are regarded as non-criteria aPL and are among the most studied non-criteria aPL. Antibodies directed against a specific epitope in the domain I (G40-R43) of ß2GPI were shown to be strongly correlated with thrombotic and obstetric events in APS. Many studies illustrated the pathogenic capacity of these antibodies, although with various results, depending on the assay used. The first studies were performed with an in-house ELISA with high specificity for aDI toward the G40-R43 epitope. More recently, a commercial chemiluminescence immunoassay for aDI IgG became obtainable for diagnostic laboratories. Although the added value of aDI on top of the criteria aPL is not clear, with opposing findings in literature, the assay might help in the diagnosis of APS, identifying the patients at risk since aDI are frequently present with high titers in triple-positive patients (positive for LA, aß2GPI, and aCL). aDI can be used as a confirmatory test and is useful for proving the specificity of the aß2GPI antibodies. In this chapter, the procedure for detecting these antibodies is outlined, using an automated chemiluminescence assay which can be used to determine the presence of IgG aDI in human samples. General guidelines that will facilitate optimal performance of the aDI assay are also provided.

The Role of Ribosomal Proteins eL15 and eL36 in the Early Steps of Yeast 60S Ribosomal Subunit Assembly.

Ribosomal proteins have important roles in maintaining the structure and function of mature ribosomes, but they also drive crucial rearrangement reactions during ribosome biogenesis. The contribution of most, but not all, ribosomal proteins to ribosome synthesis has been previously analyzed in the yeast Saccharomyces cerevisiae. Herein, we characterize the role of yeast eL15 during 60S ribosomal subunit formation. In vivo depletion of eL15 results in a shortage of 60S subunits and the appearance of half-mer polysomes. This is likely due to defective processing of the 27SA3 to the 27SBS pre-rRNA and impaired subsequent processing of both forms of 27SB pre-rRNAs to mature 25S and 5.8S rRNAs. Indeed, eL15 depletion leads to the efficient turnover of the de novo formed 27S pre-rRNAs. Additionally, depletion of eL15 blocks nucleocytoplasmic export of pre-60S particles. Moreover, we have analyzed the impact of depleting either eL15 or eL36 on the composition of early pre-60S particles, thereby revealing that the depletion of eL15 or eL36 not only affects each other's assembly into pre-60S particles but also that of neighboring ribosomal proteins, including eL8. These intermediates also lack most ribosome assembly factors required for 27SA3 and 27SB pre-rRNA processing, named A3- and B-factors, respectively. Importantly, our results recapitulate previous ones obtained upon eL8 depletion. We conclude that assembly of eL15, together with that of eL8 and eL36, is a prerequisite to shape domain I of 5.8S/25S rRNA within early pre-60S particles, through their binding to this rRNA domain and the recruitment of specific groups of assembly factors.

PEGylated Domain I of Beta-2-Glycoprotein I Inhibits Thrombosis in a Chronic Mouse Model of the Antiphospholipid Syndrome.

Antiphospholipid syndrome (APS) is an autoimmune disorder in which autoantibodies cause clinical effects of vascular thrombosis and pregnancy morbidity. The only evidence-based treatments are anticoagulant medications such as warfarin and heparin. These medications have a number of disadvantages, notably risk of haemorrhage. Therefore, there is a pressing need to develop new, more focused treatments that target the actual pathogenic disease process in APS. The pathogenic antibodies exert their effects by interacting with phospholipid-binding proteins, of which the most important is beta-2-glycoprotein I. This protein has five domains, of which the N-terminal Domain I (DI) is the main site for binding of pathogenic autoantibodies. We previously demonstrated bacterial expression of human DI and showed that this product could inhibit the ability of IgG from patients with APS (APS-IgG) to promote thrombosis in a mouse model. Since DI is a small 7kDa protein, its serum half-life would be too short to be therapeutically useful. We therefore used site-specific chemical addition of polyethylene glycol (PEG) to produce a larger variant of DI (PEG-DI) and showed that PEG-DI was equally effective as the non-PEGylated DI in inhibiting thrombosis caused by passive transfer of APS-IgG in mice. In this paper, we have used a mouse model that reflects human APS much more closely than the passive transfer of APS-IgG. In this model, the mice are immunized with human beta-2-glycoprotein I and develop endogenous anti-beta-2-glycoprotein I antibodies. When submitted to a pinch stimulus at the femoral vein, these mice develop clots. Our results show that PEG-DI inhibits production of thromboses in this model and also reduces expression of tissue factor in the aortas of the mice. No toxicity was seen in mice that received PEG-DI. Therefore, these results provide further evidence supporting possible efficacy of PEG-DI as a potential treatment for APS.

Perlecan, A Multi-Functional, Cell-Instructive, Matrix-Stabilizing Proteoglycan With Roles in Tissue Development Has Relevance to Connective Tissue Repair and Regeneration.

This review highlights the multifunctional properties of perlecan (HSPG2) and its potential roles in repair biology. Perlecan is ubiquitous, occurring in vascular, cartilaginous, adipose, lymphoreticular, bone and bone marrow stroma and in neural tissues. Perlecan has roles in angiogenesis, tissue development and extracellular matrix stabilization in mature weight bearing and tensional tissues. Perlecan contributes to mechanosensory properties in cartilage through pericellular interactions with fibrillin-1, type IV, V, VI and XI collagen and elastin. Perlecan domain I - FGF, PDGF, VEGF and BMP interactions promote embryonic cellular proliferation, differentiation, and tissue development. Perlecan domain II, an LDLR-like domain interacts with lipids, Wnt and Hedgehog morphogens. Perlecan domain III binds FGF-7 and 18 and has roles in the secretion of perlecan. Perlecan domain IV, an immunoglobulin repeat domain, has cell attachment and matrix stabilizing properties. Perlecan domain V promotes tissue repair through interactions with VEGF, VEGF-R2 and α2ß1 integrin. Perlecan domain-V LG1-LG2 and LG3 fragments antagonize these interactions. Perlecan domain V promotes reconstitution of the blood brain barrier damaged by ischemic stroke and is neurogenic and neuroprotective. Perlecan-VEGF-VEGFR2, perlecan-FGF-2 and perlecan-PDGF interactions promote angiogenesis and wound healing. Perlecan domain I, III and V interactions with platelet factor-4 and megakaryocyte and platelet inhibitory receptor promote adhesion of cells to implants and scaffolds in vascular repair. Perlecan localizes acetylcholinesterase in the neuromuscular junction and is of functional significance in neuromuscular control. Perlecan mutation leads to Schwartz-Jampel Syndrome, functional impairment of the biomechanical properties of the intervertebral disc, variable levels of chondroplasia and myotonia. A greater understanding of the functional working of the neuromuscular junction may be insightful in therapeutic approaches in the treatment of neuromuscular disorders. Tissue engineering of salivary glands has been undertaken using bioactive peptides (TWSKV) derived from perlecan domain IV. Perlecan TWSKV peptide induces differentiation of salivary gland cells into self-assembling acini-like structures that express salivary gland biomarkers and secrete α-amylase. Perlecan also promotes chondroprogenitor stem cell maturation and development of pluripotent migratory stem cell lineages, which participate in diarthrodial joint formation, and early cartilage development. Recent studies have also shown that perlecan is prominently expressed during repair of adult human articular cartilage. Perlecan also has roles in endochondral ossification and bone development. Perlecan domain I hydrogels been used in tissue engineering to establish heparin binding growth factor gradients that promote cell migration and cartilage repair. Perlecan domain I collagen I fibril scaffolds have also been used as an FGF-2 delivery system for tissue repair. With the availability of recombinant perlecan domains, the development of other tissue repair strategies should emerge in the near future. Perlecan co-localization with vascular elastin in the intima, acts as a blood shear-flow endothelial sensor that regulates blood volume and pressure and has a similar role to perlecan in canalicular fluid, regulating bone development and remodeling. This complements perlecan's roles in growth plate cartilage and in endochondral ossification to form the appendicular and axial skeleton. Perlecan is thus a ubiquitous, multifunctional, and pleomorphic molecule of considerable biological importance. A greater understanding of its diverse biological roles and functional repertoires during tissue development, growth and disease will yield valuable insights into how this impressive proteoglycan could be utilized successfully in repair biology.

Detection of anti-domain I antibodies by chemiluminescence enables the identification of high-risk antiphospholipid syndrome patients: A multicenter multiplatform study.

BACKGROUND: Classification of the antiphospholipid syndrome (APS) relies predominantly on detecting antiphospholipid antibodies (aPLs). Antibodies against a domain I (DI) epitope of anti-ß2glycoprotein I (ß2GPI) proved to be pathogenic, but are not included in the current classification criteria. OBJECTIVES: Investigate the clinical value of detecting anti-DI IgG in APS. PATIENTS/METHODS: From eight European centers 1005 patients were enrolled. Anti-cardiolipin (CL) and anti-ß2GPI were detected by four commercially available solid phase assays; anti-DI IgG by the QUANTA Flash® ß2GPI domain I assay. RESULTS: Odds ratios (ORs) of anti-DI IgG for thrombosis and pregnancy morbidity proved to be higher than those of the conventional assays. Upon restriction to patients positive for anti-ß2GPI IgG, anti-DI IgG positivity still resulted in significant ORs. When anti-DI IgG was added to the criteria aPLs or used as a substitute for anti-ß2GPI IgG/anti-CL IgG, ORs for clinical symptoms hardly improved. Upon removing anti-DI positive patients, lupus anticoagulant remained significantly correlated with clinical complications. Anti-DI IgG are mainly present in high-risk triple positive patients, showing higher levels. Combined anti-DI and triple positivity confers a higher risk for clinical symptoms compared to only triple positivity. CONCLUSIONS: Detection of anti-DI IgG resulted in higher ORs for clinical manifestations than the current APS classification criteria. Regardless of the platform used to detect anti-ß2GPI/anti-CL, addition of anti-DI IgG measured by QUANTA Flash® did not improve the clinical associations, possibly due to reduced exposure of the pathogenic epitope of DI. Our results demonstrate that anti-DI IgG potentially helps in identifying high-risk patients.

The prevalence of antiphospholipid antibodies in women with late pregnancy complications and low-risk for chromosomal abnormalities.

BACKGROUND: Antiphospholipid antibodies (aPL) are known to increase the risk of obstetrical complications. However, aPL significance and prevalence in women with late-onset pregnancy complications (LO-PC) need further clarification. OBJECTIVES: To investigate the prevalence of aPL in a cohort of women who experienced LO-PC and to compare it with a cohort of uneventful pregnancies. METHODS: One hundred pregnant women who experienced LO-PC, had a low risk for chromosomal abnormalities, and absence of fetal abnormalities were recruited from August 2018 to August 2019. One hundred women with uneventful pregnancy were included as controls. aPL testing was performed on serum samples derived from prenatal screening test and included both criteria and "extra criteria" aPL. RESULTS: Patients with LO-PC had significantly higher aPL prevalence when compared with controls (31/100 [31%] vs 10/100 [10%]; P < .001). More in detail, up to 26% of women with LO-PC were positive for one aPL, with an overall prevalence significantly higher than controls (26% vs 9%; P < .05). Among single aPL positivity, patients had significantly higher rate of positivity and titers of anticardiolipin IgG (10% vs 2%; mean ± standard deviation 11 ± 13 vs 4 ± 9.6 chemoluminescent unit; P < .05) and phosphatidylserine-prothrombin antibodies (aPS/PT) IgM (15% vs 6%; mean ± standard deviation 10.2 ± 21.7 vs 3.7 ± 13.7 U; P < .05). Testing for aPS/PT (IgM/IgG) alone allowed the identification of 17 patients negative for criteria aPL. aPL-positive patients had a significantly higher risk of preterm birth (34-36 + 6 weeks; 10% vs 8%; P < .012). CONCLUSIONS: We report a high prevalence of aPL in our cohort. Testing for both criteria and "extra criteria" aPL in women with previous LO-PC could improve the diagnostic accuracy identifying women at higher risk for recurrent pregnancy complications.