Domain 1 Research Articles

Structural basis for mouse LAG3 interactions with the MHC class II molecule I-Ab

The immune checkpoint protein, Lymphocyte activation gene-3 (LAG3), binds Major Histocompatibility Complex Class II (MHC-II) and suppresses T cell activation. Despite the recent FDA approval of a LAG3 inhibitor for the treatment of melanoma, how LAG3 engages MHC-II on the cell surface remains poorly understood. Here, we determine the 3.84 Å-resolution structure of mouse LAG3 bound to the MHC-II molecule I-Ab, revealing that domain 1 (D1) of LAG3 binds a conserved, membrane-proximal region of MHC-II spanning both the α2 and β2 subdomains. LAG3 dimerization restricts the intermolecular spacing of MHC-II molecules, which may attenuate T cell activation by enforcing suboptimal signaling geometry. The LAG3-MHC-II interface overlaps with the MHC-II-binding site of the T cell coreceptor CD4, implicating disruption of CD4-MHC-II interactions as a mechanism for LAG3 immunosuppressive function. Lastly, antibody epitope analysis indicates that multiple LAG3 inhibitors do not recognize the MHC-II-binding interface of LAG3, suggesting a role for functionally distinct mechanisms of LAG3 antagonism in therapeutic development.

The heparin-binding domain of VEGF165 directly binds to integrin αvβ3 and VEGFR2/KDR D1: a potential mechanism of negative regulation of VEGF165 signaling by αvβ3.

VEGF-A is a key cytokine in tumor angiogenesis and a major therapeutic target for cancer. VEGF165 is the predominant isoform of VEGF-A, and it is the most potent angiogenesis stimulant. VEGFR2/KDR domains 2 and 3 (D2D3) bind to the N-terminal domain (NTD, residues 1-110) of VEGF165. Since removal of the heparin-binding domain (HBD, residues 111-165) markedly reduced the mitogenic activity of the growth factor, it has been proposed that the HBD plays a critical role in the mitogenicity of VEGF165. Here, we report that αvβ3 specifically bound to the isolated VEGF165 HBD but not to VEGF165 NTD. Based on docking simulation and mutagenesis, we identified several critical amino acid residues within the VEGF165 HBD required for αvβ3 binding, i.e., Arg123, Arg124, Lys125, Lys140, Arg145, and Arg149. We discovered that VEGF165 HBD binds to the KDR domain 1 (D1) and identified that Arg123 and Arg124 are critical for KDR D1 binding by mutagenesis, indicating that the KDR D1-binding and αvβ3-binding sites overlap in the HBD. Full-length VEGF165 mutant (R123A/R124A/K125A/K140A/R145A/R149A) defective in αvβ3 and KDR D1 binding failed to induce ERK1/2 phosphorylation, integrin β3 phosphorylation, and KDR phosphorylation and did not support proliferation of endothelial cells, although the mutation did not affect the KDR D2D3 interaction with VEGF165. Since β3-knockout mice are known to show enhanced VEGF165 signaling, we propose that the binding of KDR D1 to the VEGF165 HBD and KDR D2D3 binding to the VEGF165 NTD are critically involved in the potent mitogenicity of VEGF165. We propose that binding competition between KDR and αvβ3 to the VEGF165 HBD endows integrin αvβ3 with regulatory properties to act as a negative regulator of VEGF165 signaling.

Structural insights reveal interplay between LAG-3 homodimerization, ligand binding, and function

Lymphocyte activation gene-3 (LAG-3) is an inhibitory receptor expressed on activated T cells and an emerging immunotherapy target. Domain 1 (D1) of LAG-3, which has been purported to directly interact with major histocompatibility complex class II (MHCII) and fibrinogen-like protein 1 (FGL1), has been the major focus for the development of therapeutic antibodies that inhibit LAG-3 receptor-ligand interactions and restore T cell function. Here, we present a high-resolution structure of glycosylated mouse LAG-3 ectodomain, identifying that cis-homodimerization, mediated through a network of hydrophobic residues within domain 2 (D2), is critically required for LAG-3 function. Additionally, we found a previously unidentified key protein-glycan interaction in the dimer interface that affects the spatial orientation of the neighboring D1 domain. Mutation of LAG-3 D2 residues reduced dimer formation, dramatically abolished LAG-3 binding to both MHCII and FGL1 ligands, and consequentially inhibited the role of LAG-3 in suppressing T cell responses. Intriguingly, we showed that antibodies directed against D1, D2, and D3 domains are all capable of blocking LAG-3 dimer formation and MHCII and FGL-1 ligand binding, suggesting a potential allosteric model of LAG-3 function tightly regulated by dimerization. Furthermore, our work reveals unique epitopes, in addition to D1, that can be targeted for immunotherapy of cancer and other human diseases.

Development of a Safe Driver’s Attitude Intervention Module Based on the SaringSikap Screening Method

The human factors are consistently the leading cause of road crashes in Malaysia and worldwide. This study would like to support the government's effort to reduce road crashes by proposing a comprehensive driver's intervention module based on the SaringSikap/ASDS46 screening method. A total of 240 volunteers had undergone the screening process. Domain 1 (D1) scored the highest high-risk attitude score, and Domain 2 (D2) was the lowest. This intervention will benefit all groups of road users in Malaysia. It also will assist the government in reducing road crashes in the future through education and empowerment programs.

Variation in the affinity of three representative avian adenoviruses for the cellular coxsackievirus and adenovirus receptor.

According to previous studies, three representative avian adenoviral strains utilize coxsackievirus-adenovirus receptor (CAR) as a receptor and seem to exhibit diverse binding affinities and modes. Thus, further revealing the exact molecular mechanism underlying the interaction between different FAdVs and the attachment receptor CAR is necessary. In this study, we successfully solved the crystal structure of the FAdV-4 fiber1 knob at 1.6 Å resolution. The interaction between the fibre knob and different domains of CAR was verified by confocal microscopy, coimmunoprecipitation and surface plasmon resonance (SPR) analysis. The fibre knobs of the three representative fowl adenoviruses specifically recognized CAR domain 1 (D1), but the recognition of CAR domain 2 (D2) by chicken embryo lethal orphan (CELO) strains was weak. These results provide insights into the differences in adenovirus‒host cell interactions and have important implications for the exploration of viral invasion mechanisms.

A potent and durable malaria transmission-blocking vaccine designed from a single-component 60-copy Pfs230D1 nanoparticle

Malaria transmission-blocking vaccines (TBVs) reduce disease transmission by breaking the continuous cycle of infection between the human host and the mosquito vector. Domain 1 (D1) of Pfs230 is a leading TBV candidate and comprises the majority of transmission-reducing activity (TRA) elicited by Pfs230. Here we show that the fusion of Pfs230D1 to a 60-copy multimer of the catalytic domain of dihydrolipoyl acetyltransferase protein (E2p) results in a single-component nanoparticle composed of 60 copies of the fusion protein with high stability, homogeneity, and production yields. The nanoparticle presents a potent human transmission-blocking epitope within Pfs230D1, indicating the antigen is correctly oriented on the surface of the nanoparticle. Two vaccinations of New Zealand White rabbits with the Pfs230D1 nanoparticle elicited a potent and durable antibody response with high TRA when formulated in two distinct adjuvants suitable for translation to human use. This single-component nanoparticle vaccine may play a key role in malaria control and has the potential to improve production pipelines and the cost of manufacturing of a potent and durable TBV.

Pfs47 as a Malaria Transmission-Blocking Vaccine Target.

Transmission-blocking vaccines (TBVs), pioneered by Richard Carter and others, aim to prevent parasite development in the mosquito vector and are a promising new tool for malaria elimination. Pfs47, recently identified as a TBV target, is a three-domain 6-cysteine protein on the surface of Plasmodium falciparum sexual stages. Pfs47 allows the parasite to evade mosquito immunity and is key for P. falciparum infection of the dominant malaria vectors Anopheles gambiae, Anopheles dirus, and Anopheles albimanus. Antibodies against Pfs47 domain 2 (D2) have significant transmission-blocking activity that prevents Plasmodium ookinete development and is independent of human complement. Strong transmission-blocking activity has been mapped to a region of 52 amino acids in Pfs47 D2. Efforts to optimize the immunogenicity of the Pfs47 D2 antigen with a viral-like particle have been successful, and the efficacy of a P47-based TBV was confirmed invivo with Pbs47, the orthologue of Pfs47 in the mouse malaria parasite Plasmodium berghei. The current evidence warrants further development and clinical testing of a Pfs47-based TBV.

An anti-picornaviral strategy based on the crystal structure of foot-and-mouth disease virus 2C protein.

The foot-and-mouth disease virus (FMDV) 2C protein shares conserved motifs with enterovirus 2Cs despite low sequence identity. Here, we determine the crystal structure of an FMDV 2C fragment to 1.83Å resolution, which comprises an ATPase domain, a region equivalent to the enterovirus 2C zinc-finger (ZFER), and a C-terminal domain harboring a loop (PBL) that occupies a hydrophobic cleft (Pocket) in an adjacent 2C molecule. Mutations at ZFER, PBL, and Pocket affect FMDV 2C ATPase activity and are lethal to FMDV infectious clones. Because the PBL-Pocket interaction between FMDV 2C molecules is essential for its functions, we design an anti-FMDV peptide derived from PBL (PBL-peptide). PBL-peptide inhibits FMDV 2C ATPase activity, binds FMDV 2C with nanomolar affinity, and disrupts FMDV 2C oligomerization. FMDV 2C targets lipid droplets (LDs) and induces LD clustering in cells, and PBL-peptide disrupts FMDV 2C-induced LD clustering. Finally, we demonstrate that PBL-peptide exhibits anti-FMDV activity in cells.

Systematic review search methods evaluated using the Preferred Reporting of Items for Systematic Reviews and Meta-Analyses and the Risk Of Bias In Systematic reviews tool.

To evaluate the methodological and reporting characteristics of search methods of systematic reviews (SRs) using the Preferred Reporting of Systematic Reviews and Meta-Analyses (PRISMA) checklist and the Risk Of Bias In Systematic reviews (ROBIS) tool. A sample of 505 SRs published in 2016 was taken from KSR Evidence, a database of SRs, and analyzed to assess compliance with Information sources and Search of the PRISMA checklist. Domain 2 (D2) (Identification and Selection of Studies) of the ROBIS tool was used to judge the risk of bias in search methods. Regarding Information sources and Search of PRISMA, twenty percent of SRs which claimed to be PRISMA-compliant in their methods, were compliant; twenty-four percent of SRs published in journals that require PRISMA reporting were compliant; nineteen percent in total were found to be compliant. Twenty-eight percent of SRs were judged to be at a low risk of bias in D2 and so searched widely with an effective strategy and, finally, ten percent were both compliant with the reporting of Information sources and with Search of PRISMA and were judged to be at a low risk of bias in D2. Ninety percent of SRs are failing to report search methods adequately and to conduct comprehensive searches using a wide range of resources. Editors of journals and peer reviewers need to ensure that they understand the requirements of PRISMA and that compliance is adhered to. Additionally, the comprehensiveness of search methods for SRs needs to be given more critical consideration.

Anti-domain 1 \u03b22 glycoprotein antibodies increase expression of tissue factor on monocytes and activate NK Cells and CD8+ cells in vitro

Backgroundβ2-Glycoprotein I (β2GPI) represents the major antigenic target for antiphospholipid antibodies (aPL), with domain 1 (D1) being identified as a risk factor for thrombosis and pregnancy complications in APS. We aimed to analyse the ability of aPL, and particularly anti-D1 β2GPI, to stimulate prothrombotic and proinflammatory activity of immune cells in vitro.MethodsPeripheral blood mononuclear cells (PBMCs) from 11 healthy individuals were incubated with: (1) “anti-D1(+)”—pooled plasma derived from patients suspected of having APS contained anticardiolipin antibodies (aCL), lupus anticoagulant (LA), anti-β2GPI and anti-D1 β2GPI; (2) “anti-D1(−)”—pooled plasma from patients suspected of having APS contained aCL, LA, anti-β2GPI, and negative for anti-D1 β2GPI; (3) “seronegative”—negative for aPL.ResultsThe presence of anti-D1(+) and anti-D1(−) plasma resulted in increased HLA-DR and CD11b on monocytes. While only anti-D1(+) plasma markedly increased the percentage and median fluorescence intensity (MFI) of CD142 (tissue factor, TF) on monocytes in comparison with those cultured with anti-D1(−) and seronegative plasma. Anti-D1(+) plasma resulted in increased percentage and MFI of activation marker CD69 on NK and T cytotoxic cells. Expression of IgG receptor FcγRIII(CD16) on monocytes and NK cells was down-regulated by the anti-D1(+) plasma.ConclusionsTaking together, our study shows the ability of patient-derived aPL to induce immune cell activation and TF expression on monocytes. For the first time, we demonstrated the influence of anti-D1 β2GPI on the activation status of monocytes, NK and cytotoxic T cells. Our findings further support a crucial role of D1 epitope in the promotion of thrombosis and obstetrical complications in APS.

Anti-beta-2-glycoprotein I domain 1 identifies antiphospholipid antibodies-related injuries in patients with concomitant lupus nephritis.

In this study we aimed to evaluate the usefulness of domain profiling of Beta-2-glycoprotein I(β2GPI)-Domain-1 (D1)antibodies in relation to antiphospholipid antibodies (aPL)-related nephropathy (aPL-N) in patients with biopsy-proven lupus nephritis (LN). Of 124 consecutive patients (96 women, mean age 45.5 ± 12.3years, mean disease duration 14.7 ± 9.6years) fulfilling the 1982 criteria for systemic lupus erythematosus (SLE), we identified 39 patients (mean age 39.84 ± 8.6years, mean disease duration 11.3 ± 7.7years) with the following characteristics: (a) biopsy-proven LN; (b) no previous diagnosis of antiphospholipid syndrome (APS) according to the current classification criteria. Patients with both LN and aPL-N had higher median aβ2GPI-D1 antibody titres (220.1 CU, 25-75th IQ 29.1-334.2) as compared those with LN alone (46.5 CU, 25-75th IQ 12.5-75.1) (p = 0.0087). Median aβ2GPI-D1 antibody titres were higher in patients with acute thrombotic microangiopathy(aTMA) (N = 7) (250.1 CU, 25-75th IQ 61.2-334.2) vs. with LN alone (46.5 CU, 25-75th IQ 12.5-75.1 CU) (p = 0.0009). Having a Global Antiphospholipid SyndromeScore > 10 confers an increased probability of having acute features of aTMA (OR 6.25, 95%CI 1.2-31.8). As compared to other aPL, aβ2GPI-D1 antibodies have the best diagnostic accuracy for aTMA as evaluated by performances in Area Under the Curves in a ROC analysis. aβ2GPI-D1 antibodiesdetection might provide a second-line assay to be performed in aβ2GPI positive patients with LN, allowing more accurate stratification of the renal vascular involvement risk, thus potentially leading toa more tailored management.

The IgA Isotype of Anti-β2 Glycoprotein I Antibodies Recognizes Epitopes in Domains 3, 4, and 5 That Are Located in a Lateral Zone of the Molecule (L-Shaped).

Background: Antiphospholipid syndrome (APS) is characterized by thrombosis and/or pregnancy morbidity with presence of anti-phospholipid antibodies (aPL). The APS classification criteria only consider the aPL of IgG/IgM isotype, however testing of aPL of IgA isotype is recommended when APS is suspected and consensus aPL are negative. IgA anti-βeta-2 glycoprotein-I (B2GP1) has been clearly related with occurrence of thrombotic events. Antibodies anti-B2GP1 of IgG/M isotypes recognize an epitope in Domain 1 (R39-G43), the epitopes that recognize IgA anti-B2GP1 antibodies are not well-identified.Aim: To determine the zones of B2GP1 recognized by antibodies of IgA isotype from patients with APS symptomatology and positive for IgA anti-B2GP1.Methods: IgA antibodies to Domain-1(D1) and Domain-4/5(D4/5) of B2GP1 (ELISA) and epitope mapping on oligopeptide arrays of B2GP1 were evaluated in sera from a group of 93 patients with at least one thrombotic and with isolated positivity for IgA anti-B2GP1 antibodies (negative for other aPL).Results: A total of 47 patients (50.5%) were positive for anti-D4/5 and 23(25%) were positive for anti-D1. When peptide arrays were analyzed, three zones of B2GP1 reactivity were identified for more than 50% of patients. The center of these zones corresponds to amino acids 140(D3), 204(D4), and 264(D5). The peptides recognized on D3 and D4 contain amino acid sequences sharing high homology with proteins of microorganism that were previously related with a possible APS infectious etiology. In the three-dimensional structure of B2GP1, the three peptides, as the R39-G43 epitope, are located on the right side of the molecule (L-shape). The left side (J-shape) does not bind the antibodies.Conclusions: Patients with thrombotic APS clinical-criteria, and isolated IgA anti-B2GP1 positivity appear to preferentially bind, not to the D1 or D4/5 domains of B2GP1, but rather to three sites in D3, D4, and D5. The sites on D3 and D4 were previously described as the target identified by human monoclonal antibodies derived from patients that were capable of inducing APS in animal models. The localization of these epitopes opens a new route to explore to increase understanding of the patholophysiology of the APS and to propose new alternatives and therapeutic targets.

Antibody targeting of a specific region of Pfs47 blocks Plasmodium falciparum malaria transmission

Transmission-blocking vaccines are based on eliciting antibody responses in the vertebrate host that disrupt parasite development in the mosquito vector and prevent malaria transmission. The surface protein Pfs47 is present in Plasmodium falciparum gametocytes and female gametes. The potential of Pfs47 as a vaccine target was evaluated. Soluble full-length recombinant protein, consisting of three domains, was expressed in E. coli as a thioredoxin fusion (T-Pfs47). The protein was immunogenic, and polyclonal and monoclonal antibodies (mAb) were obtained, but they did not confer transmission blocking activity (TBA). All fourteen mAb targeted either domains 1 or 3, but not domain 2 (D2), and immune reactivity to D2 was also very low in polyclonal mouse IgG after T-Pfs47 immunization. Disruption of the predicted disulfide bond in D2, by replacing cysteines for alanines (C230A and C260A), allowed expression of recombinant D2 protein in E. coli. A combination of mAbs targeting D2, and deletion proteins from this domain, allowed us to map a central 52 amino acid (aa) region where antibody binding confers strong TBA (78-99%). This 52 aa antigen is immunogenic and well conserved, with only seven haplotypes world-wide that share 96–98% identity. Neither human complement nor the mosquito complement-like system are required for the observed TBA. A dramatic reduction in ookinete numbers and ookinete-specific transcripts was observed, suggesting that the antibodies are interacting with female gametocytes and preventing fertilization.

Anti-phospholipid IgG antibodies detected by line immunoassay differentiate patients with anti-phospholipid syndrome and other autoimmune diseases

PurposeAnti-phospholipid antibodies (aPL) analyzed by line immunoassay (LIA) can recognize beta2-glycoprotein I (β2GPI) domain 1 (D1) epitopes depending on β2GPI binding to distinct phospholipids. The aPL LIA was compared with consensus ELISA to investigate whether both techniques can discriminate anti-phospholipid syndrome (APS) patients from aPL-positive, systemic autoimmune rheumatic diseases (SARD) patients without clinical symptoms of APS and controls.MethodsThirty-four APS patients (14 arterial/venous thrombosis, 16 pregnancy morbidity, and 4 both), 41 patients with SARD lacking clinical APS criteria but demonstrating positivity for anti-β2GPI (aβ2GPI) IgG, and 20 healthy subjects (HS) were tested for aPL to cardiolipin (aCL), phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol (aPG), phosphatidylinositol, phosphatidylserine, β2GPI, prothrombin, and annexin V by LIA. Samples were also tested for aCL, aβ2GPI, aβ2GPI-domain 1 (aD1), and aβ2GPI-domains 4–5 (aD4–5) by ELISA and for lupus anti-coagulant.ResultsComparison of LIA with ELISA revealed a good agreement for the consensus criteria aPL aβ2GPI and aCL IgG (kappa = 0.69, 0.68, respectively) and a moderate agreement for IgM (kappa = 0.52, 0.49, respectively). Regarding ELISA, aD1/aD4–5 demonstrated the best performance of differentiating APS from asymptomatic SARD [area under the curve (AUC): 0.76]. aPG IgG had the best performance by LIA (AUC: 0.72) not significantly different from aD1/aD4–5. There was a good agreement for aPG IgG with aD1/aD4–5 (kappa = 0.71).ConclusionsaD1/aD4–5 (ELISA) and aPG IgG (LIA) differentiate APS from SARD patients. PG appears to interact with β2GPI of APS patients and exposes D1 thereof for disease-specific aPL binding in LIA.

Computational Design of Antiangiogenic Peptibody by Fusing Human IgG1 Fc Fragment and HRH Peptide: Structural Modeling, Energetic Analysis, and Dynamics Simulation of Its Binding Potency to VEGF Receptor.

Peptibodies represent a new class of biological therapeutics with combination of peptide activity and antibody-like properties. Previously, we discovered a novel peptide HRH that exhibited a dose-dependent angiogenesis-suppressing effect by targeting vascular endothelial growth factor receptors (VEGFRs). Here, we computationally designed an antiangiogenic peptibody, termed as PbHRH, by fusing the HRH peptide to human IgG1 Fc fragment using the first approved peptibody drug Romiplostim as template. The biologically active peptide of Romiplostim is similar with HRH peptide; both of them have close sequence lengths and can fold into a α-helical conformation in free state. Molecular dynamics simulations revealed that the HRH functional domain is highly flexible, which is functionally independent of Fc fragment in the designed PbHRH peptibody. Subsequently, the intermolecular interactions between VEGFR-1 domain 2 (D2) and PbHRH were predicted, clustered and refined into three representatives. Conformational analysis and energetic evaluation unraveled that the PbHRH can adopt multiple binding modes to block the native VEGF-A binding site of VEGFR-1 D2 with its HRH functional domain, although the binding effectiveness of HRH segments in peptibody context seems to be moderately decreased relative to that of free HRH peptide. Overall, it is suggested that integrating HRH peptide into PbHRH peptibody does not promote the direct intermolecular interaction between VEGFR-1 D2 and HRH. Instead, the peptibody may indirectly help to improve the pharmacokinetic profile and bioavailability of HRH.

Overall Structural Model of NS5A Protein from Hepatitis C Virus and Modulation by Mutations Confering Resistance of Virus Replication to Cyclosporin A.

Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is a RNA-binding phosphoprotein composed of a N-terminal membrane anchor (AH), a structured domain 1 (D1), and two intrinsically disordered domains (D2 and D3). The knowledge of the functional architecture of this multifunctional protein remains limited. We report here that NS5A-D1D2D3 produced in a wheat germ cell-free system is obtained under a highly phosphorylated state. Its NMR analysis revealed that these phosphorylations do not change the disordered nature of D2 and D3 domains but increase the number of conformers due to partial phosphorylations. By combining NMR and small angle X-ray scattering, we performed a comparative structural characterization of unphosphorylated recombinant D2 domains of JFH1 (genotype 2a) and the Con1 (genotype 1b) strains produced in Escherichia coli. These analyses highlighted a higher intrinsic folding of the latter, revealing the variability of intrinsic conformations in HCV genotypes. We also investigated the effect of D2 mutations conferring resistance of HCV replication to cyclophilin A (CypA) inhibitors on the structure of the recombinant D2 Con1 mutants and their binding to CypA. Although resistance mutations D320E and R318W could induce some local and/or global folding perturbation, which could thus affect the kinetics of conformer interconversions, they do not significantly affect the kinetics of CypA/D2 interaction measured by surface plasmon resonance (SPR). The combination of all our data led us to build a model of the overall structure of NS5A, which provides a useful template for further investigations of the structural and functional features of this enigmatic protein.

Characterization of Metarhizium viride Mycosis in Veiled Chameleons (Chamaeleo calyptratus), Panther Chameleons (Furcifer pardalis), and Inland Bearded Dragons (Pogona vitticeps)

Metarhizium viride has been associated with fatal systemic mycoses in chameleons, but subsequent data on mycoses caused by this fungus in reptiles are lacking. The aim of this investigation was therefore to obtain information on the presence of M. viride in reptiles kept as pets in captivity and its association with clinical signs and pathological findings as well as improvement of diagnostic procedures. Beside 18S ribosomal DNA (rDNA) (small subunit [SSU]) and internal transcribed spacer region 1 (ITS-1), a fragment of the large subunit (LSU) of 28S rDNA, including domain 1 (D1) and D2, was sequenced for the identification of the fungus and phylogenetic analysis. Cultural isolation and histopathological examinations as well as the pattern of antifungal drug resistance, determined by using agar diffusion testing, were additionally used for comparison of the isolates. In total, 20 isolates from eight inland bearded dragons (Pogona vitticeps), six veiled chameleons (Chamaeleo calyptratus), and six panther chameleons (Furcifer pardalis) were examined. Most of the lizards suffered from fungal glossitis, stomatitis, and pharyngitis or died due to visceral mycosis. Treatment with different antifungal drugs according to resistance patterns in all three different lizard species was unsuccessful. Sequence analysis resulted in four different genotypes of M. viride based on differences in the LSU fragment, whereas the SSU and ITS-1 were identical in all isolates. Sequence analysis of the SSU fragment revealed the first presentation of a valid large fragment of the SSU of M. viride According to statistical analysis, genotypes did not correlate with differences in pathogenicity, antifungal susceptibility, or species specificity.

High thioredoxin-1 levels in rheumatoid arthritis patients diminish binding and signalling of the monoclonal antibody Tregalizumab.

The humanized non-depleting anti-CD4 monoclonal antibody Tregalizumab (BT-061) is able to selectively activate the suppressive function of regulatory T cells and has been investigated up to phase IIb in clinical trials in patients suffering from rheumatoid arthritis (RA). A pharmacokinetic–pharmacodynamic model based on clinical data from RA and healthy volunteers, which used the cell surface CD4 downmodulation as marker of activity, confirmed a stronger effect in healthy volunteers compared with RA patients. We tried to understand this phenomenon and evaluated the influence of the small oxidoreductase thioredoxin-1 (Trx1). To counteract oxidative stress that is strongly associated with RA pathophysiology, the organism employs Trx1. Therefore, increased expression and secretion of Trx1 is found in the synovial fluid and plasma of RA patients. Moreover, the binding site of Tregalizumab is in close proximity to a disulphide bond in domain 2 (D2) of CD4, which is a known target for a reduction by oxidoreductase Trx1. With the experiments reported herein, we demonstrated that specific reduction of the D2 disulphide bond by Trx1 led to diminished binding of Tregalizumab to recombinant human soluble CD4 and membrane-bound CD4 on T cells. Moreover, we showed that this caused changes in the Tregalizumab-induced CD4 signalling pathway via the lymphocyte-specific protein tyrosine kinase p56Lck and CD4 downmodulation. In summary, we provide evidence that high Trx1 levels in RA patients compared with healthy subjects are a potential reason for diminished binding of Tregalizumab to CD4-positive T cells and offer an explanation for the observed decreased CD4 downmodulation in RA patients in comparison to healthy subjects.

Thrombomodulin Attenuates Inflammatory Damage Due to Liver Ischemia and Reperfusion Injury in Mice in Toll-Like Receptor 4-Dependent Manner.

Liver ischemia reperfusion injury (IRI) is an important problem in liver transplantation. Thrombomodulin (TM), an effective drug for disseminated intravascular coagulation, is also known to exhibit an anti-inflammatory effect through binding to the high-mobility group box 1 protein (HMGB-1) known as a proinflammatory mediator. We examined the effect of recombinant human TM (rTM) on a partial warm hepatic IRI model in wild-type (WT) and toll-like receptor 4 (TLR-4) KO mice focusing on the HMGB-1/TLR-4 axis. As in vitro experiments, peritoneal macrophages were stimulated with recombinant HMGB-1 protein. The rTM showed a protective effect on liver IRI. The rTM diminished the downstream signals of TLR-4 and also HMGB-1 expression in liver cells, as well as release of HMGB-1 from the liver. Interestingly, neither rTM treatment in vivo nor HMGB-1 treatment in vitro showed any effect on TLR-4 KO mice. Parallel in vitro studies have confirmed that rTM interfered with the interaction between HMGB-1 and TLR-4. Furthermore, the recombinant N-terminal lectin-like domain 1 (D1) subunit of TM (rTMD1) also ameliorated liver IRI to the same extent as whole rTM. Not only rTM but also rTMD1 might be a novel and useful medicine for liver transplantation. This is the first report clarifying that rTM ameliorates inflammation such as IRI in a TLR-4 pathway-dependent manner.

Human Choline Kinase-α Promotes Hepatitis C Virus RNA Replication through Modulation of Membranous Viral Replication Complex Formation.

ABSTRACTHepatitis C virus (HCV) infection reorganizes cellular membranes to create an active viral replication site named the membranous web (MW). The role that human choline kinase-α (hCKα) plays in HCV replication remains elusive. Here, we first showed that hCKα activity, not the CDP-choline pathway, promoted viral RNA replication. Confocal microscopy and subcellular fractionation of HCV-infected cells revealed that a small fraction of hCKα colocalized with the viral replication complex (RC) on the endoplasmic reticulum (ER) and that HCV infection increased hCKα localization to the ER. In the pTM-NS3-NS5B model, NS3-NS5B expression increased the localization of the wild-type, not the inactive D288A mutant, hCKα on the ER, and hCKα activity was required for effective trafficking of hCKα and NS5A to the ER. Coimmunoprecipitation showed that hCKα was recruited onto the viral RC presumably through its binding to NS5A domain 1 (D1). hCKα silencing or treatment with CK37, an hCKα activity inhibitor, abolished HCV-induced MW formation. In addition, hCKα depletion hindered NS5A localization on the ER, interfered with NS5A and NS5B colocalization, and mitigated NS5A-NS5B interactions but had no apparent effect on NS5A-NS4B and NS4B-NS5B interactions. Nevertheless, hCKα activity was not essential for the binding of NS5A to hCKα or NS5B. These findings demonstrate that hCKα forms a complex with NS5A and that hCKα activity enhances the targeting of the complex to the ER, where hCKα protein, not activity, mediates NS5A binding to NS5B, thereby promoting functional membranous viral RC assembly and viral RNA replication. IMPORTANCE HCV infection reorganizes the cellular membrane to create an active viral replication site named the membranous web (MW). Here, we report that human choline kinase-α (hCKα) acts as an essential host factor for HCV RNA replication. A fraction of hCKα colocalizes with the viral replication complex (RC) on the endoplasmic reticulum (ER) in HCV-infected cells. NS3-NS5B expression increases ER localization of wild-type, but not D288A mutant, hCKα, and hCKα activity facilitates the transport of itself and NS5A to the ER. Silencing or inactivation of hCKα abrogates MW formation. Moreover, hCKα is recruited by NS5A independent of hCKα activity, presumably through binding to NS5A D1. hCKα activity then mediates the ER targeting of the hCKα-NS5A complex. On the ER membrane, hCKα protein, per se, induces NS5A binding to NS5B, thereby promoting membranous RC formation and viral RNA replication. Our study may benefit the development of hCKα-targeted anti-HCV therapeutics.