An engineered disulfide bond reversibly traps the IgE-Fc3-4 in a closed, nonreceptor binding conformation

IgE antibodies interact with the high affinity IgE Fc receptor, FcεRI, and activate inflammatory pathways associated with the allergic response. The IgE-Fc region, comprising the C-terminal domains of the IgE heavy chain, binds FcεRI and can adopt different conformations ranging from a closed form incompatible with receptor binding to an open, receptor-bound state. A number of intermediate states are also observed in different IgE-Fc crystal forms. To further explore this apparent IgE-Fc conformational flexibility and to potentially trap a closed, inactive state, we generated a series of disulfide bond mutants. Here we describe the structure and biochemical properties of an IgE-Fc mutant that is trapped in the closed, non-receptor binding state via an engineered disulfide at residue 335 (Cys-335). Reduction of the disulfide at Cys-335 restores the ability of IgE-Fc to bind to its high affinity receptor, FcεRIα. The structure of the Cys-335 mutant shows that its conformation is within the range of previously observed, closed form IgE-Fc structures and that it retains the hydrophobic pocket found in the hinge region of the closed conformation. Locking the IgE-Fc into the closed state with the Cys-335 mutation does not affect binding of two other IgE-Fc ligands, omalizumab and DARPin E2_79, demonstrating selective blocking of the high affinity receptor binding.

Phenotype-genotype correlation in eight Polish patients with inherited Factor XIII deficiency: identification of three novel mutations

Inherited factor XIII (FXIII) deficiency is known as one of the most rare blood coagulation disorder in humans. In the present study, phenotype and genotype of eight FXIII deficient Polish patients from five unrelated families were compared. The patients presented with a severe phenotype demonstrated by a high incidence of intracerebral haemorrhages (seven of eight patients), haemarthrosis (six patients) and bleeding due to trauma (five patients). Introduction of regular substitution with FXIII concentrate prevented spontaneous bleeding in seven patients. In all patients, mutations within the F13A gene have been identified revealing four missense mutations (Arg77Cys, Arg260Cys, Ala378Pro, Gly420Ser), one nonsense mutation (Arg661X), one splice site mutation (IVS5-1 G>A) and one small deletion (c.499-512del). One homozygous large deletion involving exon 15 was detected by failure of PCR product. The corresponding mutations resulted in severely reduced FXIII activity and FXIII A-subunit antigen concentration, while FXIII B-subunit antigen remained normal or mildly decreased. Structural analysis demonstrated that the novel Ala378Pro mutation may cause a disruption of the FXIII catalytic triad leading to a non-functional protein which presumably undergoes premature degradation. In conclusion, the severe phenotype with high incidence of intracranial bleeding and haemarthrosis was in accordance with laboratory findings on FXIII and with severe molecular defects of the F13A gene.

Tracking virus-specific CD4+ T cells during and after acute hepatitis C virus infection

BACKGROUND: CD4+ T cell help is critical in maintaining antiviral immune responses and such help has been shown to be sustained in acute resolving hepatitis C. In contrast, in evolving chronic hepatitis C CD4+ T cell helper responses appear to be absent or short-lived, using functional assays. METHODOLOGY/PRINCIPAL FINDINGS: Here we used a novel HLA-DR1 tetramer containing a highly targeted CD4+ T cell epitope from the hepatitis C virus non-structural protein 4 to track number and phenotype of hepatitis C virus specific CD4+ T cells in a cohort of seven HLA-DR1 positive patients with acute hepatitis C in comparison to patients with chronic or resolved hepatitis C. We observed peptide-specific T cells in all seven patients with acute hepatitis C regardless of outcome at frequencies up to 0.65% of CD4+ T cells. Among patients who transiently controlled virus replication we observed loss of function, and/or physical deletion of tetramer+ CD4+ T cells before viral recrudescence. In some patients with chronic hepatitis C very low numbers of tetramer+ cells were detectable in peripheral blood, compared to robust responses detected in spontaneous resolvers. Importantly we did not observe escape mutations in this key CD4+ T cell epitope in patients with evolving chronic hepatitis C. CONCLUSIONS/SIGNIFICANCE: During acute hepatitis C a CD4+ T cell response against this epitope is readily induced in most, if not all, HLA-DR1+ patients. This antiviral T cell population becomes functionally impaired or is deleted early in the course of disease in those where viremia persists.

High expression of neuropeptide Y1 receptors in ewing sarcoma tumors

PURPOSE: Peptide receptors are frequently overexpressed in human tumors, allowing receptor-targeted scintigraphic imaging and therapy with radiolabeled peptide analogues. Neuropeptide Y (NPY) receptors are new candidates for these applications, based on their high expression in specific cancers. Because NPY receptors are expressed in selected sarcoma cell lines and because novel treatment options are needed for sarcomas, this study assessed the NPY receptor in primary human sarcomas. EXPERIMENTAL DESIGN: Tumor tissues of 88 cases, including Ewing sarcoma family of tumors (ESFT), synovial sarcomas, osteosarcomas, chondrosarcomas, liposarcomas, angiosarcomas, rhabdomyosarcomas, leiomyosarcomas, and desmoid tumors, were investigated for NPY receptor protein with in vitro receptor autoradiography using (125)I-labeled NPY receptor ligands and for NPY receptor mRNA expression with in situ hybridization. RESULTS: ESFT expressed the NPY receptor subtype Y1 on tumor cells in remarkably high incidence (84%) and density (mean, 5,314 dpm/mg tissue). Likewise, synovial sarcomas expressed Y1 on tumor cells in high density (mean, 7,497 dpm/mg; incidence, 40%). The remaining tumors expressed NPY receptor subtypes Y1 or Y2 at lower levels. Moreover, many of the sarcomas showed Y1 expression on intratumoral blood vessels. In situ hybridization for Y1 mRNA confirmed the autoradiography results. CONCLUSIONS: NPY receptors are novel molecular markers for human sarcomas. Y1 may inhibit growth of specific sarcomas, as previously shown in an in vivo mouse model of human ESFT. The high Y1 expression on tumor cells of ESFT and synovial sarcomas and on blood vessels in many other sarcomas represents an attractive basis for an in vivo tumor targeting.

Distinct microRNA Expression Profile in Prostate Cancer Patients with Early Clinical Failure and the Impact of let-7 as Prognostic Marker in High-Risk Prostate Cancer

Background The identification of additional prognostic markers to improve risk stratification and to avoid overtreatment is one of the most urgent clinical needs in prostate cancer (PCa). MicroRNAs, being important regulators of gene expression, are promising biomarkers in various cancer entities, though the impact as prognostic predictors in PCa is poorly understood. The aim of this study was to identify specific miRNAs as potential prognostic markers in high-risk PCa and to validate their clinical impact. Methodology and Principal Findings We performed miRNA-microarray analysis in a high-risk PCa study group selected by their clinical outcome (clinical progression free survival (CPFS) vs. clinical failure (CF)). We identified seven candidate miRNAs (let-7a/b/c, miR-515-3p/5p, -181b, -146b, and -361) that showed differential expression between both groups. Further qRT-PCR analysis revealed down-regulation of members of the let-7 family in the majority of a large, well-characterized high-risk PCa cohort (n = 98). Expression of let-7a/b/and -c was correlated to clinical outcome parameters of this group. While let-7a showed no association or correlation with clinical relevant data, let-7b and let-7c were associated with CF in PCa patients and functioned partially as independent prognostic marker. Validation of the data using an independent high-risk study cohort revealed that let-7b, but not let-7c, has impact as an independent prognostic marker for BCR and CF. Furthermore, we identified HMGA1, a non-histone protein, as a new target of let-7b and found correlation of let-7b down-regulation with HMGA1 over-expression in primary PCa samples. Conclusion Our findings define a distinct miRNA expression profile in PCa cases with early CF and identified let-7b as prognostic biomarker in high-risk PCa. This study highlights the importance of let-7b as tumor suppressor miRNA in high-risk PCa and presents a basis to improve individual therapy for high-risk PCa patients.

Kidney disease in antiretroviral-naïve HIV-positive adults with high CD4 counts: prevalence and predictors of kidney disease at enrolment in the INSIGHT Strategic Timing of AntiRetroviral Treatment (START) trial.

OBJECTIVES HIV infection has been associated with an increased risk of chronic kidney disease (CKD). Little is known about the prevalence of CKD in individuals with high CD4 cell counts prior to initiation of antiretroviral therapy (ART). We sought to address this knowledge gap. METHODS We describe the prevalence of CKD among 4637 ART-naïve adults (mean age 36.8 years) with CD4 cell counts > 500 cells/μL at enrolment in the Strategic Timing of AntiRetroviral Treatment (START) study. CKD was defined by estimated glomerular filtration rate (eGFR) < 60 mL/min/1.73 m(2) and/or dipstick urine protein ≥ 1+. Logistic regression was used to identify baseline characteristics associated with CKD. RESULTS Among 286 [6.2%; 95% confidence interval (CI) 5.5%, 6.9%] participants with CKD, the majority had isolated proteinuria. A total of 268 participants had urine protein ≥ 1+, including 41 with urine protein ≥ 2+. Only 22 participants (0.5%) had an estimated glomerular filtration rate < 60 mL/min/1.73 m(2) , including four who also had proteinuria. Baseline characteristics independently associated with CKD included diabetes [adjusted odds ratio (aOR) 1.73; 95% CI 1.05, 2.85], hypertension (aOR 1.82; 95% CI 1.38, 2.38), and race/ethnicity (aOR 0.59; 95% CI 0.37, 0.93 for Hispanic vs. white). CONCLUSIONS We observed a low prevalence of CKD associated with traditional CKD risk factors among ART-naïve clinical trial participants with CD4 cell counts > 500 cells/μL.

Targeting membrane-bound viral RNA synthesis reveals potent inhibition of diverse coronaviruses including the middle East respiratory syndrome virus.

Coronaviruses raise serious concerns as emerging zoonotic viruses without specific antiviral drugs available. Here we screened a collection of 16671 diverse compounds for anti-human coronavirus 229E activity and identified an inhibitor, designated K22, that specifically targets membrane-bound coronaviral RNA synthesis. K22 exerts most potent antiviral activity after virus entry during an early step of the viral life cycle. Specifically, the formation of double membrane vesicles (DMVs), a hallmark of coronavirus replication, was greatly impaired upon K22 treatment accompanied by near-complete inhibition of viral RNA synthesis. K22-resistant viruses contained substitutions in non-structural protein 6 (nsp6), a membrane-spanning integral component of the viral replication complex implicated in DMV formation, corroborating that K22 targets membrane bound viral RNA synthesis. Besides K22 resistance, the nsp6 mutants induced a reduced number of DMVs, displayed decreased specific infectivity, while RNA synthesis was not affected. Importantly, K22 inhibits a broad range of coronaviruses, including Middle East respiratory syndrome coronavirus (MERS-CoV), and efficient inhibition was achieved in primary human epithelia cultures representing the entry port of human coronavirus infection. Collectively, this study proposes an evolutionary conserved step in the life cycle of positive-stranded RNA viruses, the recruitment of cellular membranes for viral replication, as vulnerable and, most importantly, druggable target for antiviral intervention. We expect this mode of action to serve as a paradigm for the development of potent antiviral drugs to combat many animal and human virus infections.

Proteome-wide screening of the European porcine reproductive and respiratory syndrome virus reveals a broad range of T cell antigen reactivity.

The porcine reproductive and respiratory syndrome virus (PRRSV) is a rapidly evolving and diversifying pathogen necessitating the development of improved vaccines. Immunity to PRRSV is not well understood although there are data suggesting that virus-specific T cell IFN-γ responses play an important role. We therefore aimed to better characterise the T cell response to genotype 1 (European) PRRSV by utilising a synthetic peptide library spanning the entire proteome and a small cohort of pigs rendered immune to PRRSV-1 Olot/91 by repeated experimental infection. Using an IFN-γ ELISpot assay as a read-out, we were able to identify 9 antigenic regions on 5 of the viral proteins and determine the corresponding responder T cell phenotype. The diversity of the IFN-γ response to PRRSV proteins suggests that antigenic regions are scattered throughout the proteome and no one single antigen dominates the T cell response. To address the identification of well-conserved T cell antigens, we subsequently screened groups of pigs infected with a closely related avirulent PRRSV-1 strain (Lelystad) and a divergent virulent subtype 3 strain (SU1-Bel). Whilst T cell responses from both groups were observed against many of the antigens identified in the first study, animals infected with the SU1-Bel strain showed the greatest response against peptides representing the non-structural protein 5. The proteome-wide peptide library screening method used here, as well as the antigens identified, warrant further evaluation in the context of next generation vaccine development.

Phosphatase and tensin homolog regulates stability and activity of EphB1 receptor

Deregulation of receptor tyrosine kinases (RTKs) is linked to a broad range of cancers, stressing the necessity of studying their regulatory pathways. We and others demonstrated previously that c-Cbl is necessary for the lysosomal degradation of erythropoietin-producing hepatocellular B1 (EphB1) carcinoma and epidermal growth factor receptor (EGFR) RTKs. Moreover, the tumor suppressor phosphatase and tensin homolog (PTEN) was shown to modulate c-Cbl-dependent EGFR degradation. We therefore investigated the involvement of PTEN in EphB1 signaling and degradation. We used PTEN mutants, PTEN, and NHERF1 small interfering RNA in CHO-EphB1 and SW480 cells endogenously expressing EphB1 to delineate EphB1-PTEN interactions. PTEN was constitutively associated with c-Cbl, protecting it from degradation. EphB1 stimulation triggered ∼50% serine-threonine PTEN dephosphorylation and PTEN-Cbl complex disruption, a process requiring PTEN protein phosphatase activity. Both proteins independently translocated to EphB1, with PTEN in association with the scaffold protein NHERF1. Biologically, PTEN lipid phosphatase activity impairs EphB1-dependent cell adhesion and chemotaxis. This study demonstrates for the first time in mammalian cells that the Eph receptor and PTEN associate and influence their signaling. Moreover, it contributes to the emerging concept that PTEN regulates expression of RTKs through modulation of their degradation. Finally, it reveals a new role for PTEN protein phosphatase activity involved in this process.

Regulation of human GH receptor gene transcription by 20 and 22 kDa GH in a human hepatoma cell line.

The human GH gene is 1.7 kilobase pairs (kb) in length and is composed of five exons and four introns. This gene is expressed in the pituitary gland and encodes a 22 kDa protein. In addition to this predominant (75%) form, 5-10% of pituitary GH is present as a 20 kDa protein that has an amino acid (aa) sequence identical to the 22 kDa form except for a 15 aa internal deletion of residues 32-46 as a result of an alternative splicing event. Because it has been reported that non-22-kDa GH isoforms might be partly responsible for short stature and growth retardation in children, the aim of this study was to compare the impact of both 22 kDa and 20 kDa GH on GH receptor gene (GH receptor/GH binding protein (GHR/GHBP)) expression. Various concentrations of 20 kDa and 22 kDa GH (0, 2, 5, 12.5, 25, 50 and 150 ng/ml) were added to human hepatoma (HuH7) cells cultured in serum-free hormonally defined medium for 0, 1 and 2 h. Thereafter GHR/GHBP mRNA expression was measured by quantitative PCR. Addition of either 20 kDa or 22 kDa GH, at low or normal physiological concentrations (0, 2, 5, 12.5, 25 or 50 ng/ml) induced a dose-dependent increase in GHR/GHBP expression. However, a supraphysiological concentration of 20 kDa GH (150 ng/ml) resulted in a significantly lower (P<0.05) downregulation of GHR/GHBP gene transcription compared with the downregulation achieved by this concentration of 22 kDa GH. This difference might be explained by a decreased ability to form a 1 : 1 complex with GHR and/or GHBP, which normally occurs at high concentrations of GH. Nuclear run-on experiments and GHBP determinations confirmed the changes in GHR/GHBP mRNA levels. In conclusion, we report that both 20 kDa and 22 kDa GH, in low and normal physiological concentrations, have the same effect on regulation of GHR/GHBP gene transcription in a human hepatoma cell line. At a supraphysiological concentration of 150 ng/ml, however, 20 kDa GH has a less self-inhibitory effect than the 22 kDa form.

Hypoxia aggravates non-alcoholic steatohepatitis in mice lacking hepatocellular PTEN

The metabolic disorders that predispose patients to NASH (non-alcoholic steatohepatitis) include insulin resistance and obesity. Repeated hypoxic events, such as occur in obstructive sleep apnoea syndrome, have been designated as a risk factor in the progression of liver disease in such patients, but the mechanism is unclear, in particular the role of hypoxia. Therefore we studied the influence of hypoxia on the development and progression of steatohepatitis in an experimental mouse model. Mice with a hepatocellular-specific deficiency in the Pten (phosphatase and tensin homologue deleted on chromosome 10) gene, a tumour suppressor, were exposed to a 10% O2 (hypoxic) or 21% O2 (control) atmosphere for 7 days. Haematocrit, AST (aspartate aminotransferase), glucose, triacylglycerols (triglycerides) and insulin tolerance were measured in blood. Histological lesions were quantified. Expression of genes involved in lipogenesis and mitochondrial beta-oxidation, as well as FOXO1 (forkhead box O1), hepcidin and CYP2E1 (cytochrome P450 2E1), were analysed by quantitative PCR. In the animals exposed to hypoxia, the haematocrit increased (60+/-3% compared with 50+/-2% in controls; P<0.01) and the ratio of liver weight/body weight increased (5.4+/-0.2% compared with 4.7+/-0.3% in the controls; P<0.01). Furthermore, in animals exposed to hypoxia, steatosis was more pronounced (P<0.01), and the NAS [NAFLD (non-alcoholic fatty liver disease) activity score] (8.3+/-2.4 compared with 2.3+/-10.7 in controls; P<0.01), serum AST, triacylglycerols and glucose were higher. Insulin sensitivity decreased in mice exposed to hypoxia relative to controls. The expression of the lipogenic genes SREBP-1c (sterol-regulatory-element-binding protein-1c), PPAR-gamma (peroxisome-proliferator-activated receptor-gamma), ACC1 (acetyl-CoA carboxylase 1) and ACC2 (acetyl-CoA carboxylase 2) increased significantly in mice exposed to hypoxia, whereas mitochondria beta-oxidation genes [PPAR-alpha (peroxisome-proliferator-activated receptor-alpha) and CPT-1 (carnitine palmitoyltransferase-1)] decreased significantly. In conclusion, the findings of the present study demonstrate that hypoxia alone aggravates and accelerates the progression of NASH by up-regulating the expression of lipogenic genes, by down-regulating genes involved in lipid metabolism and by decreasing insulin sensitivity.

Acute Regulation of Renal Na+/H+ Exchanger NHE3 by Dopamine: Role of Protein Phosphatase 2A

Nephrogenic dopamine is a potent natriuretic paracrine/autocrine hormone that is central for mammalian sodium homeostasis. In the renal proximal tubule, dopamine induces natriuresis partly via inhibition of the sodium/proton exchanger NHE3. The signal transduction pathways and mechanisms by which dopamine inhibits NHE3 are complex and incompletely understood. This manuscript describes the role of the serine/threonine protein phosphatase 2A (PP2A) in the regulation of NHE3 by dopamine. The PP2A regulatory subunit B56 delta (coded by the Ppp2r5d gene) directly associates with more than one region of the carboxy-terminal hydrophilic putative cytoplasmic domain of NHE3 (NHE3-cyto), as demonstrated by yeast-two-hybrid, co-immunoprecipitation, blot overlay and in vitro pull-down assays. Phosphorylated NHE3-cyto is a substrate for purified PP2A in an in vitro dephosphorylation reaction. In cultured renal cells, inhibition of PP2A by either okadaic acid or by overexpression of the simian virus 40 (SV40) small t antigen blocks the ability of dopamine to inhibit NHE3 activity and to reduce surface NHE3 protein. Dopamine-induced NHE3 redistribution is also blocked by okadaic acid ex vivo in rat kidney cortical slices. These studies demonstrate that PP2A is an integral and critical participant in the signal transduction pathway between dopamine receptor activation and NHE3 inhibition. Key words: Natriuresis, Sodium transport, Signal transduction.

Tyrosine kinase modulation of protein kinase C activity regulates G protein-linked Ca2+ signaling in leukemic hematopoietic cells

We have used a recombinant mouse pre-B cell line (TonB210.1, expressing Bcr/Abl under the control of an inducible promoter) and several human leukemia cell lines to study the effect of high tyrosine kinase activity on G protein-coupled receptor (GPCR) agonist-stimulated cellular Ca(2+) release and store-operated Ca(2+) entry (SOCE). After induction of Bcr/Abl expression, GPCR-linked SOCE increased. The effect was reverted in the presence of the specific Abl inhibitor imatinib (1microM) and the Src inhibitor PP2 (10microM). In leukemic cell lines constitutively expressing high tyrosine kinase activity, Ca(2+) transients were reduced by imatinib and/or PP2. Ca(2+) transients were enhanced by specific inhibitors of PKC subtypes and this effect was amplified by tyrosine kinase inhibition in Bcr/Abl expressing TonB210.1 and K562 cells. Under all conditions Ca(2+) transients were essentially blocked by the PKC activator PMA. In Bcr/Abl expressing (but not in native) TonB210.1 cells, tyrosine kinase inhibitors enhanced PKCalpha catalytic activity and PKCalpha co-immunoprecipitated with Bcr/Abl. Unlike native TonB210.1 cells, Bcr/Abl expressing cells showed a high rate of cell death if Ca(2+) influx was reduced by complexing extracellular Ca(2+) with BAPTA. Our data suggest that tonic inhibition of PKC represents a mechanism by which high tyrosine kinase activity can enhance cellular Ca(2+) transients and thus exert profound effects on the proliferation, apoptosis and chemotaxis of leukemic cells.

Caveolin-1 is required for signaling and membrane targeting of EphB1 receptor tyrosine kinase

Eph receptor tyrosine kinases are key players during the development of the embryonic vasculature; however, their role and regulation in adult angiogenesis remain to be defined. Caveolae are flask-shaped invaginations of the cell membrane; their major structural protein, caveolin-1, has been shown to regulate signaling molecules localized in these micro-domains. The interaction of caveolin-1 with several of these proteins is mediated by the binding of its scaffolding domain to a region containing hydrophobic amino acids within these proteins. The presence of such a motif within the EphB1 kinase domain prompted us to investigate the caveolar localization and regulation of EphB1 by caveolin-1. We report that EphB1 receptors are localized in caveolae, and directly interact with caveolin-1 upon ligand stimulation. This interaction, as well as EphB1-mediated activation of extracellular-signal-regulated kinase (ERK), was abrogated by overexpression of a caveolin-1 mutant lacking a functional scaffolding domain. Interaction between Ephs and caveolin-1 is not restricted to the B-subclass of receptors, since we show that EphA2 also interacts with caveolin-1. Furthermore, we demonstrate that the caveolin-binding motif within the kinase domain of EphB1 is primordial for its correct membrane targeting. Taken together, our findings establish caveolin-1 as an important regulator of downstream signaling and membrane targeting of EphB1.

Differential expression of the receptor tyrosine kinase EphB4 and its ligand Ephrin-B2 during human placental development

Normal placentation involves the development of an utero-placental circulation following the migration of the extravillous cytotrophoblasts into the decidua and invasion of the spiral arteries, which are thereby transformed into large vessels of low resistance. Given the documented role of the receptor tyrosine kinase EphB4 and its ligand ephrin-B2 in the establishment of the embryonal vascular network, we hypothesized that these molecules are also instrumental in the development of the human placenta. Monitoring the expression during placental development revealed that in first trimester and term placentae both molecules are equally expressed at the RNA level. In contrast, the protein levels were significantly reduced during gestation. Immunohistochemistry revealed a distinct localization of the EphB4 and ephrin-B2 proteins. EphB4 was predominantly expressed in the villous syncytial trophoblast layer and in a subset of intravillous capillaries. Prominent expression was also observed in the extravillous cytotrophoblast giant cells. In contrast, ephrin-B2 expression was detected in the villous cytotrophoblast and syncytial trophoblast cell layers, as well as initially in all intravillous capillaries. Strong expression was also observed in extravillous anchoring cytotrophoblast cells. Hypoxia is a major inducer of placental development. In vitro studies employing trophoblast-derived cell lines revealed that predominantly ephrin-B2 expression is induced by hypoxia, however, in an Hif-1alpha independent manner. These experiments suggest that EphB4 and ephrin-B2 are instrumental in the establishment of a functional placental structure and of the utero-placental circulation.