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Abstract
In search of new prognostic markers, many mutation analyses of the HBV genome were performed. However, the Kozak sequence preceding precore was covered only infrequently in these analyses. In this study, HBV core promoter/precore region was sequenced in serum samples of European inactive HBV carriers (n=560). Quadruple mutation GCAC1809-1812TTCT was found with a high prevalence of 42% in the Kozak sequence preceding precore among all HBV genotypes. GCAC1809-1812TTCT was strongly associated with coexistence of basal core promoter (BCP) double mutation A1762T/G1764A and lower HBV DNA levels (p<0.0001). In vitro GCAC1809-1812TTCT leads to drastically diminished synthesis of pregenomic(pg)RNA, precore mRNA, core, HBsAg and HBeAg. Calculation of the pgRNA secondary structure suggests a destabilization of the pgRNA structure by A1762T/G1764A that is compensated by GCAC1809-1812TTCT. In 125 patients with HBV-related cirrhosis, GCAC1809-1812TTCT was not detected. While a strong association of GCAC1809-1812TTCT with inactive carrier status (p<0.0001) was observed, BCP double mutation was strongly correlated with cirrhosis (p<0.0001), but this was only observed in absence of GCAC1809-1812TTCT. In conclusion, our data reveal that GCAC1809-1812TTCT is highly prevalent in inactive carriers, and acts as a compensatory mutation for BCP double mutation. GCAC1809-1812TTCT seems to be a biomarker of good prognosis in HBV infection.
Authors
Kai-Henrik Peiffer, Catrina Spengler, Michael Basic, Bingfu Jiang, Lisa Kuhnhenn, Wiebke Obermann, Tobias Zahn, Mirco Glitscher, Alessandro Loglio, Floriana Facchetti, Gert Carra, Alica Kubesch, Johannes Vermehren, Viola Knop, Christiana Graf, Julia Dietz, Fabian Finkelmeier, Eva Herrmann, Jonel Trebicka, Arnold Grünweller, Stefan Zeuzem, Christoph Sarrazin, Pietro Lampertico, Eberhard Hildt
Abstract
Heart failure is often accompanied by titin-dependent myocardial stiffness. Phosphorylation of titin by cGMP-dependent protein kinase (PKG)I increases cardiomyocyte distensibility. The upstream pathways stimulating PKGI-mediated titin phosphorylation are unclear. We studied whether C-type natriuretic peptide (CNP), via its guanylyl cyclase-B (GC-B) receptor and cGMP/PKGI signalling, modulates titin-based ventricular compliance. To dissect GC-B-mediated effects of endogenous CNP in cardiomyocytes, we generated mice with cardiomyocyte (CM)-restricted GC-B deletion. The impact on heart morphology and function, myocyte passive tension and titin isoform expression and phosphorylation was studied at baseline and after increased afterload induced by transverse aortic constriction (TAC).Pressure-overload increased left ventricular, especially endothelial CNP expression, with an early peak after 3 days. Concomitantly, titin phosphorylation at Ser4080, the site phosphorylated by PKGI, was augmented. Notably, in CM GC-B KO mice this titin response was abolished. TAC-induced hypertrophy and fibrosis were not different between genotypes. However, the KO mice presented mild systolic and diastolic dysfunction together with myocyte stiffness, which were not observed in control littermates. In vitro, recombinant PKGI rescued reduced Titin-Ser4080 phosphorylation and reverted passive stiffness of GC-B-deficient cardiomyocytes. CNP-induced activation of GC-B/cGMP/PKGI signalling in cardiomyocytes provides a protecting regulatory circuit preventing titin-based myocyte stiffening during early phases of pressure-overload.
Authors
Konstanze Michel, Melissa Herwig, Franziska Werner, Katarina Špiranec Spes, Marco Abeßer, Kai Schuh, Swati Dabral, Andreas Mügge, Hideo A. Baba, Boris V. Skryabin, Nazha Hamdani, Michaela Kuhn
Abstract
The challenge of discovering a completely new human tumor virus of unknown phylogeny or sequence depends on detecting viral molecules and differentiating them from host molecules in the virus-associated neoplasm. We developed differential peptide subtraction (DPS) using differential mass-spectrometry (dMS) followed by targeted analysis to facilitate this discovery. We validated this approach by analyzing Merkel cell carcinoma (MCC), an aggressive human neoplasm, in which ~80% of cases are caused by the human Merkel cell polyomavirus (MCV). Approximately 20% of MCC have a high mutational burden and are negative for MCV, but are microscopically indistinguishable from virus positive cases. Using 23 (12 MCV positive, 11 MCV negative) formalin-fixed MCC, DPS identified both viral and human biomarkers (MCV Large T antigen, CDKN2AIP, SERPINB5 and TRIM29) that discriminates MCV positive and negative MCC. Statistical analysis of 498,131 dMS features not matching the human proteome by DPS revealed 562 (0.11%) to be up-regulated in virus-infected samples. Remarkably, four (20%) of the top 20 candidate MS spectra originated from MCV T oncoprotein peptides and confirmed by reverse translation degenerate oligonucleotide sequencing. DPS is a robust proteomic approach to identify novel viral sequences in infectious tumors when nucleic acid-based methods are not feasible.
Authors
Tuna Toptan, Pamela S. Cantrell, Xuemei Zeng, Yang Liu, Mai Sun, Nathan A. Yates, Yuan Chang, Patrick S. Moore
Abstract
Infections caused by multi-drug resistant Staphylococcus aureus, especially MRSA, are responsible for high mortality and morbidity worldwide. Resistant lineages were previously confined to hospitals, but are now also causing infections among healthy individuals in the community. It is therefore imperative to explore therapeutic avenues that are less prone to raise drug resistance compared to today’s antibiotics. An opportunity to achieve this ambitious goal could be provided by targeted antimicrobial photodynamic therapy (aPDT), which relies on the combination of a bacteria-specific targeting agent and light-induced generation of reactive oxygen species by an appropriate photosensitizer. Here we conjugated the near-infrared photosensitizer IRDye700DX to a fully human monoclonal antibody, specific for the invariantly expressed staphylococcal antigen IsaA. The resulting immunoconjugate 1D9-700DX was characterized biochemically and in preclinical infection models. As demonstrated in vitro, in vivo, and in a human post-mortem orthopedic implant infection model, targeted aPDT with 1D9-700DX is highly effective. Importantly, combined with the non-toxic aPDT-enhancing agent potassium iodide, 1D9-700DX overcomes the antioxidant properties of human plasma and fully eradicates high titers of MRSA. We show that the developed immunoconjugate 1D9-700DX targets MRSA and kills it upon illumination with red light, without causing collateral damage to human cells.
Authors
Mafalda Bispo, Andrea Anaya-Sanchez, Sabrina Suhani, Elisa J.M. Raineri, Marina López-Álvarez, Marjolein Heuker, Wiktor Szymański, Francisco Romero Pastrana, Girbe Buist, Alexander R. Horswill, Kevin P. Francis, Gooitzen M. van Dam, Marleen van Oosten, Jan Maarten van Dijl
Abstract
Intratumoral immune infiltrate was recently reported in Gastrointestinal Stromal Tumors (GIST). However, what tumor-intrinsic factors dictate GIST immunogenicity is still largely undefined. To shed light on this issue a large cohort (82 samples) of primary untreated GIST, representative of major clinicopathological variables, was investigated by an integrated immunohistochemical, transcriptomic and computational approach. Our results indicate that tumor genotype, location and malignant potential concur to shape the immunogenicity of primary naïve GIST. Immune infiltration was greater in overt GIST than in lesions with limited malignant potential (miniGIST), in KIT/PDGFRA mutated than in KIT/PDGFRA wild-type tumors and in PDGFRA versus KIT mutated GIST. Within the KIT mutated subset, a higher degree of immune colonization was detected in the intestine. Immune hot tumors showed expression patterns compatible with a potentially proficient but curbed antigen-specific immunity, hinting at sensitivity to immunomodulatory treatments. Poorly infiltrated GIST, primarily KIT/PDGFRA wild-type intestinal tumors, showed activation of Hedgehog and WNT/β-catenin immune excluding pathways. This finding discloses a potential therapeutic vulnerability, as the targeting of these pathways might prove effective by both inhibiting pro-oncogenic signals and fostering anti-tumor immune responses. Finally, an intriguing anticorrelation between immune infiltration and ANO1/DOG1 expression was observed, suggesting an immunomodulatory activity for anoctamin-1.
Authors
Daniela Gasparotto, Marta Sbaraglia, Sabrina Rossi, Davide Baldazzi, Monica Brenca, Alessia Mondello, Federica Nardi, Dominga Racanelli, Matilde Cacciatore, Angelo Paolo Dei Tos, Roberta Maestro
Abstract
Plasma antimalarial antibody can mediate anti-parasite immunity but has not previously been characterized at the molecular level. Here, we develop an innovative strategy to characterize humoral responses by integrating profiles of plasma immunoglobulins (IG) or antibodies with those expressed on B cells as part of BcR. We applied this strategy to define plasma IG and determine variable V gene usage after vaccination with the Plasmodium falciparum zygote antigen Pfs25. First, using proteomic tools coupled with bulk immunosequencing data, we determined human F(ab′)2 peptide sequences from plasma IG of adults who received four doses of Pfs25-EPA/Alhydrogel. Specifically, Pfs25 antigen-specific F(ab′)2 peptides (Pfs25-IG) were aligned to cDNA sequences of IGH complementarity determining region 3 (CDR3) from a dataset generated by total peripheral B cell immunosequencing of the entire vaccinated population. IGHV4 was the most commonly identified IGHV subgroup of Pfs25-IG, a pattern that was corroborated by VH/VL sequencing of Pfs25-specific single B cells from five vaccinees and by matching plasma Pfs25-IG peptides and V-(D)-J sequences of Pfs25-specific single B cells from the same donor. Among 13 recombinant human mAbs generated from IG sequences of Pfs25-specific single B cells, a single IGHV4 mAb displayed strong neutralizing activity, reducing the number of P. falciparum oocysts in infected mosquitoes by more than 80% at 100 μg/mL. Our approach characterizes the human plasma antibody repertoire in response to the Pfs25-EPA/Alhydrogel vaccine and will be useful to study circulating antibodies in response to other vaccines as well as those induced during infections or autoimmune disorders.
Authors
Camila H. Coelho, Steven T. Nadakal, Patricia A. Gonzales Hurtado, Robert Morrison, Jacob D. Galson, Jillian Neal, Yimin Wu, C. Richter King, Virginia Price, Kazutoyo Miura, Sharon Wong-Madden, Justin Y.A. Dortichamou, David L. Narum, Nicholas J. MacDonald, Maryonne Snow-Smith, Marissa Vignali, Justin J. Taylor, Marie-Paule Lefranc, Johannes Trück, Carole A. Long, Issaka Sagara, Michal Fried, Patrick E. Duffy
Abstract
WNK1 is an atypical kinase protein ubiquitously expressed in humans and mice. A mutation in its encoding gene causes hypertension in humans which is associated with abnormal ion homeostasis. WNK1 is critical for in vitro decidualization in human endometrial stromal cells, thereby demonstrating its importance in female reproduction. Using a mouse model, WNK1 was ablated in the female reproductive tract to define its in vivo role in uterine biology. Loss of WNK1 altered uterine morphology, causing endometrial epithelial hyperplasia, adenomyotic features and a delay in embryo implantation, ultimately resulting in compromised fertility. Combining transcriptomic, proteomic and interactomic analyses revealed a novel regulatory pathway whereby WNK1 represses AKT phosphorylation through the phosphatase PP2A in endometrial cells from both humans and mice. We show that WNK1 interacts with PPP2R1A, the alpha isoform of the PP2A scaffold subunit. This maintains the levels of PP2A subunits and stabilizes its activity, which then dephosphorylates AKT. Therefore, loss of WNK1 reduced PP2A activity, causing AKT hypersignaling. Using FOXO1 as a readout of AKT activity, we demonstrate that there was escalated FOXO1 phosphorylation and nuclear exclusion, leading to a disruption in the expression of genes that are crucial for embryo implantation.
Authors
Ru-pin Alicia Chi, Tianyuan Wang, Chou-Long Huang, San-Pin Wu, Steven Young, John Lydon, Francesco DeMayo
Abstract
Rapid and specific antibody testing is crucial for improved understanding, control, and treatment of COVID-19 pathogenesis. Herein, we describe and apply a rapid, sensitive, and accurate virus neutralization assay for SARS-CoV-2 antibodies. The new assay is based on an HIV-1 lentiviral vector that contains a secreted intron Gaussia luciferase or secreted Nano-luciferase reporter cassette, pseudotyped with the SARS-CoV-2 spike (S) glycoprotein, and is validated with a plaque reduction assay using an authentic, infectious SARS-CoV-2 strain. The new assay was used to evaluate SARS-CoV-2 antibodies in serum from individuals with a broad range of COVID-19 symptoms, including intensive care unit (ICU) patients, health care workers (HCWs), and convalescent plasma donors. The highest neutralizing antibody titers were observed among ICU patients, followed by general hospitalized patients, HCWs and convalescent plasma donors. Our study highlights a wide phenotypic variation in human antibody responses against SARS-CoV-2, and demonstrates the efficacy of a novel lentivirus pseudotype assay for high-throughput serological surveys of neutralizing antibody titers in large cohorts.
Authors
Cong Zeng, John P. Evans, Rebecca Pearson, Panke Qu, Yi-Min Zheng, Richard T. Robinson, Luanne Hall-Stoodley, Jacob S. Yount, Sonal Pannu, Rama K. Mallampalli, Linda Saif, Eugene Oltz, Gerard Lozanski, Shan-Lu Liu
Abstract
While the RV144 HIV vaccine trial lead to moderately reduced risk of HIV acquisition, emerging data from the repeat failure of the HVTN702 trial point to the critical need to re-examine the relationships between previously identified correlates of reduced risk of protection in the RV144 study. Specifically, the induction of V2-binding, non-IgA, IgG3 antibody responses with non-neutralizing functions were linked to reduced risk of infection in RV144 vaccinees. While each of these features was individually linked to reduced risk of infection, the relationships and interactions between these humoral immune signatures remain unclear. Thus, here we comprehensively profiled the humoral immune response in 300 RV144 vaccinees to specifically decipher the relationships between humoral biomarkers of protection and susceptibility. Here, we found that vaccine-specific IgG1, IgG3, and IgA were highly correlated. However, ratios of IgG1:IgG3:IgA provided new insights into subclass/isotype polyclonal functional regulation. For instance, in the absence of high IgG1 levels, IgG3 antibodies exhibited limited functional activity, pointing to IgG3 as a critical contributor, but not sole driver, of more effective antiviral humoral immunity. Moreover, in contrast to previous findings, higher IgA levels were linked to enhanced antibody effector function, including neutrophil phagocytosis (ADNP), complement deposition (ADCD) and NK degranulation (CD107a). Several IgA-associated functions were increased in infected vaccinees in a case:control dataset, suggesting that rather than blocking, IgA may have driven deleterious functions, thereby compromising immunity. These data highlight the interplay between IgG1, IgG3 and IgA, pointing to the critical need to deeply profile the relationships between subclass/isotype selection.
Authors
Stephanie Fischinger, Sepideh Dolatshahi, Madeleine F. Jennewein, Supachai Rerks-Ngarm, Punnee Pitisuttithum, Sorachai Nitayaphan, Nelson L. Michael, Sandhya Vasan, Margaret E Ackerman, Hendrik Streeck, Galit Alter
Abstract
Long non-coding RNAs (lncRNAs) play important roles in regulating diverse cellular processes in the vessel wall, including atherosclerosis. RNAseq profiling of intimal lesions revealed a lncRNA, VINAS (Vascular INfllammation and Atherosclerosis lncRNA Sequence), that is enriched in the aortic intima and regulates vascular inflammation. Aortic intimal expression of VINAS fell with atherosclerotic progression and rose with regression. VINAS knockdown reduced atherosclerotic lesion formation by 55% in LDLR-/- mice, independent of effects on circulating lipids, by decreasing inflammation in the vessel wall. Loss- and gain-of-function studies in vitro demonstrated that VINAS serves as a critical regulator of inflammation by modulating NF-κB and MAPK signaling pathways. VINAS knockdown decreased the expression of key inflammatory markers, such as MCP-1, TNF-α, IL-1β , COX-2, in endothelial (EC), vascular smooth muscle cells, and bone marrow-derived macrophages. Moreover, VINAS silencing decreased expression of leukocyte adhesion molecules VCAM-1, E-selectin, and ICAM-1 and reduced monocyte adhesion to ECs. DEPDC4, an evolutionary conserved human ortholog of VINAS with ~74% homology, shows similar regulation in human and pig atherosclerotic specimens. DEPDC4 knockdown replicated VINAS’ anti-inflammatory effects in human ECs. These findings reveal a novel lncRNA that regulates vascular inflammation, with broad implications for vascular diseases.
Authors
Viorel Simion, Haoyang Zhou, Jacob B. Pierce, Dafeng Yang, Stefan Haemmig, Yevgenia Tesmenitsky, Galina Sukhova, Peter H. Stone, Peter Libby, Mark W. Feinberg
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