Accumulation of mutations in antibody and CD8 T cell epitopes in a B cell depleted lymphoma patient with chronic SARS-CoV-2 infection.

Antibodies against the spike protein of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) can drive adaptive evolution in immunocompromised patients with chronic infection. Here we longitudinally analyze SARS-CoV-2 sequences in a B cell-depleted, lymphoma patient with chronic, ultimately fatal infection, and identify three mutations in the spike protein that dampen convalescent plasma-mediated neutralization of SARS-CoV-2. Additionally, four mutations emerge in non-spike regions encoding three CD8 T cell epitopes, including one nucleoprotein epitope affected by two mutations. Recognition of each mutant peptide by CD8 T cells from convalescent donors is reduced compared to its ancestral peptide, with additive effects resulting from double mutations. Querying public SARS-CoV-2 sequences shows that these mutations have independently emerged as homoplasies in circulating lineages. Our data thus suggest that potential impacts of CD8 T cells on SARS-CoV-2 mutations, at least in those with humoral immunodeficiency, warrant further investigation to inform on vaccine design.

Association between IgG responses against the nucleocapsid proteins of alphacoronaviruses and COVID-19 severity.

Understanding immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is crucial to contain the COVID-19 pandemic. Using a multiplex approach, serum IgG responses against the whole SARS-CoV-2 proteome and the nucleocapsid proteins of endemic human coronaviruses (HCoVs) were measured in SARS-CoV-2-infected donors and healthy controls. COVID-19 severity strongly correlated with IgG responses against the nucleocapsid (N) of SARS-CoV-2 and possibly with the number of viral antigens targeted. Furthermore, a strong correlation between COVID-19 severity and serum responses against N of endemic alpha- but not betacoronaviruses was detected. This correlation was neither caused by cross-reactivity of antibodies, nor by a general boosting effect of SARS-CoV-2 infection on pre-existing humoral immunity. These findings raise the prospect of a potential disease progression marker for COVID-19 severity that allows for early stratification of infected individuals.

The molecular ontogeny of follicular lymphoma: gene mutations succeeding the BCL2 translocation define common precursor cells.

Relapsed follicular lymphoma (FL) can arise from common progenitor cells (CPCs). Conceptually, CPC-defining mutations are somatic alterations shared by the initial and relapsed tumours, mostly B-cell leukaemia/lymphoma 2 (BCL2)/immunoglobulin heavy locus (IGH) translocations and other recurrent gene mutations. Through complementary approaches for highly sensitive mutation detection, we do not find CPC-defining mutations in highly purified BCL2/IGH-negative haematopoietic progenitor cells in clinical remission samples from three patients with relapsed FL. Instead, we find cells harbouring the same BCL2/IGH translocation but lacking CREB binding protein (CREBBP), lysine methyltransferase 2D (KMT2D) and other recurrent gene mutations. Thus, (i) the BCL2/IGH translocation can precede CPC-defining mutations in human FL, and (ii) BCL2/IGH-translocated cells can persist in clinical remission.

A central role of IKK2 and TPL2 in JNK activation and viral B-cell transformation.

IκB kinase 2 (IKK2) is well known for its pivotal role as a mediator of the canonical NF-κB pathway, which has important functions in inflammation and immunity, but also in cancer. Here we identify a novel and critical function of IKK2 and its co-factor NEMO in the activation of oncogenic c-Jun N-terminal kinase (JNK) signaling, induced by the latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV). Independent of its kinase activity, the TGFß-activated kinase 1 (TAK1) mediates LMP1 signaling complex formation, NEMO ubiquitination and subsequent IKK2 activation. The tumor progression locus 2 (TPL2) kinase is induced by LMP1 via IKK2 and transmits JNK activation signals downstream of IKK2. The IKK2-TPL2-JNK axis is specific for LMP1 and differs from TNFα, Interleukin-1 and CD40 signaling. This pathway mediates essential LMP1 survival signals in EBV-transformed human B cells and post-transplant lymphoma, and thus qualifies as a target for treatment of EBV-induced cancer.

An Exploratory Step Toward Measuring the "Meaning of Life" in Patients with Tinnitus and in Cochlear Implant Users.

BACKGROUND: Many questionnaires attempt to quantify the "quality of life." However, we believe understanding the quality of life is complex, and many widely used questionnaires do not capture the broad range of factors that we believe are important. Many do not include questions about communicating. PURPOSE: We developed a preliminary questionnaire designed to measure "The Meaning of Life" from a broader perspective. RESEARCH DESIGN: We reviewed other scales and sought input from individuals with disabilities and developed an initial 23-item questionnaire. STUDY SAMPLE: As a first step, we sampled 116 adults with tinnitus and 196 with cochlear implants (CIs). Individuals who were participating in our CI or tinnitus research programs participated. DATA COLLECTION AND ANALYSIS: To compare differences between the two participant groups, independent sample f-tests were completed for specific items on the questionnaire and for the total score. We compared age and gender differences across all participants using f-tests. Statistical significance was defined as p < 0.05. An exploratory factor analysis was conducted to examine the relationship among the questionnaire items using oblique rotation to produce correlated factors. Extracted factors with an eigenvalue >1.0 were retained according to the Kaiser-Guttman rule. RESULTS: Four factors were prominent in this initial sample, which we labeled (1) friendship and positive outlook, (2) physical health, (3) hearing and mental health, and (4) satisfaction with life. Participants with tinnitus reported more trouble sleeping than participants with CI, whereas both groups had lower scores on hearing. Older patients reported more difficulty with remembering things but were more satisfied with their financial situation. Female participants reportedly had more hobbies and were more satisfied with their sex lives than male participants. CONCLUSIONS: This exploratory study intended to take a broader look at quality of life scales. Further work is needed with a larger sample including younger and older participants with and without disabilities.

Tinnitus: How Partners Can Help?

Purpose Tinnitus can be distressing for sufferers, but for those who do not have tinnitus, it is difficult to understand what it is like. We attempted to gain an understanding of the knowledge and misconceptions of sufferers and their partners about tinnitus. Method Two different websites were created with surveys, 1 for tinnitus sufferers and the other for their partners. A mass e-mail was sent to employees and students at The University of Iowa inviting people with tinnitus and their significant others to fill out the online surveys. Those with tinnitus were asked to rate how their tinnitus affected their thoughts and emotions, sleep, concentration, and hearing. They were also invited to fill out the Iowa Tinnitus Primary Function Questionnaire (12-item version; Tyler et al., 2014 ). Partners completed a similar survey that asked how tinnitus affected the sufferer in these domains. Open-ended questions were also included to obtain more specific feedback from the participants regarding their experiences and how tinnitus affects their lifestyle and relationships. Results Two hundred twenty-two replies were obtained from 197 tinnitus sufferers and 25 partners of those with tinnitus. Partners and sufferers were not completely in agreement regarding their knowledge about tinnitus or familiar with the impact that this symptom may have on the sufferers. Sufferers showed more confidence in their hearing ability, regardless of tinnitus, than their partners. Furthermore, sufferers and partners do not generally talk about tinnitus to each other. Conclusion We conclude that both sufferers and partners would benefit from receiving counseling to address many misunderstandings regarding tinnitus and its consequences in their everyday life activities.

Intracellular signaling prevents effective blockade of oncogenic gp130 mutants by neutralizing antibodies.

BACKGROUND: Short in-frame deletions in the second extracellular domain of the cytokine receptor gp130 are the leading cause of inflammatory hepatocellular adenomas (IHCAs). The deletions render gp130 constitutively active. In this study we investigate the intracellular signaling potential of one of the most potent constitutively active gp130 mutants (CAgp130) found in IHCAs. RESULTS: Trafficking and signaling of CAgp130 were studied in stably transfected cell lines that allowed the inducible expression of CAgp130 fused to fluorescent proteins such as YFP and mCherry. In contrast to the predominantly highly glycosylated gp130 wild type (WTgp130), CAgp130 is preferentially found in the less glycosylated high-mannose form. Accordingly, the mutated receptor is retained intracellularly and therefore less prominently expressed at the cell surface. CAgp130 persistently activates Stat3 despite the presence of the feedback inhibitor SOCS3 but fails to activate Erk1/2. De novo synthesized CAgp130 signals already from the ER-Golgi compartment before having reached the plasma membrane. Cell surface expressed and endocytosed CAgp130 do not significantly contribute to signaling. As a consequence, Stat3 activation through CAgp130 cannot be inhibited by neutralizing gp130 antibodies but through overexpression of a dominant-negative Stat3 mutant. CONCLUSION: CAgp130 and WTgp130 differ significantly with respect to glycosylation, trafficking and signaling. As a consequence of intracellular signaling pharmacological inhibition of CAgp130 will not be achieved by targeting the receptor extracellularly but by compounds that act from within the cell.

Dominant-negative activity of the STAT3-Y705F mutant depends on the N-terminal domain.

BACKGROUND: STAT3 is a transcription factor of central importance in chronic inflammation and cancer. In response to cytokine stimulation STAT3 is phosphorylated on a single tyrosine residue at position 705, dimerizes and accumulates in the nucleus to induce target gene expression. The substitution of tyrosine 705 to phenylalanine leads to a dominant-negative STAT3 mutant (STAT3-YF) which influences the activation of WT-STAT3 in stimulated cells through a mechanism that is not completely understood. In this study we analyzed the molecular mechanism of STAT3-YF dominant-negative activity in IL-6-induced STAT3 signaling and the relevance of the N-terminal domain. RESULTS: Expression of STAT3-YF-YFP impairs tyrosine phosphorylation, nuclear translocation and the transcriptional activity of WT-STAT3 in IL-6-stimulated cells. The fluorescently labelled STAT3-YF mutant binds to a phosphorylated gp130 receptor-peptide comparable to WT-STAT3-YFP. STAT3-YF-YFP forms homodimers as well as heterodimers with WT-STAT3 in the presence and absence of IL-6. The preformed heterodimers in unstimulated cells are detectable by colocalization of STAT3-CFP with STAT3-YF-YFP fused to a nuclear localization signal. STAT3/STAT3-YF heterodimers are not able to bind to DNA in stimulated cells, but the presence of the mutant reduces DNA-binding of WT-STAT3 homodimers. STAT3-YF-ΔN-YFP lacking the N-terminal domain forms no dimers and only marginally affects the activity of WT-STAT3. CONCLUSION: Our findings demonstrate that dominant-negative STAT3-YF affects the activation of WT-STAT3 at multiple levels. Unexpectedly, the N-terminal domain of STAT3-YF plays an important role for the dominant-negative effect. We show that (i) STAT3-YF competes with WT-STAT3 in binding to activated gp130-receptors, (ii) the formation of WT-STAT3/STAT3-YF heterodimers in IL-6-stimulated cells results in inactive, semiphosphorylated dimers which do not bind to DNA and thus fail to induce target gene expression, (iii) the N-terminal domain-mediated formation of preformed STAT3/STAT3-YF heterodimers in unstimulated cells which affects the IL-6-induced homodimerization of WT-STAT3 contributes to the dominant-negative effect of STAT3-YF. These findings will contribute to our understanding of naturally occuring dominant-negative STAT3 mutants that cause the hyper-IgE syndrome.

Dynamics and non-canonical aspects of JAK/STAT signalling.

The Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway directly links ligand-binding to a membrane-bound receptor with the activation of a transcription factor. This signalling module enables the cell to rapidly initiate a transcriptional response to external stimulation. The main components of this evolutionary conserved module are cytokines that specifically bind to cytokine receptors leading to the activation of receptor-associated Janus tyrosine kinases (JAKs). The receptor-bound JAKs activate STAT transcription factors through phosphorylation of a single tyrosine residue. Activated STAT dimers translocate into the nucleus to induce target gene expression. In this article we will review current opinions on the molecular mechanism and on intracellular dynamics of JAK/STAT signalling with a special focus on the cytokine receptor glycoprotein 130 (gp130) and STAT3. In particular we will concentrate on non-canonical aspects of Jak/STAT signalling including preassembled receptor complexes, preformed STAT dimers, STAT trafficking and non-canonical functions of STATs.