INTRODUCTION CSF analysis and other measurements of amyloidosis, such as amyloid-binding positron emission tomography (PET) studies, are limited by cost and availability. There is a need for a more practical Aβ biomarker for central nervous system (CNS) amyloid deposition. METHODS We adapted our previously reported Stable Isotope Labeling Kinetics (SILK) protocol to analyze the turnover kinetics and concentrations of Aβ38, Aβ40, and Aβ42 in human plasma. RESULTS Aβ isoforms have a half-life of approximately three hours in plasma. Aβ38 demonstrated faster turnover kinetics compared to Aβ40 and Aβ42. Faster fractional turnover of Aβ42 relative to Aβ40 and lower Aβ42 and Aβ42/Aβ40 concentrations in amyloid positive participants were observed. DISCUSSION Blood plasma Aβ42 shows similar amyloid-associated alterations as we have previously reported in CSF, suggesting a blood-brain transportation mechanism of Aβ. The stability and sensitivity of plasma Aβ measurements suggests this may be a useful screening test for CNS amyloidosis.

NCT01829932.

A novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was recently identified as the causative agent for the coronavirus disease 2019 (COVID-19) outbreak that has generated a global health crisis. We use a combination of genomic analysis and sensitive profile-based sequence and structure analysis to understand the potential pathogenesis determinants of this virus. As a result, we identify several fast-evolving genomic regions that might be at the interface of virus-host interactions, corresponding to the receptor binding domain of the Spike protein, the three tandem Macro fold domains in ORF1a, and the uncharacterized protein ORF8. Further, we show that ORF8 and several other proteins from alpha- and beta-CoVs belong to novel families of immunoglobulin (Ig) proteins. Among them, ORF8 is distinguished by being rapidly evolving, possessing a unique insert, and having a hypervariable position among SARS-CoV-2 genomes in its predicted ligand-binding groove. We also uncover numerous Ig domain proteins from several unrelated metazoan viruses, which are distinct in sequence and structure but share comparable architectures to those of the CoV Ig domain proteins. Hence, we propose that SARS-CoV-2 ORF8 and other previously unidentified CoV Ig domain proteins fall under the umbrella of a widespread strategy of deployment of Ig domain proteins in animal viruses as pathogenicity factors that modulate host immunity. The rapid evolution of the ORF8 Ig domain proteins points to a potential evolutionary arms race between viruses and hosts, likely arising from immune pressure, and suggests a role in transmission between distinct host species. IMPORTANCE The ongoing COVID-19 pandemic strongly emphasizes the need for a more complete understanding of the biology and pathogenesis of its causative agent SARS-CoV-2. Despite intense scrutiny, several proteins encoded by the genomes of SARS-CoV-2 and other SARS-like coronaviruses remain enigmatic. Moreover, the high infectivity and severity of SARS-CoV-2 in certain individuals make wet-lab studies currently challenging. In this study, we used a series of computational strategies to identify several fast-evolving regions of SARS-CoV-2 proteins which are potentially under host immune pressure. Most notably, the hitherto-uncharacterized protein encoded by ORF8 is one of them. Using sensitive sequence and structural analysis methods, we show that ORF8 and several other proteins from alpha- and beta-coronavirus comprise novel families of immunoglobulin domain proteins, which might function as potential immune modulators to delay or attenuate the host immune response against the viruses.

Background Open label placebos with patient education are effective in reducing chronic pain, and recent studies on their effect on pain have established interest in this field. Nevertheless, data on their effect on acute pain are scarce, and on hyperalgesia and allodynia, absent. This study assessed the effect of open label placebos on acute pain in healthy adult males and the influence of placebo education. Methods Thirty-two healthy males were included in this prospective, randomized, assessor-blinded crossover, single-center study assessing pain intensities (via numeric rating scale), area of hyperalgesia (von Frey filament), and allodynia (dry cotton swab) in a pain model utilizing intracutaneous electrical stimulation. The authors compared the effect of intravenous open label placebo on pain compared to no treatment. The authors further examined the effect of placebo on hyperalgesia and allodynia, and the influence of education (short vs. detailed) before placebo application. Saliva cortisol concentrations were also measured. Results Pain ratings (median, first to third quartile) were 21% lower during placebo treatment compared to no treatment, 4.0 (3.2 to 4.9) versus 5.1 (4.7 to 5.4), respectively (P = 0.001). The areas of hyperalgesia and allodynia were lower during placebo treatment compared to no treatment (hyperalgesia, 30 cm2 [17 to 47] vs. 55 cm2 [42 to 68], P = 0.003; allodynia, 24 cm2 [11 to 39] vs. 45 cm2 [31 to 62], P = 0.007). This corresponds to reductions of 47%. The extent of placebo education had no effect on pain. Saliva cortisol decreased significantly over time and was under the limit of detectability in the majority of participants in postbaseline measurements in both treatment branches. Baseline cortisol was not associated with the placebo effect or strength applied of current to reach defined pain ratings. Conclusions Open label placebos might play a role in multimodal analgesic concepts. Editor’s Perspective What We Already Know about This Topic What This Article Tells Us That Is New

Cavernous sinus syndrome (CSS) is characterized by deficits in more than one of the cranial nerves (CN) that traverse the cavernous sinus at the base of the cranial vault: CN 111 (oculomotor), IV (trochlear), VI (abducens), and the first two branches of CN V (trigeminal). Records from 4 dogs and 8 cats with CSS diagnosed over a 14-year period were reviewed. The most common clinical signs were ophthalmoparesis or ophthalmoplegia, mydriasis with no direct or consensual pupillary light reflexes, ptosis, decreased corneal sensation, and decreased retractor oculi reflex. All cats had initial signs referable to a left CSS lesion (one had bilateral CSS), whereas in all dogs the lesions were localized to the right cavernous sinus. Median ages at diagnosis were 9 and 10 years of age for dogs and cats, respectively. Cerebel lomedullary cisternae cerebrospinal fluid analysis in 6 aniavernous sinus syndrome (CSS) is a recognized syn-C drome in people characterized by variable impairment of multiple cranial nerves (CN) including the oculomotor (III), trochlear (IV), abducens (VI), and the first two branches of the trigeminal nerve (CN V)." The cavernous sinus is a paired venous sinus that lies on each side of the floor in the middle cranial fossa and runs from the orbital fissure to the petro-occipital canal! CN 111, IV, VI, and the ophthalmic branch of V exit the skull through the orbital fissure on their way to innervation of the muscles of the eye.4 The maxillary branch of CN V passes in the dura mater of the lateral wall of the cavernous sinus and exits through the round f~r a m e n .~ This intimate association of CN 111, IV, VI, and the ophthalmic and maxillary branches of CN V is the reason for multiple cranial nerve deficits due to inflammatory or mass lesions in this area.In people, CSS is associated with several disease processes. Neoplasia accounted for approximately 70% of the cases in one study.5 Other causes include infectious and noninfectious inflammatory:,' v a~c u l a r ?~,~ and traumatic lesions.' Only sporadic individual cases of CSS have been described in animals . *-' * This retrospective study describes CSS in 4 dogs and 8 cats. The purpose of this article is to familiarize the clinician with the common clinical signs, diagnostic tests, clinical outcome, and review of applicable neuro-ophthalmology associated with CSS in these species. Materials and Methods Criteria for EnrollmentMedical records from The Ohio State University Veterinary Teaching Hospital were reviewed for all small animals diagnosed with CSS from 1979 to 1993. The criterion for a diagnosis of CSS was a deficit in more than one cranial nerve closely associated with the cavernous sinus: CN 111, IV, VI, and the maxillary and ophthalmic branches of CN V. Diagnostic EvaluationThe diagnostic evaluation of each animal varied. All animals had complete physical, ophthalmologic, and neurological examinations. Serological, biochemical, and hematologic evaluation performed on one or more animals included CBC, serum chemistry profile, feline Provoca...

The coronavirus, SARS-CoV-2, responsible for the ongoing COVID-19 pandemic, has emphasized the need for a better understanding of the evolution of virus-host interactions. ORF3a in both SARS-CoV-1 and SARS-CoV-2 are ion channels (viroporins) implicated in virion assembly and membrane budding. Using sensitive profile-based homology detection methods, we unify the SARS-CoV ORF3a family with several families of viral proteins, including ORF5 from MERS-CoVs, proteins from beta-CoVs (ORF3c), alpha-CoVs (ORF3b), most importantly, the Matrix (M) proteins from CoVs, and more distant homologs from other nidoviruses. We present computational evidence that these viral families might utilize specific conserved polar residues to constitute an aqueous pore within the membrane-spanning region. We reconstruct an evolutionary history of these families and objectively establish the common origin of the M proteins of CoVs and Toroviruses. We also show that the divergent ORF3 clade (ORF3a/ORF3b/ORF3c/ORF5 families) represents a duplication stemming from the M protein in alpha- and beta-CoVs. By phyletic profiling of major structural components of primary nidoviruses, we present a hypothesis for their role in virion assembly of CoVs, ToroVs and Arteriviruses. The unification of diverse M/ORF3 ion channel families in a wide range of nidoviruses, especially the typical M protein in CoVs, reveal a conserved, previously under-appreciated role of ion channels in virion assembly and membrane budding. We show that M and ORF3 are under different evolutionary pressures; in contrast to the slow evolution of M as core structural component, the ORF3 clade is under selection for diversification, which suggests it might act at the interface with host molecules and/or immune attack.

Keywords: CD28 r CTLA-4 r Dendritic cell r Treg cell r T-cell anergy Additional supporting information may be found in the online version of this article at the publisher's web-site IntroductionThe term clonal anergy is used to define a specific, functionally unresponsive state of CD4 + T lymphocytes (initially characterized in Th1 T-cell clones, i.e., previously activated T cells) achieved by strong TCR/CD3 engagement (signal 1) in the absence of CD28 costimulation in vitro (signal 2) [1,2]. Although a variety of experimental approaches have been used to induce T-cell anergy inCorrespondence: Prof. Manfred B. Lutz e-mail: m.lutz@vim.uni-wuerzburg.de vitro, the most consistent hallmarks are defective IL-2 production and lack of proliferation upon TCR restimulation even in the presence of costimulation [2][3][4]. The phenotype of clonal T-cell anergy could be reversed by addition of exogenous IL-2 as CD4 + T-cell clones express high-affinity IL-2 receptors (IL-2Rs) [2]. Reversal of the CD4 + T-cell anergic state can also be achieved by exposure to polyclonal mitogens bypassing TCR signaling, such as phorbol 12-myristate 13-acetate (PMA) and the calcium ionophore ionomycin. However, in contrast to antigen-experienced T-cell clones, naive CD4 + T cells are resistant to anergy induction in vivo and in vitro upon anti-CD3 TCR ligation in the absence of costimulation [5]. Indeed, naive CD4 + T cells appeared to be also dependent on B7 costimulation-driven CTLA-4 engagement for anergy induction [6]. CTLA-4 expression is obligatory for tolerance induction in vivo, also termed adaptive tolerance [2], as judged by studies using T cells from CTLA-4-deficient mice or antibody-mediated blocking experiments [7,8]. The precise role and signaling mechanisms of CTLA-4 for induction of anergy in naive T cells is still a matter of debate [9]. Earlier reports suggested that CTLA-4 signaling prevents cell cycle progression through regulation of the cyclin-dependent kinase inhibitors p27 Kip1 and p21 Cip1 [7,10], although we found that this may not be a strict requirement [11]. Engagement of CTLA-4 has also been shown to block IL-2 production and IL-2R expression at least in part through decreased NFAT translocation to the nucleus [10,12,13]. Conversely, anergy induction in antigen-experienced T cells can be induced by Ca 2+ /calcineurin-dependent signaling through ionomycin only thereby triggering downstream NFATc2, but not its transcriptional binding partner 14,15]. Rao and colleagues identified an NFAT-dependent transcriptional program inducing various anergy-associated genes that crucially contribute to anergy induction in vitro and in vivo [4,14,[16][17][18]. Several analyses revealed a dominant role for the early growth response genes 2 (Egr2) and Egr-3 for expression of the anergy-associated transcriptional program, as identified in clonal anergy and in in vivo anergy models [19][20][21][22][23]. Therefore, Egr2 serves as one of the best markers for anergic T cells. After anergy induction, the question remains which functional...

Background Zinc (Zn) and copper (Cu) are trace elements that serve as cofactors in catalytic processes with impact on immune responses. In patients with inflammatory bowel disease (IBD), decreased levels of serum Zn and Cu have been observed. Here, we investigated the effect of inflammation on serum concentrations of these trace elements in patients with IBD. Methods In this cross-sectional study, 98 patients with Crohn disease (CD) and 56 with ulcerative colitis (UC) were prospectively enrolled. Disease activity parameters, such as C-reactive protein (CRP) and fecal calprotectin (FC) were compared to serum Zn, Cu, and Cu/Zn ratio. Results Zinc insufficiency was observed in 11.2% of patients with CD and 14.3% with UC, Cu insufficiency in 20.4% with CD and 7.1% with UC. Anemia, hypoalbuminemia, increased FC, and elevated CRP were more frequently present in Zn-insufficient patients with IBD. In contrast, lower serum CRP values and a trend to lower FC were observed in Cu-insufficient patients. In multiple linear regression models adjusted for age, gender, and serum albumin, CRP positively correlated with serum Cu (P < 0.001) and the Cu/Zn ratio in both CD and UC (P < 0.001) but not with serum Zn concentrations. FC levels correlated only with the Cu/Zn ratio in patients with UC (P < 0.038). Conclusion Systemic inflammation inversely affected the serum Zn and Cu concentrations and, consequently, resulted in an increased Cu/Zn ratio.