<p><div>(<em>b</em>)BACKGROUND</<em>b</em>)</div>Cystic fi<em>b</em>rosis is caused <em>b</em>y mutations in the gene encoding the cystic fi<em>b</em>rosis transmem<em>b</em>rane conductance regulator (CFTR) protein, and nearly 90% of patients have at least one copy of the Phe508del <i>CFTR</i> mutation. In a phase 2 trial involving patients who were heterozygous for the Phe508del <i>CFTR</i> mutation and a minimal-function mutation (Phe508del-minimal function genotype), the next-generation CFTR corrector elexacaftor, in com<em>b</em>ination with tezacaftor and ivacaftor, improved Phe508del CFTR function and clinical outcomes.</p><p><div>(<em>b</em>)METHODS</<em>b</em>)</div>We conducted a phase 3, randomized, dou<em>b</em>le-<em>b</em>lind, place<em>b</em>o-controlled trial to confirm the efficacy and safety of elexacaftor-tezacaftor-ivacaftor in patients 12 years of age or older with cystic fi<em>b</em>rosis with Phe508del-minimal function genotypes. Patients were randomly assigned to receive elexacaftor-tezacaftor-ivacaftor or place<em>b</em>o for 24 weeks. The primary end point was a<em>b</em>solute change from <em>b</em>aseline in percentage of predicted forced expiratory volume in 1 second (FEV<su<em>b</em>)1</su<em>b</em>)) at week 4.</p><p><div>(<em>b</em>)RESULTS</<em>b</em>)</div>A total of 403 patients underwent randomization and received at least one dose of active treatment or place<em>b</em>o. Elexacaftor-tezacaftor-ivacaftor, relative to place<em>b</em>o, resu<em>lt</em>ed in a percentage of predicted FEV<su<em>b</em>)1</su<em>b</em>) that was 13.8 points higher at 4 weeks and 14.3 points higher through 24 weeks, a rate of pulmonary exacer<em>b</em>ations that was 63% lower, a respiratory domain score on the Cystic Fi<em>b</em>rosis Questionnaire-Revised (range, 0 to 100, with higher scores indicating a higher patient-reported quality of life with regard to respiratory symptoms; minimum clinically important difference, 4 points) that was 20.2 points higher, and a sweat chloride concentration that was 41.8 mmol per liter lower (P&<em>lt</em>;0.001 for all comparisons). Elexacaftor-tezacaftor-ivacaftor was generally safe and had an accepta<em>b</em>le side-effect profile. Most patients had adverse events that were mild or moderate. Adverse events leading to discontinuation of the trial regimen occurred in 1% of the patients in the elexacaftor-tezacaftor-ivacaftor group.</p><A<em>b</em>stractText>Elexacaftor-tezacaftor-ivacaftor was efficacious in patients with cystic fi<em>b</em>rosis with Phe508del-minimal function genotypes, in whom previous CFTR modulator regimens were ineffective. (Funded <em>b</em>y Vertex Pharmaceuticals; VX17-445-102 ClinicalTrials.gov num<em>b</em>er, NCT03525444.).</A<em>b</em>stractText>

<A<em>b</em>stractText>Senescent cells, which can release factors that cause inflammation and dysfunction, the senescence-associated secretory phenotype (SASP), accumulate with ageing and at etiological sites in mu<em>lt</em>iple chronic diseases. Senolytics, including the com<em>b</em>ination of Dasatini<em>b</em> and Quercetin (D + Q), selectively eliminate senescent cells <em>b</em>y transiently disa<em>b</em>ling pro-survival networks that defend them against their own apoptotic environment. In the first clinical trial of senolytics, D + Q improved physical function in patients with idiopathic pulmonary fi<em>b</em>rosis (IPF), a fatal senescence-associated disease, <em>b</em>ut to date, no peer-reviewed study has directly demonstrated that senolytics decrease senescent cells in humans.</A<em>b</em>stractText><p><div>(<em>b</em>)METHODS</<em>b</em>)</div>In an open la<em>b</em>el Phase 1 pilot study, we administered 3 days of oral D 100 mg and Q 1000 mg to su<em>b</em>jects with dia<em>b</em>etic kidney disease (N = 9; 68·7 ± 3·1 years old; 2 female; BMI:33·9 ± 2·3 kg/m²; eGFR:27·0 ± 2·1 mL/min/1·73m²). Adipose tissue, skin <em>b</em>iopsies, and <em>b</em>lood were collected <em>b</em>efore and 11 days after completing senolytic treatment. Senescent cell and macrophage/Langerhans cell markers and circulating SASP factors were assayed.</p><p><div>(<em>b</em>)FINDINGS</<em>b</em>)</div>D + Q reduced adipose tissue senescent cell <em>b</em>urden within 11 days, with decreases in p16^INK4A-and p21^CIP1-expressing cells, cells with senescence-associated <em>β</em>-galactosidase activity, and adipocyte progenitors with limited replicative potential. Adipose tissue macrophages, which are attracted, anchored, and activated <em>b</em>y senescent cells, and crown-like structures were decreased. Skin epidermal p16^INK4A+ and p21^CIP1+ cells were reduced, as were circulating SASP factors, including IL-1α, IL-6, and MMPs-9 and -12.</p><A<em>b</em>stractText>"Hit-and-run" treatment with senolytics, which in the case of D + Q have elimination half-lives &<em>lt</em>;11 h, significantly decreases senescent cell <em>b</em>urden in humans. FUND: NIH and Foundations. ClinicalTrials.gov Identifier: NCT02848131. Senescence, Frai<em>lt</em>y, and Mesenchymal Stem Cell Functionality in Chronic Kidney Disease: Effect of Senolytic Agents.</A<em>b</em>stractText>

The formation of lymph nodes (LN) and Peyer's patches (PP) can be distinguished by the requirement of RANK for LN but not IL-7R(alpha), which is essential for PP development. However, lymphotoxin-alphabeta (LT(alpha)beta) signaling is required for both organs. The cellular basis underlying this dichotomy was revealed by the finding that the fetal IL-7R(alpha)(+) population responded equally well to IL-7 and RANKL to express LT(alpha)beta. IL-7R(alpha)(+) cells harvested from TRAF6(-/-) embryos expressed LTalphabeta in response to IL-7 but not RANKL, demonstrating that the RANK-TRAF6 signaling pathway regulates LT(alpha)beta expression in LN but not in PP. Soluble IL-7 administered to TRAF6(-/-) embryos was sufficient to restore LN genesis indicating the functional similarities of the IL-7R(alpha)(+) inducer cells for LN and PP genesis.

Purpose: The immunomodulatory effect of lenvatinib (a multikinase inhibitor) on tumor microenvironments may contribute to antitumor activity when combined with programmed death receptor-1 (PD-1) signaling inhibitors in hepatocellular carcinoma (HCC). We report results from a phase Ib study of lenvatinib plus pembrolizumab (an anti-PD-1 antibody) in unresectable HCC (uHCC).

Patients and methods: In this open-label multicenter study, patients with uHCC received lenvatinib (bodyweight ≥ 60 kg, 12 mg; &lt; 60 kg, 8 mg) orally daily and pembrolizumab 200 mg intravenously on day 1 of a 21-day cycle. The study included a dose-limiting toxicity (DLT) phase and an expansion phase (first-line patients). Primary objectives were safety/tolerability (DLT phase), and objective response rate (ORR) and duration of response (DOR) by modified RECIST (mRECIST) and RECIST version 1.1 (v1.1) per independent imaging review (IIR; expansion phase).

Results: A total of 104 patients were enrolled. No DLTs were reported (n = 6) in the DLT phase; 100 patients (expansion phase; included n = 2 from DLT phase) had received no prior systemic therapy and had Barcelona Clinic Liver Cancer stage B (n = 29) or C disease (n = 71). At data cutoff, 37% of patients remained on treatment. Median duration of follow-up was 10.6 months (95% CI, 9.2 to 11.5 months). Confirmed ORRs by IIR were 46.0% (95% CI, 36.0% to 56.3%) per mRECIST and 36.0% (95% CI, 26.6% to 46.2%) per RECIST v1.1. Median DORs by IIR were 8.6 months (95% CI, 6.9 months to not estimable [NE]) per mRECIST and 12.6 months (95% CI, 6.9 months to NE) per RECIST v1.1. Median progression-free survival by IIR was 9.3 months per mRECIST and 8.6 months per RECIST v1.1. Median overall survival was 22 months. Grade ≥ 3 treatment-related adverse events occurred in 67% (grade 5, 3%) of patients. No new safety signals were identified.

Conclusion: Lenvatinib plus pembrolizumab has promising antitumor activity in uHCC. Toxicities were manageable, with no unexpected safety signals.

The TNF receptor superfamily members are all type I membrane glycoproteins with typical homology in the extracellular domain of variable numbers of cysteine-rich repeats (overall homologies, 25% to 30%). In contrast, the TNF ligand superfamily members (with the exception of LT alpha) are type II membrane glycoproteins with homology to TNF in the extracellular domain (overall homologies, 20%). TNF and LT alpha are trimeric proteins and are composed of beta-strands forming a beta-jellyroll. The homology of the beta-strand regions for the TNF ligand superfamily members suggest a similar beta-sandwich structure and possible trimeric or multimeric complex formation for most or all members. A genetic linkage, as evidence for evolutionary relatedness, is found by chromosomal cluster of TNFR p80, CD30, 4-1BB, and OX40 for 1p36; TNFR p60, TNFR-RP, and CD27 for 12p13; TNF, LT alpha, and LT beta for 6 (MHC locus); CD27L and 4-1BBL for 19p13; and FASL and OX40L for 1q25. Of the TNF ligand superfamily, TNF, LT alpha, and LT beta and their receptors (TNFR p60, TNFR p80, and TNFR-RP) interact in a complex fashion of cross-binding. However, the other family members presently have a one ligand/one receptor binding principle (CD27/CD27L, CD30/CD30L, CD40/CD40L, 4-1BB/4-1BBL, OX40/gp34, and FAS/FASL). In general, the members of the TNF ligand superfamily mediate interaction between different hematopoietic cells, such as T cell/B cell, T cell/monocyte, and T cell/T cell. Signals can be transduced not only through the receptors but also through at least some of the ligands. The transduced signals can be stimulatory or inhibitory depending on the target cell or the activation state. Taken together, TNF superfamily ligands show for the immune response an involvement in the induction of cytokine secretion and the upregulation of adhesion molecules, activation antigens, and costimulatory proteins, all known to amplify stimulatory and regulatory signals. On the other hand, differences in the distribution, kinetics of induction, and requirements for induction support a defined role for each of the ligands for T-cell-mediated immune responses. The shedding of members of the TNF receptor superfamily could limit the signals mediated by the corresponding ligands as a functional regulatory mechanism. Induction of cytotoxic cell death, observed for TNF, LT alpha, CD30L, CD95L, and 4-1BBL, is another common functional feature of this cytokine family. Further studies have to identify unique versus redundant biologic and physiologic functions for each of the TNF superfamily ligands.(ABSTRACT TRUNCATED AT 400 WORDS)

<A<em>b</em>stractText>Since the out<em>b</em>reak of Coronavirus Disease 2019 (COVID-19) in China in Decem<em>b</em>er 2019, considera<em>b</em>le attention has <em>b</em>een focused on its elucidation. However, it is also important for clinicians and epidemiologists to differentiate COVID-19 from other respiratory infectious diseases, such as influenza viruses.</A<em>b</em>stractText><A<em>b</em>stractText>The aim of the study was to explore the different clinical presentations <em>b</em>etween COVID-19 and influenza A (H1N1) pneumonia in patients with acute respiratory distress syndrome (ARDS).</A<em>b</em>stractText><A<em>b</em>stractText>and Methods: This was a retrospective case-control study. We compared two independent cohorts of ARDS patients infected with either COVID-19 (n=73) or H1N1 (n=75). We analyzed and compared their clinical manifestations, imaging characteristics, treatments, and prognosis.</A<em>b</em>stractText><p><div>(<em>b</em>)RESULTS</<em>b</em>)</div>The median age of COVID-19 patients was higher than that of H1N1 patients, and there was a higher proportion of males among COVID-19 patients (p&<em>lt</em>;0.05). COVID-19 patients exhi<em>b</em>ited higher proportions of non-productive coughs, fatigue, and gastrointestinal symptoms than those of H1N1 patients (p&<em>lt</em>;0.05). H1N1 patients had higher sequential organ failure assessment (SOFA) scores than COVID-19 patients (p&<em>lt</em>;0.05). The PaO<su<em>b</em>)2</su<em>b</em>)/FiO<su<em>b</em>)2</su<em>b</em>) of 198.2 mmHg in COVID-19 patients was significantly higher than the PaO<su<em>b</em>)2</su<em>b</em>)/FiO<su<em>b</em>)2</su<em>b</em>) of 107.0 mmHg of H1N1 patients (p&<em>lt</em>;0.001). Ground-glass opacities was more common in COVID-19 patients than in H1N1 patients (p&<em>lt</em>;0.001). There was a greater variety of antiviral therapies administered to COVID-19 patients than to H1N1 patients. The in-hospital mortality of COVID-19 patients was 28.8%, while that of H1N1 patients was 34.7% (p=0.483). SOFA-score adjusted mortality of H1N1 patients was significantly higher than that of COVID-19 patients with the rate ratio was 2.009 (95% CI [1.563, 2.583], p&<em>lt</em>;0.001).</p><A<em>b</em>stractText>There were many differences <em>b</em>etween COVID-19 and H1N1-induced ARDS patients in clinical presentations. Compared with H1N1, patients with COVID-19 induced ARDS had lower severity of illness scores at presentation and lower SOFA-score adjusted mortality.</A<em>b</em>stractText>

<p><div>(<em>b</em>)BACKGROUND</<em>b</em>)</div>Patients with relapsed or refractory acute myeloid leukemia (AML) with mutations in the FMS-like tyrosine kinase 3 gene (<i>FLT3</i>) infrequently have a response to salvage chemotherapy. Gi<em>lt</em>eritini<em>b</em> is an oral, potent, selective FLT3 inhi<em>b</em>itor with single-agent activity in relapsed or refractory <i>FLT3</i>-mutated AML.</p><p><div>(<em>b</em>)METHODS</<em>b</em>)</div>In a phase 3 trial, we randomly assigned adu<em>lt</em>s with relapsed or refractory <i>FLT3</i>-mutated AML in a 2:1 ratio to receive either gi<em>lt</em>eritini<em>b</em> (at a dose of 120 mg per day) or salvage chemotherapy. The two primary end points were overall survival and the percentage of patients who had complete remission with full or partial hematologic recovery. Secondary end points included event-free survival (freedom from treatment failure [i.e., relapse or lack of remission] or death) and the percentage of patients who had complete remission.</p><A<em>b</em>stractText>Of 371 eligi<em>b</em>le patients, 247 were randomly assigned to the gi<em>lt</em>eritini<em>b</em> group and 124 to the salvage chemotherapy group. The median overall survival in the gi<em>lt</em>eritini<em>b</em> group was significantly longer than that in the chemotherapy group (9.3 months vs. 5.6 months; hazard ratio for death, 0.64; 95% confidence interval [CI], 0.49 to 0.83; P&<em>lt</em>;0.001). The median event-free survival was 2.8 months in the gi<em>lt</em>eritini<em>b</em> group and 0.7 months in the chemotherapy group (hazard ratio for treatment failure or death, 0.79; 95% CI, 0.58 to 1.09). The percentage of patients who had complete remission with full or partial hematologic recovery was 34.0% in the gi<em>lt</em>eritini<em>b</em> group and 15.3% in the chemotherapy group (risk difference, 18.6 percentage points; 95% CI, 9.8 to 27.4); the percentages with complete remission were 21.1% and 10.5%, respectively (risk difference, 10.6 percentage points; 95% CI, 2.8 to 18.4). In an analysis that was adjusted for therapy duration, adverse events of grade 3 or higher and serious adverse events occurred less frequently in the gi<em>lt</em>eritini<em>b</em> group than in the chemotherapy group; the most common adverse events of grade 3 or higher in the gi<em>lt</em>eritini<em>b</em> group were fe<em>b</em>rile neutropenia (45.9%), anemia (40.7%), and throm<em>b</em>ocytopenia (22.8%).</A<em>b</em>stractText><p><div>(<em>b</em>)CONCLUSIONS</<em>b</em>)</div>Gi<em>lt</em>eritini<em>b</em> resu<em>lt</em>ed in significantly longer survival and higher percentages of patients with remission than salvage chemotherapy among patients with relapsed or refractory <i>FLT3</i>-mutated AML. (Funded <em>b</em>y Astellas Pharma; ADMIRAL ClinicalTrials.gov num<em>b</em>er, NCT02421939.).</p>

(<em>b</em>)Introduction:</<em>b</em>) A recently emerging respiratory disease named coronavirus disease 2019 (COVID-19) has quickly spread across the world. This disease is initiated <em>b</em>y severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and uncontrolled cytokine storm, <em>b</em>ut it remains unknown as to whether a ro<em>b</em>ust anti<em>b</em>ody response is related to clinical deterioration and poor outcome in COVID-19 patients. (<em>b</em>)Methods:</<em>b</em>) Anti-SARS-CoV-2 IgG and IgM anti<em>b</em>odies were determined <em>b</em>y chemiluminescence analysis (CLIA) in COVID-19 patients at a single center in Wuhan. Median IgG and IgM levels in acute and convalescent-phase sera (within 35 days) for all included patients were calculated and compared <em>b</em>etween severe and non-severe patients. Immune response phenotyping <em>b</em>ased on the late IgG levels and neutrophil-to-lymphocyte ratio (NLR) was characterized to stratified patients into different disease severities and outcomes. (<em>b</em>)Resu<em>lt</em>s:</<em>b</em>) A total of 222 patients were included in this study. IgG was first detected on day 4 of illness, and its peak levels occurred in the fourth week. Severe cases were more frequently found in patients with high IgG levels, compared to those with low IgG levels (51.8 vs. 32.3%; <i>p</i> = 0.008). Severity rates for patients with NLR^hiIgG^hi, NLR^hiIgG^lo, NLR^loIgG^hi, and NLR^loIgG^lo phenotype were 72.3, 48.5, 33.3, and 15.6%, respectively (<i>p</i> &<em>lt</em>; 0.0001). Furthermore, severe patients with NLR^hiIgG^hi, NLR^hiIgG^lo had higher inflammatory cytokines levels including IL-2, IL-6 and IL-10, and decreased CD4+ T cell count compared to those with NLR^loIgG^lo phenotype (<i>p</i> &<em>lt</em>; 0.05). Recovery rates for severe patients with NLR^hiIgG^hi, NLR^hiIgG^lo, NLR^loIgG^hi, and NLR^loIgG^lo phenotype were 58.8% (20/34), 68.8% (11/16), 80.0% (4/5), and 100% (12/12), respectively (<i>p</i> = 0.0592). Dead cases only occurred in NLR^hiIgG^hi and NLR^hiIgG^lo phenotypes. (<em>b</em>)Conclusions:</<em>b</em>) COVID-19 severity is associated with increased IgG response, and an immune response phenotyping <em>b</em>ased on the late IgG response and NLR could act as a simple complementary tool to discriminate <em>b</em>etween severe and non-severe COVID-19 patients, and further predict their clinical outcome.

Keywords: COVID-19; IgG; clinical outcome; disease severity; neutrophil-to-lymphocyte ratio.

Recently, the heat-labile enterotoxin (LT) of Escherichia coli has been purified to homogeneity and partially characterized (Clements and Finkelstein, Infect. Immun. 24:760-769, 1979). This study extends our observations on the physicochemical properties of LT. The toxin has an isoelectric point of pH 8.0, as compared with choleragen and choleragenoid, which have isoelectric points of pH 6.75 and 7.75, respectively. Sedimentation equilibrium measurements established an approximate molecular weight for LT of 91,440. LT had an even more marked affinity than choleragen for agarose-containing matrixes in gel filtration. Of several mono- and disaccharides tested, only galactose and lactose were highly efficient in removing 125I-labeled LT from agarose-containing columns. LT dissociated into subunits (designated A and B) during gel filtration in the presence of 5 M guanidine. These subunits were immunologically distinct and possessed unique and shared antigenic determinants to the corresponding A and B subunits of choleragen. During gel filtration of LT at pH 6.5 and room temperature, a spontaneously occurring toxoid of LT, analogous to choleragenoid, was discovered and designated "coligenoid." This product contains only the B subunits of the toxin. A partial amino acid sequence of the B subunit of LT revealed a remarkable homology to the primary structure of cholera toxin B. Within the first 20 amino acids of the two chains, only 5 differ, and these differences may be attributable to single base substitutions.

Heat-labile Escherichia coli enterotoxin (LT) has the innate property of being a strong mucosal immunogen and adjuvant. In the attempt to reduce toxicity and maintain the useful immunological properties, several LT mutants have been produced. Some of these are promising mucosal adjuvants. However, so far, only those that were still toxic maintained full adjuvanticity. In this paper we describe a novel LT mutant with greatly reduced toxicity that maintains most of the adjuvanticity. The new mutant (LTR72), that contains a substitution Ala ->> Arg in position 72 of the A subunit, showed only 0.6% of the LT enzymatic activity, was 100,000-fold less toxic than wild-type LT in Y1 cells in vitro, and was at least 20 times less effective than wild-type LT in the rabbit ileal loop assay in vivo. At a dose of 1 microg, LTR72 exhibited a mucosal adjuvanticity, similar to that observed with wild-type LT, better than that induced by the nontoxic, enzymatically inactive LTK63 mutant, and much greater than that of the recombinant B subunit. This trend was consistent for both the amounts and kinetics of the antibody induced, and priming of antigen-specific T lymphocytes. The data suggest that the innate high adjuvanticity of LT derives from the independent contribution of the nontoxic AB complex and the enzymatic activity. LTR72 optimizes the use of both properties: the enzymatic activity for which traces are enough, and the nontoxic AB complex, the effect of which is dose dependent. In fact, in dose-response experiments in mice, 20 microg of LTR72 were a stronger mucosal adjuvant than wild-type LT. This suggests that LTR72 may be an excellent candidate to be tested in clinical trials.

An assay was developed to detect the cytotoxic effects of cytokines on rat pancreatic islet cells in monolayer culture. Cell lysis was detected by a 51Cr-release assay after 4 days of incubation with various cytokines. When tested alone, murine (rat and mouse) interferon-gamma (mIFN-gamma) produced a small dose-dependent lysis of islet cells; human IFN-gamma, mouse IFN-alpha/beta, interleukins 1 and 2 (IL-1 and IL-2), tumor necrosis factor (TNF), and lymphotoxin (LT) were inactive. When added together, the following combinations of cytokines showed synergistic cytotoxic effects: TNF (or LT) plus IL-1, TNF (or LT) plus mIFN-gamma, and IL-1 plus mIFN-gamma. These results indicate that the cytokine products of mononuclear cells of the immune system, IFN-gamma, TNF, LT, and IL-1 have strong synergistic cytotoxic effects on islet cells and therefore may act as direct chemical mediators of islet beta-cell destruction in type I (insulin-dependent) diabetes.

Although self-renewal is the central property of stem cells, the underlying mechanism remains inadequately defined. Using a hematopoietic stem and progenitor cell (HSPC)-specific conditional induction line, we generated a compound genetic model bearing both Pten deletion and β-catenin activation. These double mutant mice exhibit a novel phenotype, including expansion of phenotypic long-term hematopoietic stem cells (LT-HSCs) without extensive differentiation. Unexpectedly, constitutive activation of β-catenin alone results in apoptosis of HSCs. However, together, the Wnt/β-catenin and PTEN/PI3k/Akt pathways interact to drive phenotypic LT-HSC expansion by inducing proliferation while simultaneously inhibiting apoptosis and blocking differentiation, demonstrating the necessity of complementary cooperation between the two pathways in promoting self-renewal. Mechanistically, β-catenin activation reduces multiple differentiation-inducing transcription factors, blocking differentiation partially through up-regulation of Inhibitor of differentiation 2 (Id2). In double mutants, loss of Pten enhances the HSC anti-apoptotic factor Mcl-1. All of these contribute in a complementary way to HSC self-renewal and expansion. While permanent, genetic alteration of both pathways in double mutant mice leads to expansion of phenotypic HSCs, these HSCs cannot function due to blocked differentiation. We developed a pharmacological approach to expand normal, functional HSCs in culture using factors that reversibly activate both Wnt/β-catenin and PI3K/Akt signaling simultaneously. We show for the first time that activation of either single pathway is insufficient to expand primitive HSCs, but in combination, both pathways drive self-renewal and expansion of HSCs with long-term functional capacity.

Hybridization probes derived from the A and B subunit genes of the heat-labile enterotoxin (LT) of Escherichia coli were used to analyze DNA from Vibrio cholera strain 569B for cholera toxin gene sequences. Southern blot analysis indicated that the cholera toxin A and B subunit genes were each duplicated in the strain. One of the two toxin subunit gene pairs was cloned as a 5.1-kilobase DNA insert in plasmid pBR322. E. coli cells carrying the recombinant plasmid pJM17 were shown to produce cholera toxin, which was found to be largely cell associated. Protein chemical analysis indicated that the toxin was in its unnicked form and required additional proteolytic processing by trypsin to exhibit full toxicity in tissue culture. The alteration in E. coli of the secretion and proteolytic processing of cholera toxin parallels that previously observed for LT. An in vitro generated insertion mutation in the A subunit gene on pJM17 was shown to abolish production of the A chain but still allow production of the B chain. These observations, together with restriction mapping data, have demonstrated that the cholera toxin and LT genes are very similar in their genetic organization.

(<em>b</em>)O<em>b</em>jective:</<em>b</em>) To investigate the mental hea<em>lt</em>h of clinical first-line medical staff in COVID-19 epidemic and provide theoretical <em>b</em>asis for psychological intervention. (<em>b</em>)Method:</<em>b</em>) The mental hea<em>lt</em>h status of the first-line medical staff was investigated <em>b</em>y Self-rating Anxiety Acale (SAS) and Post-Traumatic Stress Disorder Self-rating Scale(PTSD-SS). From Fe<em>b</em>ruary 7 to 14, 2020, 246 medical staff were investigated who participated in the treatment of COVID-19 using cluster sampling , and received 230 responses, with a recovery rate of 93.5%. (<em>b</em>)Resu<em>lt</em>s:</<em>b</em>) The incidence of anxiety in medical staff was 23.04% (53/230), and the score of SAS was (42.91 ± 10.89). Among them, the incidence of severe anxiety, moderate anxiety and mild anxiety were 2.17% (5/230), 4.78% (11/230) and 16.09% (37/230), respectively. The incidence of anxiety in female medical staff was higher than that in male [25.67% (48/187) <i>vs</i> 11.63% (5/43), <i>Z</i>=-2.008, <i>P</i>=0.045], the score of SAS in female medical staff was higher than that in male [(43.78±11.12) <i>vs</i> (39.14 ± 9.01), <i>t</i> =-2.548, <i>P</i>=0.012]. The incidence of anxiety in nurses was higher than that in doctors [26.88% (43/160) <i>vs</i> 14.29% (10/70), <i>Z</i>=-2.066, <i>P</i>=0.039], and the score of SAS in nurses was higher than that in doctors [(44.84±10.42) <i>vs</i> (38.50±10.72), <i>t</i> =-4.207, <i>P</i>&<em>lt</em>;0.001]. The incidence of stress disorder in medical staff was 27.39% (63/230), and the score of PTSD-SS was (42.92 ± 17.88). The score of PTSD-SS in female medical staff was higher than that of male [(44.30±18.42) <i>vs</i>(36.91 ± 13.95), <i>t</i>=-2.472, <i>P</i>=0.014]. (<em>b</em>)Conclusions:</<em>b</em>) In COVID-19 epidemic, the incidence of anxiety and stress disorder is high among medical staff. Medical institutions should strengthen the training of psychological skills of medical staff. Special attention should <em>b</em>e paid to the mental hea<em>lt</em>h of female nurses.

<A<em>b</em>stractText>We estimated the accuracy of Fi<em>b</em>roScan vi<em>b</em>ration-controlled transient elastography controlled attenuation parameter (CAP) and liver stiffness measurement (LSMs) in assessing steatosis and fi<em>b</em>rosis in patients with suspected nonalcoholic liver disease (NAFLD).</A<em>b</em>stractText><A<em>b</em>stractText>We collected data from 450 consecutive adu<em>lt</em>s who underwent liver <em>b</em>iopsy analysis for suspected NAFLD at 7 centers in the United Kingdom from March 2014 through January 2017. Fi<em>b</em>roScan examinations with M or XL pro<em>b</em>e were completed within the 2 weeks of the <em>b</em>iopsy analysis (404 had a valid examination). The <em>b</em>iopsies were scored <em>b</em>y 2 <em>b</em>linded expert pathologists according to nonalcoholic steatohepatitis clinical research network criteria. Diagnostic accuracy was estimated using the area under the receiver operating characteristic curves (AUROCs) for the categories of steatosis and fi<em>b</em>rosis. We assessed effects of disease prevalence on positive and negative predictive values. For LSM, the effects of histological parameters and pro<em>b</em>e type were appraised using mu<em>lt</em>ivaria<em>b</em>le analysis.</A<em>b</em>stractText><p><div>(<em>b</em>)RESULTS</<em>b</em>)</div>Using <em>b</em>iopsy analysis as the reference standard, we found that CAP identified patients with steatosis with an AUROC of 0.87 (95% confidence interval [CI] 0.82-0.92) for S≥S1, 0.77 (95% CI 0.71-0.82) for S≥S2, and 0.70 (95% CI 0.64-0.75) for S=S3. Youden cutoff values for S≥S1, S≥S2, and S≥S3 were 302 dB/m, 331 dB/m, and 337 dB/m, respectively. LSM identified patients with fi<em>b</em>rosis with AUROCs of 0.77 (95% CI 0.72-0.82) for F≥F2, 0.80 (95% CI 0.75-0.84) for F≥F3, and 0.89 (95% CI 0.84-0.93) for F=F4. Youden cutoff values for F≥F2, F≥F3, and F=F4 were 8.2 kPa, 9.7 kPa, and 13.6 kPa, respectively. Applying the optimal cutoff values, determined from this cohort, to populations of lower fi<em>b</em>rosis prevalence increased negative predictive values and reduced positive predictive values. Mu<em>lt</em>ivaria<em>b</em>le analysis found that the only parameter that significantly affected LSMs was fi<em>b</em>rosis stage (P&<em>lt</em>;10^-16); we found no association with steatosis or pro<em>b</em>e type.</p><A<em>b</em>stractText>In a prospective analysis of patients with NAFLD, we found CAP and LSM <em>b</em>y Fi<em>b</em>roScan to assess liver steatosis and fi<em>b</em>rosis, respectively, with AUROC values ranging from 0.70 to 0.89. Pro<em>b</em>e type and steatosis did not affect LSM.</A<em>b</em>stractText><A<em>b</em>stractText>ClinicalTrials.gov Identifier: NCT01985009.</A<em>b</em>stractText>

Bacillus anthracis secretes two critical virulence factors, lethal toxin (LT) and edema toxin (ET). In this study, we show that murine bone marrow-derived dendritic cells (DC) infected with B. anthracis strains secreting ET exhibit a very different cytokine secretion pattern than DC infected with B. anthracis strains secreting LT, both toxins, or a nontoxinogenic strain. ET produced during infection selectively inhibits the production of IL-12p70 and TNF-alpha, whereas LT targets IL-10 and TNF-alpha production. To confirm the direct role of the toxins, we show that purified ET and LT similarly disrupt cytokine secretion by DC infected with a nontoxinogenic strain. These effects can be reversed by specific inhibitors of each toxin. Furthermore, ET inhibits in vivo IL-12p70 and IFN-gamma secretion induced by LPS. These results suggest that ET produced during infection impairs DC functions and cooperates with LT to suppress the innate immune response. This may represent a new strategy developed by B. anthracis to escape the host immune response.

The coronavirus disease 2019 (COVID-19) epidemic affects people's health and health-related quality of life (HRQoL), especially in those who have suspected COVID-19 symptoms (S-COVID-19-S). We examined the effect of modifications of health literacy (HL) on depression and HRQoL. A cross-sectional study was conducted from 14 February to 2 March 2020. 3947 participants were recruited from outpatient departments of nine hospitals and health centers across Vietnam. The interviews were conducted using printed questionnaires including participants' characteristics, clinical parameters, health behaviors, HL, depression, and HRQoL. People with S-COVID-19-S had a higher depression likelihood (OR, 2.88; p &lt; 0.001), lower HRQoL-score (B, -7.92; p &lt; 0.001). In comparison to people without S-COVID-19-S and low HL, those with S-COVID-19-S and low HL had 9.70 times higher depression likelihood (p &lt; 0.001), 20.62 lower HRQoL-score (p &lt; 0.001), for the people without S-COVID-19-S, 1 score increment of HL resulted in 5% lower depression likelihood (p &lt; 0.001) and 0.45 higher HRQoL-score (p &lt; 0.001), while for those people with S-COVID-19-S, 1 score increment of HL resulted in a 4% lower depression likelihood (p = 0.004) and 0.43 higher HRQoL-score (p &lt; 0.001). People with S-COVID-19-S had a higher depression likelihood and lower HRQoL than those without. HL shows a protective effect on depression and HRQoL during the epidemic.

The leukotrienes and lipoxins are biologically active metabolites derived from arachidonic acid. Their diverse and potent actions are associated with specific receptors. Recent molecular techniques have established the nucleotide and amino acid sequences and confirmed the evidence that suggested the existence of different G-protein-coupled receptors for these lipid mediators. The nomenclature for these receptors has now been established for the leukotrienes. BLT receptors are activated by leukotriene B(4) and related hydroxyacids and this class of receptors can be subdivided into BLT(1) and BLT(2). The cysteinyl-leukotrienes (LT) activate another group called CysLT receptors, which are referred to as CysLT(1) and CysLT(2). A provisional nomenclature for the lipoxin receptor has also been proposed. LXA(4) and LXB(4) activate the ALX receptor and LXB(4) may also activate another putative receptor. However this latter receptor has not been cloned. The aim of this review is to provide the molecular evidence as well as the properties and significance of the leukotriene and lipoxin receptors, which has lead to the present nomenclature.

As detected by cross-sectional imaging, severe muscle depletion, which is termed sarcopenia, holds promise for prognostication in patients with cirrhosis. Our aims were to describe the prevalence and predictors of sarcopenia in patients with cirrhosis listed for liver transplantation (LT) and to determine its independent prognostic significance for the prediction of waiting-list mortality. Adults listed for LT who underwent abdominal computed tomography/magnetic resonance imaging within 6 weeks of activation were retrospectively identified. The exclusions were hepatocellular carcinoma, acute liver failure, prior LT, and listing for multivisceral transplantation or living related LT. Sixty percent of the 142 eligible patients were male, the median age was 53 years, and the median Model for End-Stage Liver Disease (MELD) score at listing was 15. Forty-one percent were sarcopenic; sarcopenia was more prevalent in males versus females (54% versus 21%, P < 0.001) and increased with the Child-Pugh class (10% for class A, 34% for class B, and 54% for class C, P = 0.007). Male sex, the dry-weight body mass index (BMI), and Child-Pugh class C cirrhosis (but not the MELD score) were independent predictors of sarcopenia. Sarcopenia was an independent predictor of mortality (hazard ratio = 2.36, 95% confidence interval = 1.23-4.53) after adjustments for age and MELD scores. In conclusion, sarcopenia is associated with increased waiting-list mortality and is poorly predicted by subjective nutritional assessment tools such as BMI and subjective global assessment. If this is validated in larger studies, the objective assessment of sarcopenia holds promise for prognostication in this patient population.

Background: Despite the high efficacy of the BNT162b2 messenger RNA vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), rare breakthrough infections have been reported, including infections among health care workers. Data are needed to characterize these infections and define correlates of breakthrough and infectivity.

Methods: At the largest medical center in Israel, we identified breakthrough infections by performing extensive evaluations of health care workers who were symptomatic (including mild symptoms) or had known infection exposure. These evaluations included epidemiologic investigations, repeat reverse-transcriptase-polymerase-chain-reaction (RT-PCR) assays, antigen-detecting rapid diagnostic testing (Ag-RDT), serologic assays, and genomic sequencing. Correlates of breakthrough infection were assessed in a case-control analysis. We matched patients with breakthrough infection who had antibody titers obtained within a week before SARS-CoV-2 detection (peri-infection period) with four to five uninfected controls and used generalized estimating equations to predict the geometric mean titers among cases and controls and the ratio between the titers in the two groups. We also assessed the correlation between neutralizing antibody titers and N gene cycle threshold (Ct) values with respect to infectivity.

Results: Among 1497 fully vaccinated health care workers for whom RT-PCR data were available, 39 SARS-CoV-2 breakthrough infections were documented. Neutralizing antibody titers in case patients during the peri-infection period were lower than those in matched uninfected controls (case-to-control ratio, 0.361; 95% confidence interval, 0.165 to 0.787). Higher peri-infection neutralizing antibody titers were associated with lower infectivity (higher Ct values). Most breakthrough cases were mild or asymptomatic, although 19% had persistent symptoms (>6 weeks). The B.1.1.7 (alpha) variant was found in 85% of samples tested. A total of 74% of case patients had a high viral load (Ct value, &lt;30) at some point during their infection; however, of these patients, only 17 (59%) had a positive result on concurrent Ag-RDT. No secondary infections were documented.

Conclusions: Among fully vaccinated health care workers, the occurrence of breakthrough infections with SARS-CoV-2 was correlated with neutralizing antibody titers during the peri-infection period. Most breakthrough infections were mild or asymptomatic, although persistent symptoms did occur.

Heat-labile enterotoxin (LT) from Escherichia coli is a bacterial protein toxin with an ABB pentamer has a membrane binding function and the A subunit is needed for enzymatic activity. The LT crystal structure has been solved using a combination of multiple isomorphous replacement, fivefold averaging and molecular dynamics refinement. Phase combination using all these sources of phase information was of crucial importance for the chain tracing. The structure has now been refined to 1.95 A resolution, resulting in a model containing 6035 protein atoms and 293 solvent molecules with a crystallographic R-factor of 18.2% and good stereochemistry. The B subunits are arranged as a highly stable pentamer with a donut shape. Each subunit takes part in approximately 30 inter-subunit hydrogen bonds and six salt bridges with its two neighbors, whilst burying a large surface area. The A subunit has higher temperature factors and less well-defined secondary structure than the B subunits. It interacts with the B pentamer mainly via the C-terminal A2 fragment, which runs through the highly charged central pore of the B subunits. The pore contains at least 66 water molecules, which fill the space left by the A2 fragment. A detailed analysis of the contacts between A and B subunits showed that most specific contacts occur at the entrance of the central pore of the B pentamer, while the contacts within the pore are mainly hydrophobic and water mediated, with the exception of two salt bridges. Only a few contacts exist between the A1 fragment and the B pentamer, showing that the A2 fragment functions as a "linker" of the A and B parts of the protein. Interacting with the A subunit by the B subunits does not cause large deviations from a common B subunit structure, and the 5-fold symmetry is well maintained. A potential NAD(+)-binding site is located in an elongated crevice at the interface of two small sheets in the A1 fragment. At the back of this crevice the functionally important Arg7 makes a hydrogen bond connecting two strands, which seems to be conserved across the ADP-ribosylating toxin family. The putative catalytic residue (A1:Glu112) is located nearby, close to a very hydrophobic region, which packs two loops together. This hydrophobic region may be important for catalysis and membrane translocation.

Pathogenesis of Bacillus anthracis is associated with the production of lethal toxin (LT), which activates the murine Nalp1b/Nlrp1b inflammasome and induces caspase-1-dependent pyroptotic death in macrophages and dendritic cells. In this study, we investigated the effect of allelic variation of Nlrp1b on the outcome of LT challenge and infection by B. anthracis spores. Nlrp1b allelic variation did not alter the kinetics or pathology of end-stage disease induced by purified LT, suggesting that, in contrast to previous reports, macrophage lysis does not contribute directly to LT-mediated pathology. However, animals expressing a LT-sensitive allele of Nlrp1b showed an early inflammatory response to LT and increased resistance to infection by B. anthracis. Data presented here support a model whereby LT-mediated activation of Nlrp1b and subsequent lysis of macrophages is not a mechanism used by B. anthracis to promote virulence, but rather a protective host-mediated innate immune response.

Uncontrolled T helper type 1 (T(H)1) and T(H)17 cells are associated with autoimmune responses. We identify surface lymphotoxin-alpha (LT-alpha) as common to T(H)0, T(H)1 and T(H)17 cells and employ a unique strategy to target these subsets using a depleting monoclonal antibody (mAb) directed to surface LT-alpha. Depleting LT-alpha-specific mAb inhibited T cell-mediated models of delayed-type hypersensitivity and experimental autoimmune encephalomyelitis. In collagen-induced arthritis (CIA), preventive and therapeutic administration of LT-alpha-specific mAb inhibited disease, and immunoablated T cells expressing interleukin-17 (IL-17), interferon-gamma and tumor necrosis factor-alpha (TNF-alpha), whereas decoy lymphotoxin-beta receptor (LT-betaR) fusion protein had no effect. A mutation in the Fc tail, rendering the antibody incapable of Fcgamma receptor binding and antibody-dependent cellular cytotoxicity activity, abolished all in vivo effects. Efficacy in CIA was preceded by a loss of rheumatoid-associated cytokines IL-6, IL-1beta and TNF-alpha within joints. These data indicate that depleting LT-alpha-expressing lymphocytes with LT-alpha-specific mAb may be beneficial in the treatment of autoimmune disease.