Our aim is evaluating the changes in weight and dietary habits in a sample of outpatients with obesity after 1 month of enforced lockdown during the COVID-19 pandemic in Northern Italy. In this observational retrospective study, the patients of our Obesity Unit were invited to answer to a 12-question multiple-choice questionnaire relative to weight changes, working activity, exercise, dietary habits, and conditions potentially impacting on nutritional choices. A multivariate regression analysis was performed to evaluate the associations among weight/BMI changes and the analyzed variables. A total of 150 subjects (91.5%) completed the questionnaire. Mean self-reported weight gain was ≈1.5 kg (p < 0.001). Lower exercise, self-reported boredom/solitude, anxiety/depression, enhanced eating, consumption of snacks, unhealthy foods, cereals, and sweets were correlated with a significantly higher weight gain. Multiple regression analyses showed that increased education (inversely, β = -1.15; 95%CI -2.13, -0.17, p = 0.022), self-reported anxiety/depression (β = 1.61; 0.53, 2.69, p = 0.004), and not consuming healthy foods (β = 1.48; 0.19, 2.77, p = 0.026) were significantly associated with increased weight gain. The estimated direct effect of self-reported anxiety/depression on weight was 2.07 kg (1.07, 3.07, p < 0.001). Individuals with obesity significantly gained weight 1 month after the beginning of the quarantine. The adverse mental burden linked to the COVID-19 pandemic was greatly associated with increased weight gain.

Keywords: COVID-19 infection; dietary habits; lockdown; obesity.

<A<em>b</em>stractText>Evidence suggests that participation in physical activity may support young people's current and future mental hea<em>lt</em>h. A<em>lt</em>hough previous reviews have examined the relationship <em>b</em>etween physical activity and a range of mental hea<em>lt</em>h outcomes in children and adolescents, due to the large increase in pu<em>b</em>lished studies there is a need for an update and quantitative synthesis of effects.</A<em>b</em>stractText><A<em>b</em>stractText>The o<em>b</em>jectives of this study were to determine the effect of physical activity interventions on mental hea<em>lt</em>h outcomes <em>b</em>y conducting a systematic review and meta-analysis, and to systematically synthesize the o<em>b</em>servational evidence (<em>b</em>oth longitudinal and cross-sectional studies) regarding the associations <em>b</em>etween physical activity and sedentary <em>b</em>ehavior and mental hea<em>lt</em>h in preschoolers (2-5 years of age), children (6-11 years of age) and adolescents (12-18 years of age).</A<em>b</em>stractText><A<em>b</em>stractText>A systematic search of the Pu<em>b</em>Med and We<em>b</em> of Science electronic data<em>b</em>ases was performed from January 2013 to April 2018, <em>b</em>y two independent researchers. Meta-analyses were performed to examine the effect of physical activity on mental hea<em>lt</em>h outcomes in randomized controlled trials (RCTs) and non-RCTs (i.e. quasi-experimental studies). A narrative synthesis of o<em>b</em>servational studies was conducted. Studies were included if they included physical activity or sedentary <em>b</em>ehavior data and at least one psychological ill-<em>b</em>eing (i.e. depression, anxiety, stress or negative affect) or psychological well-<em>b</em>eing (i.e. self-esteem, self-concept, self-efficacy, self-image, positive affect, optimism, happiness and satisfaction with life) outcome in preschoolers, children or adolescents.</A<em>b</em>stractText><p><div>(<em>b</em>)RESULTS</<em>b</em>)</div>A total of 114 original articles met all the eligi<em>b</em>ility criteria and were included in the review (4 RCTs, 14 non-RCTs, 28 prospective longitudinal studies and 68 cross-sectional studies). Of the 18 intervention studies, 12 (3 RCTs and 9 non-RCTs) were included in the meta-analysis. There was a small <em>b</em>ut significant overall effect of physical activity on mental hea<em>lt</em>h in children and adolescents aged 6-18 years (effect size 0.173, 95% confidence interval 0.106-0.239, p &<em>lt</em>; 0.001, percentage of total varia<em>b</em>ility attri<em>b</em>uted to <em>b</em>etween-study heterogeneity [I²] = 11.3%). When the analyses were performed separately for children and adolescents, the resu<em>lt</em>s were significant for adolescents <em>b</em>ut not for children. Longitudinal and cross-sectional studies demonstrated significant associations <em>b</em>etween physical activity and lower levels of psychological ill-<em>b</em>eing (i.e. depression, stress, negative affect, and total psychological distress) and greater psychological well-<em>b</em>eing (i.e. self-image, satisfaction with life and happiness, and psychological well-<em>b</em>eing). Furthermore, significant associations were found <em>b</em>etween greater amounts of sedentary <em>b</em>ehavior and <em>b</em>oth increased psychological ill-<em>b</em>eing (i.e. depression) and lower psychological well-<em>b</em>eing (i.e. satisfaction with life and happiness) in children and adolescents. Evidence on preschoolers was nearly non-existent.</p><A<em>b</em>stractText>Findings from the meta-analysis suggest that physical activity interventions can improve adolescents' mental hea<em>lt</em>h, <em>b</em>ut additional studies are needed to confirm the effects of physical activity on children's mental hea<em>lt</em>h. Findings from o<em>b</em>servational studies suggest that promoting physical activity and decreasing sedentary <em>b</em>ehavior might protect mental hea<em>lt</em>h in children and adolescents. PROSPERO Registration Num<em>b</em>er: CRD42017060373.</A<em>b</em>stractText>

<A<em>b</em>stractText>No therapy has yet proven effective in COVID-19. Tocilizuma<em>b</em> (TCZ) in patients with severe COVID-19 could <em>b</em>e an effective treatment.</A<em>b</em>stractText><A<em>b</em>stractText>We conducted a retrospective case-control study in the Nord Franche-Comté Hospital, France. We compared the outcome of patients treated with TCZ and patients without TCZ considering a com<em>b</em>ined primary endpoint: death and/or ICU admissions.</A<em>b</em>stractText><p><div>(<em>b</em>)RESULTS</<em>b</em>)</div>Patients with TCZ (n=20) had a higher Charlson comor<em>b</em>idity index (5.3 [±2.4] vs 3.4 [±2.6], p=0.014), presented with more severe forms (higher level of oxygen therapy at 13 L/min vs 6 L/min, p&<em>lt</em>;0.001), and had poorer <em>b</em>iological findings (severe lymphopenia: 676/mm³ vs 914/mm³, p=0.037 and higher CRP level: 158 mg/l vs 105 mg/l, p=0.017) than patients without TCZ (n=25). However, death and/or ICU admissions were higher in patients without TCZ than in the TCZ group (72% vs 25%, p=0.002).</p><A<em>b</em>stractText>Despite the small sample size and retrospective nature of the work, this resu<em>lt</em> strongly suggests that TCZ may reduce the num<em>b</em>er of ICU admissions and/or mortality in patients with severe SARS-CoV-2 pneumonia.</A<em>b</em>stractText>

<A<em>b</em>stractText>Enzalutamide, a potent androgen-receptor inhi<em>b</em>itor, has demonstrated significant <em>b</em>enefits in metastatic and nonmetastatic castration-resistant prostate cancer. We evaluated the efficacy and safety of enzalutamide in metastatic hormone-sensitive prostate cancer (mHSPC).</A<em>b</em>stractText><A<em>b</em>stractText>ARCHES (ClinicalTrials.gov identifier: NCT02677896) is a mu<em>lt</em>inational, dou<em>b</em>le-<em>b</em>lind, phase III trial, wherein 1,150 men with mHSPC were randomly assigned 1:1 to enzalutamide (160 mg/day) or place<em>b</em>o, plus androgen deprivation therapy (ADT), stratified <em>b</em>y disease volume and prior docetaxel chemotherapy. The primary end point was radiographic progression-free survival.</A<em>b</em>stractText><p><div>(<em>b</em>)RESULTS</<em>b</em>)</div>As of Octo<em>b</em>er 14, 2018, the risk of radiographic progression or death was significantly reduced with enzalutamide plus ADT versus place<em>b</em>o plus ADT (hazard ratio, 0.39; 95% CI, 0.30 to 0.50; <i>P</i> &<em>lt</em>; .001; median not reached <i>v</i> 19.0 months). Similar significant improvements in radiographic progression-free survival were reported in prespecified su<em>b</em>groups on the <em>b</em>asis of disease volume and prior docetaxel therapy. Enzalutamide plus ADT significantly reduced the risk of prostate-specific antigen progression, initiation of new antineoplastic therapy, first symptomatic skeletal event, castration resistance, and reduced risk of pain progression. More men achieved an undetecta<em>b</em>le prostate-specific antigen level and/or an o<em>b</em>jective response with enzalutamide plus ADT (<i>P</i> &<em>lt</em>; .001). Patients in <em>b</em>oth treatment groups reported a high <em>b</em>aseline level of quality of life, which was maintained over time. Grade 3 or greater adverse events were reported in 24.3% of patients who received enzalutamide plus ADT versus 25.6% of patients who received place<em>b</em>o plus ADT, with no unexpected adverse events.</p><A<em>b</em>stractText>Enzalutamide with ADT significantly reduced the risk of metastatic progression or death over time versus place<em>b</em>o plus ADT in men with mHSPC, including those with low-volume disease and/or prior docetaxel, with a safety analysis that seems consistent with the safety profile of enzalutamide in previous clinical trials in castration-resistant prostate cancer.</A<em>b</em>stractText>

We report on the long-term intention-to-treat (ITT) outcome of 118 patients with hepatocellular carcinoma (HCC) undergoing downstaging to within Milan/United Network for Organ Sharing T2 criteria before liver transplantation (LT) since 2002 and compare the results with 488 patients listed for LT with HCC meeting T2 criteria at listing in the same period. The downstaging subgroups include 1 lesion >5 and ≤8 cm (n = 43), 2 or 3 lesions at least one >3 and ≤5 cm with total tumor diameter ≤8 cm (n = 61), or 4-5 lesions each ≤3 cm with total tumor diameter ≤8 cm (n = 14). In the downstaging group, 64 patients (54.2%) had received LT and 5 (7.5%) developed HCC recurrence. Two of the five patients with HCC recurrence had 4-5 tumors at presentation. The 1- and 2-year cumulative probabilities for dropout (competing risk) were 24.1% and 34.2% in the downstaging group versus 20.3% and 25.6% in the T2 group (P = 0.04). Kaplan-Meier's 5-year post-transplant survival and recurrence-free probabilities were 77.8% and 90.8%, respectively, in the downstaging group versus 81% and 88%, respectively, in the T2 group (P = 0.69 and P = 0.66, respectively). The 5-year ITT survival was 56.1% in the downstaging group versus 63.3% in the T2 group (P = 0.29). Factors predicting dropout in the downstaging group included pretreatment alpha-fetoprotein ≥1,000 ng/mL (multivariate hazard ratio [HR]: 2.42; P = 0.02) and Child's B versus Child's A cirrhosis (multivariate HR: 2.19; P = 0.04).

CONCLUSIONS

Successful downstaging of HCC to within T2 criteria was associated with a low rate of HCC recurrence and excellent post-transplant survival, comparable to those meeting T2 criteria without downstaging. Owing to the small number of patients with 4-5 tumors, further investigations are needed to confirm the efficacy of downstaging in this subgroup.

Using mice double deficient for tumor necrosis factor (TNF) and lymphotoxin alpha (LT alpha), we demonstrated that TNF and/or LT alpha are necessary for development of a normal splenic microarchitecture and for isotype switch after immunization with sheep red blood cells (SRBC). In the present study, we extended these observations by determining which TNF receptor (TNFR) is involved in morphological and functional differentiation of the spleen. Spleen morphology and antibody response were investigated in wild-type, TNFR1-/-, TNFR2-/- and TNF/LT alpha-/- mice immunized with SRBC. TNF/LT alpha-/- mice, which have a complete disruption of the TNF/LT alpha signaling system including the LT beta-receptor pathway, displayed an abnormal microarchitecture, and isotype switch did not take place. TNFR1-/- and TNFR2-/- mice displayed a normal spleen microarchitecture and mounted an IgM and IgG antibody response to SRBC. However, the IgG production in TNFR1-/- mice was minimal, with citers leveling off 6 d after immunization. In this strain, immunofluorescence revealed a lack of follicular dendritic cells (FDC) network, detected with FDC-M1 as well as anti-CR1, and a lack of germinal centers, detected with peanut agglutinin. In conclusion, whereas normal splenic microarchitecture and isotype switch might require the LT beta receptor, differentiation of FDC network, development of germinal centers, and full IgG response depend on signaling via TNFR1.

Bacillus anthracis lethal toxin (LT) produces symptoms of anthrax in mice and induces rapid lysis of macrophages (M phi) derived from certain inbred strains. We used nine inbred strains and two inducible nitric oxide synthase (iNOS) knockout C57BL/6J strains polymorphic for the LT M phi sensitivity Kif1C locus to analyze the role of M phi sensitivity (to lysis) in LT-mediated cytokine responses and lethality. LT-mediated induction of cytokines KC, MCP-1/JE, MIP-2, eotaxin, and interleukin-1 beta occurred only in mice having LT-sensitive M phi. However, while iNOS knockout C57BL/6J mice having LT-sensitive M phi were much more susceptible to LT than the knockout mice with LT-resistant M phi, a comparison of susceptibilities to LT in the larger set of inbred mouse strains showed a lack of correlation between M phi sensitivity and animal susceptibility to toxin. For example, C3H/HeJ mice, harboring LT-sensitive M phi and having the associated LT-mediated cytokine response, were more resistant than mice with LT-resistant M phi and no cytokine burst. Toll-like receptor 4 (Tlr4)-deficient, lipopolysaccharide-nonresponsive mice were not more resistant to LT. We also found that CAST/Ei mice are uniquely sensitive to LT and may provide an economical bioassay for toxin-directed therapeutics. The data indicate that while the cytokine response to LT in mice requires M phi lysis and while M phi sensitivity in the C57BL/6J background is sufficient for BALB/cJ-like mortality of that strain, the contribution of M phi sensitivity and cytokine response to animal susceptibility to LT differs among other inbred strains. Thus, LT-mediated lethality in mice is influenced by genetic factors in addition to those controlling M phi lysis and cytokine response and is independent of Tlr4 function.

<AbstractText>Metastatic castration-resistant prostate cancer is enriched in DNA damage response (DDR) gene aberrations. The TOPARP-<em>B</em> trial aims to prospectively validate the association between DDR gene aberrations and response to olaparib in metastatic castration-resistant prostate cancer.</AbstractText><AbstractText>In this open-label, investigator-initiated, randomised phase 2 trial following a selection (or pick-the-winner) design, we recruited participants from 17 UK hospitals. Men aged 18 years or older with progressing metastatic castration-resistant prostate cancer previously treated with one or two taxane chemotherapy regimens and with an Eastern Cooperative Oncology Group performance status of 2 or less had tumour biopsies tested with targeted sequencing. Patients with DDR gene aberrations were randomly assigned (1:1) by a computer-generated minimisation method, with balancing for circulating tumour cell count at screening, to receive 400 mg or 300 mg olaparib twice daily, given continuously in 4-week cycles until disease progression or unacceptable toxicity. Neither participants nor investigators were masked to dose allocation. The primary endpoint of confirmed response was defined as a composite of all patients presenting with any of the following outcomes: radiological objective response (as assessed by Response Evaluation Criteria in Solid Tumors 1.1), a decrease in prostate-specific antigen (PSA) of 50% or more (PSA50) from baseline, or conversion of circulating tumour cell count (from ≥5 cells per 7·5 mL blood at baseline to &<em>lt</em>;5 cells per 7·5 mL blood). A confirmed response in a consecutive assessment after at least 4 weeks was required for each component. The primary analysis was done in the evaluable population. If at least 19 (43%) of 44 evaluable patients in a dose cohort responded, then the dose cohort would be considered successful. Safety was assessed in all patients who received at least one dose of olaparib. This trial is registered at ClinicalTrials.gov, NCT01682772. Recruitment for the trial has completed and follow-up is ongoing.</AbstractText><AbstractText>711 patients consented for targeted screening between April 1, 2015, and Aug 30, 2018. 161 patients had DDR gene aberrations, 98 of whom were randomly assigned and treated (49 patients for each olaparib dose), with 92 evaluable for the primary endpoint (46 patients for each olaparib dose). Median follow-up was 24·8 months (IQR 16·7-35·9). Confirmed composite response was achieved in 25 (54·3%; 95% CI 39·0-69·1) of 46 evaluable patients in the 400 mg cohort, and 18 (39·1%; 25·1-54·6) of 46 evaluable patients in the 300 mg cohort. Radiological response was achieved in eight (24·2%; 11·1-42·3) of 33 evaluable patients in the 400 mg cohort and six (16·2%; 6·2-32·0) of 37 in the 300 mg cohort; PSA50 response was achieved in 17 (37·0%; 23·2-52·5) of 46 and 13 (30·2%; 17·2-46·1) of 43; and circulating tumour cell count conversion was achieved in 15 (53·6%; 33·9-72·5) of 28 and 13 (48·1%; 28·7-68·1) of 27. The most common grade 3-4 adverse event in both cohorts was anaemia (15 [31%] of 49 patients in the 300 mg cohort and 18 [37%] of 49 in the 400 mg cohort). 19 serious adverse reactions were reported in 13 patients. One death possibly related to treatment (myocardial infarction) occurred after 11 days of treatment in the 300 mg cohort.</AbstractText><AbstractText>Olaparib has antitumour activity against metastatic castration-resistant prostate cancer with DDR gene aberrations, supporting the implementation of genomic stratification of metastatic castration-resistant prostate cancer in clinical practice.</AbstractText><AbstractText>Cancer Research UK, AstraZeneca, Prostate Cancer UK, the Prostate Cancer Foundation, the Experimental Cancer Medicine Centres Network, and the National Institute for Hea<em>lt</em>h Research <em>B</em>iomedical Research Centres.</AbstractText>

The kinetics of pre-mRNA processing in living cells is poorly known, preventing a detailed analysis of the regulation of these reactions. Using tetracycline-regulated promoters we performed, during a transcriptional induction, a complete analysis of the maturation of two cellular mRNAs, those for LT-alpha and beta-globin. In both cases, splicing was appropriately described by first-order reactions with corresponding half-lives ranging between 0.4 and 7.5 min, depending on the intron. Transport also behaved as a first-order reaction during the early phase of beta-globin expression, with a nuclear dwelling time of 4 min. At a later time, analysis was prevented by the progressive accumulation within the nucleus of mature mRNA not directly involved in export. Our results further establish for these genes that (i) splicing components are never limiting, even when expression is induced in naive cells, (ii) there is no significant RNA degradation during splicing and transport, and (iii) precursor-to-product ratios at steady state can be used for the determination of splicing rates. Finally, the comparison between the kinetics of splicing during transcriptional induction and during transcriptional shutoff reveals a novel coupling between transcription and splicing.

Background: Public polling indicates that vaccine uptake will be suboptimal when COVID-19 vaccines become available. Formative research seeking an understanding of weak vaccination intentions is urgently needed.

Methods: Nationwide online survey of 804 U.S. English-speaking adults. Compensated participants were recruited from the U.S. through an internet survey panel of 2.5 million residents developed by a commercial survey firm. Recruitment was based on quota sampling to produce a U.S. Census-matched sample representative of the nation with regard to region of residence, sex, and age.

Results: COVID-19 vaccination intentions were weak, with 14.8% of respondents being unlikely to get vaccinated and another 23.0% unsure. Intent to vaccinate was highest for men, older people, individuals who identified as white and non-Hispanic, the affluent and college-educated, Democrats, those who were married or partnered, people with pre-existing medical conditions, and those vaccinated against influenza during the 2019-2020 flu season. In a multiple linear regression, significant predictors of vaccination intent were general vaccine knowledge (β = 0.311, p &lt; .001), rejection of vaccine conspiracies (β = -0.117, p = .003), perceived severity of COVID-19 (β = 0.273, p &lt; .001), influenza vaccine uptake (β = 0.178, p &lt; .001), having ≥ 5 pre-existing conditions (β = 0.098, p = .003), being male (β = 0.119, p &lt; .001), household income of ≥ $120,000 (β = 0.110, p = .004), identifying as a Democrat (β = 0.075, p &lt; .029), and not relying upon social media for virus information (β = -0.090, p 〈002). Intent to vaccinate was lower for Fox News (57.3%) than CNN/MSNBC viewers (76.4%) (χ2(1) = 12.68, p &lt; .001). Political party differences in threat appraisals and vaccine conspiracy beliefs are described.

Conclusions: Demographic characteristics, vaccine knowledge, perceived vulnerability to COVID-19, risk factors for COVID-19, and politics likely contribute to vaccination hesitancy.

Keywords: COVID-19; Conspiracy beliefs; Coronavirus; Media; Social media; Vaccine.

OBJECTIVE

Although hepatitis B and C have been the main drivers of hepatocellular carcinoma (HCC), nonalcoholic steatohepatitis (NASH) has recently become an important cause of HCC. The aim of this study was to assess the causes of HCC among liver transplant (LT) candidates in the United States.

METHODS

The Scientific Registry of Transplant Recipients (2002-2016) was used to estimate the trends in prevalence of HCC in LT candidates with the most common types of chronic liver disease: alcoholic liver disease (ALD), chronic hepatitis B (CHB), chronic hepatitis C, and NASH.

RESULTS

158,347 adult LT candidates were included. Of these, 26,121 (16.5%) had HCC; this proportion increased from 6.4% (2002) to 23.0% (2016) (trend P < .0001). Over the study period, CHC remained the most common etiology for HCC (65%). The proportions of HCC accounted for by CHC and ALD remained stable (both trend P>> .10), the proportion of CHB decreased 3.1-fold (P < .0001), while the proportion of NASH in HCC increased 7.7-fold (from 2.1% to 16.2%; P < .0001). Furthermore, since 2002, the prevalence of HCC in LT candidates with NASH increased 11.8-fold, while this rate increased 6.0-fold in CHB, 3.4-fold in ALD, and 2.3-fold in CHC (all P < .0001); the increasing trend in NASH was steeper than that for any other etiology (P < .0001 in a trend regression model). The proportion of LT candidates with HCC who ultimately received a transplant or died while waiting did not differ between etiologies (P>> .05).

CONCLUSIONS

Nonalcoholic steatohepatitis is the most rapidly growing cause of HCC among US patients listed for liver transplantation.

Background: Idecabtagene vicleucel (ide-cel, also called bb2121), a B-cell maturation antigen-directed chimeric antigen receptor (CAR) T-cell therapy, has shown clinical activity with expected CAR T-cell toxic effects in patients with relapsed and refractory multiple myeloma.

Methods: In this phase 2 study, we sought to confirm the efficacy and safety of ide-cel in patients with relapsed and refractory myeloma. Patients with disease after at least three previous regimens including a proteasome inhibitor, an immunomodulatory agent, and an anti-CD38 antibody were enrolled. Patients received ide-cel target doses of 150 × 10⁶ to 450 × 10⁶ CAR-positive (CAR+) T cells. The primary end point was an overall response (partial response or better); a key secondary end point was a complete response or better (comprising complete and stringent complete responses).

Results: Of 140 patients enrolled, 128 received ide-cel. At a median follow-up of 13.3 months, 94 of 128 patients (73%) had a response, and 42 of 128 (33%) had a complete response or better. Minimal residual disease (MRD)-negative status (<10^-5 nucleated cells) was confirmed in 33 patients, representing 26% of all 128 patients who were treated and 79% of the 42 patients who had a complete response or better. The median progression-free survival was 8.8 months (95% confidence interval, 5.6 to 11.6). Common toxic effects among the 128 treated patients included neutropenia in 117 patients (91%), anemia in 89 (70%), and thrombocytopenia in 81 (63%). Cytokine release syndrome was reported in 107 patients (84%), including 7 (5%) who had events of grade 3 or higher. Neurotoxic effects developed in 23 patients (18%) and were of grade 3 in 4 patients (3%); no neurotoxic effects higher than grade 3 occurred. Cellular kinetic analysis confirmed CAR+ T cells in 29 of 49 patients (59%) at 6 months and 4 of 11 patients (36%) at 12 months after infusion.

Conclusions: Ide-cel induced responses in a majority of heavily pretreated patients with refractory and relapsed myeloma; MRD-negative status was achieved in 26% of treated patients. Almost all patients had grade 3 or 4 toxic effects, most commonly hematologic toxic effects and cytokine release syndrome. (Funded by bluebird bio and Celgene, a Bristol-Myers Squibb company; KarMMa ClinicalTrials.gov number, NCT03361748.).

Heat-labile enterotoxins (LTs) from porcine EWD299) and human (H74-114) enterotoxigenic strains of Escherichia coli were isolated by a single-step galactose elution procedure. Although both strains had similar amounts of LT in their whole-cell lysates, H74-114 yielded a smaller quantity of purified LT than did EWD299. Immunodiffusion studies with specific antisera revealed that although the two LTs shared major antigenic determinants each also had unique antigens. Both also had shared and unique specificities in comparison with the cholera enterotoxin (choleragen). Differences also exist in the apparent molecular weights of their B-subunit oligomers (coligenoid) as well as in the B-subunit monomers. The monomer molecular weights are 11,500 for EWD299 porcine LT and 12,700 for H74-114 human LT. The results suggest that either this isolated human LT has a tetrameric coligenoid or it moves differently in sodium dodecyl sulfate gels for other reasons. The A-subunits of both LTs were similar in size (28,000 daltons), and both LTs were activated by mild proteolytic processing. Amino acid analysis showed a threefold increase in the level of tryptophan and two- and fourfold decreases in the levels of glutamic acid and methionine, respectively, in H74-114 LT compared with EWD299 LT. These structural and antigenic differences may prove to be significant in immunoprophylaxis of the cholera-coli family of enterotoxins. Further studies to define the extent of evolutionary drift of these toxins are needed.

Heat-labile enterotoxin (LT) is an important virulence factor expressed by enterotoxigenic Escherichia coli. The route of LT secretion through the outer membrane and the cellular and extracellular localization of secreted LT were examined. Using a fluorescently labeled receptor, LT was found to be specifically secreted onto the surface of wild type enterotoxigenic Escherichia coli. The main terminal branch of the general secretory pathway (GSP) was necessary and sufficient to localize LT to the bacterial surface in a K-12 strain. LT is a heteromeric toxin, and we determined that its cell surface localization was mediated by the its B subunit independent of an intact G(M1) ganglioside binding site and that LT binds lipopolysaccharide and G(M1) concurrently. The majority of LT secreted into the culture supernatant by the GSP in E. coli associated with vesicles. Only a mutation in hns, not overexpression of the GSP or LT, caused an increase in vesicle yield, supporting a specific vesicle formation machinery regulated by the nucleoid-associated protein HNS. We propose a model in which LT is secreted by the GSP across the outer membrane, secreted LT binds lipopolysaccharide via a G(M1)-independent binding region on its B subunit, and LT on the surface of released outer membrane vesicles interacts with host cell receptors, leading to intoxication. These data explain a novel mechanism of vesicle-mediated receptor-dependent delivery of a bacterial toxin into a host cell.

<A<em>b</em>stractText>Data regarding the efficacy of treatment with i<em>b</em>rutini<em>b</em>-rituxima<em>b</em>, as compared with standard chemoimmunotherapy with fludara<em>b</em>ine, cyclophosphamide, and rituxima<em>b</em>, in patients with previously untreated chronic lymphocytic leukemia (CLL) have <em>b</em>een limited.</A<em>b</em>stractText><A<em>b</em>stractText>In a phase 3 trial, we randomly assigned (in a 2:1 ratio) patients 70 years of age or younger with previously untreated CLL to receive either i<em>b</em>rutini<em>b</em> and rituxima<em>b</em> for six cycles (after a single cycle of i<em>b</em>rutini<em>b</em> alone), followed <em>b</em>y i<em>b</em>rutini<em>b</em> until disease progression, or six cycles of chemoimmunotherapy with fludara<em>b</em>ine, cyclophosphamide, and rituxima<em>b</em>. The primary end point was progression-free survival, and overall survival was a secondary end point. We report the resu<em>lt</em>s of a planned interim analysis.</A<em>b</em>stractText><p><div>(<em>b</em>)RESULTS</<em>b</em>)</div>A total of 529 patients underwent randomization (354 patients to the i<em>b</em>rutini<em>b</em>-rituxima<em>b</em> group, and 175 to the chemoimmunotherapy group). At a median follow-up of 33.6 months, the resu<em>lt</em>s of the analysis of progression-free survival favored i<em>b</em>rutini<em>b</em>-rituxima<em>b</em> over chemoimmunotherapy (89.4% vs. 72.9% at 3 years; hazard ratio for progression or death, 0.35; 95% confidence interval [CI], 0.22 to 0.56; P&<em>lt</em>;0.001), and the resu<em>lt</em>s met the protocol-defined efficacy threshold for the interim analysis. The resu<em>lt</em>s of the analysis of overall survival also favored i<em>b</em>rutini<em>b</em>-rituxima<em>b</em> over chemoimmunotherapy (98.8% vs. 91.5% at 3 years; hazard ratio for death, 0.17; 95% CI, 0.05 to 0.54; P&<em>lt</em>;0.001). In a su<em>b</em>group analysis involving patients without immunoglo<em>b</em>ulin heavy-chain varia<em>b</em>le region (<i>IGHV</i>) mutation, i<em>b</em>rutini<em>b</em>-rituxima<em>b</em> resu<em>lt</em>ed in <em>b</em>etter progression-free survival than chemoimmunotherapy (90.7% vs. 62.5% at 3 years; hazard ratio for progression or death, 0.26; 95% CI, 0.14 to 0.50). The 3-year progression-free survival among patients with <i>IGHV</i> mutation was 87.7% in the i<em>b</em>rutini<em>b</em>-rituxima<em>b</em> group and 88.0% in the chemoimmunotherapy group (hazard ratio for progression or death, 0.44; 95% CI, 0.14 to 1.36). The incidence of adverse events of grade 3 or higher (regardless of attri<em>b</em>ution) was similar in the two groups (in 282 of 352 patients [80.1%] who received i<em>b</em>rutini<em>b</em>-rituxima<em>b</em> and in 126 of 158 [79.7%] who received chemoimmunotherapy), whereas infectious complications of grade 3 or higher were less common with i<em>b</em>rutini<em>b</em>-rituxima<em>b</em> than with chemoimmunotherapy (in 37 patients [10.5%] vs. 32 [20.3%], P&<em>lt</em>;0.001).</p><A<em>b</em>stractText>The i<em>b</em>rutini<em>b</em>-rituxima<em>b</em> regimen resu<em>lt</em>ed in progression-free survival and overall survival that were superior to those with a standard chemoimmunotherapy regimen among patients 70 years of age or younger with previously untreated CLL. (Funded <em>b</em>y the National Cancer Institute and Pharmacyclics; E1912 ClinicalTrials.gov num<em>b</em>er, NCT02048813.).</A<em>b</em>stractText>

The non-obese diabetic (NOD) mouse develops insulin-dependent diabetes mellitus (IDDM) with mononuclear cell infiltration of the islets of Langerhans and selective destruction of the insulin-producing beta-cells, as in humans. Most infiltrating cells are T lymphocytes, and most of these carry the CD4 antigen. Adoptive transfer of T cells from diabetic NOD mice into irradiated NOD or athymic nude NOD mice induces diabetes. Susceptibility to IDDM in NOD mice is polygenic, with one gene linked to the major histocompatibility complex class II locus, which in NOD mice expresses a unique I-A molecule but no I-E. Speculation exists as to the role of the I-A molecule in the diabetes susceptibility of NOD mice, especially regarding the significance of specific unique residues. To examine the role of the NOD I-A molecule in IDDM pathogenesis, we made NOD/Lt mice transgenic for I-Ak by microinjecting I-Ak alpha- and beta-genes into fertilized NOD/Lt eggs. Insulitis was markedly reduced and diabetes prevented in NOD/Lt mice expressing I-Ak.

Our previous experiments indicated that GTPases, other than RhoA, are important for the maintenance of endothelial barrier integrity in both intact microvessels of rats and mice and cultured mouse myocardial endothelial (MyEnd) cell monolayers. In the present study, we inhibited the endothelial GTPase Rac by Clostridium sordellii lethal toxin (LT) and investigated the relation between the degree of inhibition of Rac by glucosylation and increased endothelial barrier permeability. In rat venular microvessels, LT (200 ng/ml) increased hydraulic conductivity from a control value of 2.5 +/- 0.6 to 100.8 +/- 18.7 x 10-7 cm x s(-1) x cm H2O(-1) after 80 min. In cultured MyEnd cells exposed to LT (200 ng/ml), up to 60% of cellular Rac was glucosylated after 90 min, resulting in depolymerization of F-actin and interruptions of junctional distribution of vascular endothelial cadherin (VE-cadherin) and beta-catenin as well as the formation of intercellular gaps. To understand the mechanism by which inhibition of Rac caused disassembly of adherens junctions, we used laser tweezers to quantify VE-cadherin-mediated adhesion. LT and cytochalasin D, an actin depolymerizing agent, both reduced adhesion of VE-cadherin-coated microbeads to the endothelial cell surface, whereas the inhibitor of Rho kinase Y-27632 did not. Stabilization of actin filaments by jasplakinolide completely blocked the effect of cytochalasin D but not of LT on bead adhesion. We conclude that Rac regulates endothelial barrier properties in vivo and in vitro by 1) modulation of actin filament polymerization and 2) acting directly on the tether between VE-cadherin and the cytoskeleton.

Although the essential role of tumor necrosis factor (TNF) in the control of intracellular bacterial infection is well established, it is uncertain whether the related cytokines lymphotoxin-alpha (LTalpha3) and lymphotoxin-beta (LTbeta) have independent roles in this process. Using C57Bl/6 mice in which the genes for these cytokines have been disrupted, we have examined the relative contribution of secreted LTalpha3 and membrane-bound LTbeta in the host response to aerosol Mycobacterium tuberculosis infection. To overcome the lack of peripheral lymph nodes in LTalpha-/- and LTbeta-/- mice, bone marrow chimeric mice were constructed. LT-/- chimeras, which lack both secreted LTalpha3 and membrane-bound LTbeta (LTbetaLTbetaLTbeta-/- chimeras, which lack only the membrane-bound LTbeta, controlled the infection in a comparable manner to wild-type (WT) chimeric mice. T cell responses to mycobacterial antigens and macrophage responses in LTalpha-/- chimeras were equivalent to those of WT chimeras, but in LTalpha-/- chimeras, granuloma formation was abnormal. LTalpha-/- chimeras recruited normal numbers of T cells into their lungs, but the lymphocytes were restricted to perivascular and peribronchial areas and were not colocated with macrophages in granulomas. Therefore, LTalpha3is essential for the control of pulmonary tuberculosis, and its critical role lies not in the activation of T cells and macrophages per se but in the local organization of the granulomatous response.

The lymphotoxin-alpha beta complex (LT alpha beta) is found on the surface of activated lymphocytes and binds to a specific receptor called the LT beta receptor (LT beta R). In the mouse, signaling through this pathway is important for lymph node development and splenic organization, yet the biochemical properties of murine LT alpha and LT beta are essentially unknown. Here we have used soluble receptor-Ig forms of LT beta R and TNF-R55 and mAbs specific for murine LT alpha, LT beta, and LT beta R to characterize the appearance of surface LT alpha beta complexes and LT beta R on several common murine cell lines. Cells that bound LT beta R also bound anti-LT alpha and anti-LT beta mAbs in a FACS analysis. The ability of these reagents to discriminate between surface TNF and LT was verified by analysis of surface TNF-positive, LPS-activated murine RAW 264.7 monocytic cells. Primary mouse leukocytes from spleen, thymus, lymph node, and peritoneum were activated in vitro, and CD4+ and CD8+ T cells as well as B cells expressed surface LT ligand but not the LT beta R. Conversely, elicited peritoneal monocytes/macrophages were surface LT negative yet LT beta R positive. This study shows that on mononuclear cells, surface LT complexes and receptor are expressed similarly in mice and man, and the tools described herein form the foundation for study of the functional roles of the LT system in the mouse.

<A<em>b</em>stractText>The BCL2 inhi<em>b</em>itor venetoclax has shown activity in patients with chronic lymphocytic leukemia (CLL), <em>b</em>ut its efficacy in com<em>b</em>ination with other agents in patients with CLL and coexisting conditions is not known.</A<em>b</em>stractText><A<em>b</em>stractText>In this open-la<em>b</em>el, phase 3 trial, we investigated fixed-duration treatment with venetoclax and o<em>b</em>inutuzuma<em>b</em> in patients with previously untreated CLL and coexisting conditions. Patients with a score of greater than 6 on the Cumulative Illness Rating Scale (scores range from 0 to 56, with higher scores indicating more impaired function of organ systems) or a calculated creatinine clearance of less than 70 ml per minute were randomly assigned to receive venetoclax-o<em>b</em>inutuzuma<em>b</em> or chloram<em>b</em>ucil-o<em>b</em>inutuzuma<em>b</em>. The primary end point was investigator-assessed progression-free survival. The safety of each regimen was also evaluated.</A<em>b</em>stractText><p><div>(<em>b</em>)RESULTS</<em>b</em>)</div>In total, 432 patients (median age, 72 years; median Cumulative Illness Rating Scale score, 8; median creatinine clearance, 66.4 ml per minute) underwent randomization, with 216 assigned to each group. After a median follow-up of 28.1 months, 30 primary end-point events (disease progression or death) had occurred in the venetoclax-o<em>b</em>inutuzuma<em>b</em> group and 77 had occurred in the chloram<em>b</em>ucil-o<em>b</em>inutuzuma<em>b</em> group (hazard ratio, 0.35; 95% confidence interval [CI], 0.23 to 0.53; P&<em>lt</em>;0.001). The Kaplan-Meier estimate of the percentage of patients with progression-free survival at 24 months was significantly higher in the venetoclax-o<em>b</em>inutuzuma<em>b</em> group than in the chloram<em>b</em>ucil-o<em>b</em>inutuzuma<em>b</em> group: 88.2% (95% CI, 83.7 to 92.6) as compared with 64.1% (95% CI, 57.4 to 70.8). This <em>b</em>enefit was also o<em>b</em>served in patients with <i>TP53</i> deletion, mutation, or <em>b</em>oth and in patients with unmutated immunoglo<em>b</em>ulin heavy-chain genes. Grade 3 or 4 neutropenia occurred in 52.8% of patients in the venetoclax-o<em>b</em>inutuzuma<em>b</em> group and in 48.1% of patients in the chloram<em>b</em>ucil-o<em>b</em>inutuzuma<em>b</em> group, and grade 3 or 4 infections occurred in 17.5% and 15.0%, respectively. All-cause mortality was 9.3% in the venetoclax-o<em>b</em>inutuzuma<em>b</em> group and 7.9% in the chloram<em>b</em>ucil-o<em>b</em>inutuzuma<em>b</em> group. These differences were not significant.</p><A<em>b</em>stractText>Among patients with untreated CLL and coexisting conditions, venetoclax-o<em>b</em>inutuzuma<em>b</em> was associated with longer progression-free survival than chloram<em>b</em>ucil-o<em>b</em>inutuzuma<em>b</em>. (Funded <em>b</em>y F. Hoffmann-La Roche and A<em>b</em><em>b</em>Vie; ClinicalTrials.gov num<em>b</em>er, NCT02242942.).</A<em>b</em>stractText>

Background: Chronic liver disease (CLD) and cirrhosis are associated with immune dysregulation leading to concerns that these patients may be at risk of adverse outcomes following SARS-CoV-2 infection. However, the impact of COVID-19 among patients with pre-existing liver disease remains ill-defined.

Methods: Data for CLD patients with SARS-CoV-2 were collected by two international registries. Comparisons were made with non-CLD patients with SARS-CoV-2 from a UK hospital network.

Results: Between 25th March and 8th July 2020, 745 CLD patients were reported from 29 countries including 386 with cirrhosis and 359 without. Mortality was 32% in patients with cirrhosis compared with 8% in those without (p&lt;0.001). Mortality in cirrhosis patients increased according to Child-Turcotte-Pugh class (CTP-A (19%), CTP-B (35%), CTP-C (51%)) and the main cause of death was respiratory failure (71%). After adjusting for baseline characteristics, factors associated with death in the total CLD cohort were age (OR 1.02; 1.01-1.04), CTP-A (OR 1.90; 1.03-3.52), CTP-B (OR 4.14; 2.4-7.65), CTP-C cirrhosis (OR 9.32; 4.80-18.08) and alcohol related liver disease (ALD) (OR 1.79; 1.03-3.13). When comparing CLD versus non-CLD (n=620) in propensity-score-matched analysis there were significant increases in mortality with CTP-B +20.0% (8.8%-31.3%) and CTP-C cirrhosis +38.1% (27.1%-49.2%). Acute hepatic decompensation occurred in 46% of patients with cirrhosis, of which 21% had no respiratory symptoms. 50% of those with hepatic decompensation had acute-on-chronic liver failure.

Conclusions: This is the largest reported cohort of CLD and cirrhosis patients with SARS-CoV-2 infection to date. We demonstrate that baseline liver disease stage and ALD are independent risk factor for death from COVID-19. These data have important implications for the risk stratification of patients with CLD across the globe during the COVID-19 pandemic.

Keywords: COVID-19; SARS-CoV-2; acute-on-chronic liver failure; chronic liver disease; cirrhosis.

Targeted inactivation of genes in the tumor necrosis factor (TNF)/lymphotoxin (LT) ligand and receptor system has recently revealed essential roles for these molecules in lymphoid tissue development and organization. Lymphotoxin-alphabeta (LTalphabeta)/lymphotoxin-beta receptor (LTbeta-R) signaling is critical for the organogenesis of lymph nodes and Peyer's patches and for the structural compartmentalization of the splenic white pulp into distinct B and T cell areas and marginal zones. Moreover, an essential role has been demonstrated for TNF/p55 tumor necrosis factor receptor (p55TNF-R) signaling in the formation of splenic B lymphocyte follicles, follicular dendritic cell networks, and germinal centers. In contrast to a previously described essential role for the p55TNF-R in Peyer's patch organogenesis, we show in this report that Peyer's patches are present in both TNF and p55TNF-R knockout mice, demonstrating that these molecules are not essential for the organogenesis of this lymphoid organ. Furthermore, we show that in the absence of TNF/p55TNF-R signaling, lymphocytes segregate normally into T and B cell areas and a normal content and localization of dendritic cells is observed in both lymph nodes and Peyer's patches. However, although B cells are found to home normally within Peyer's patches and in the outer cortex area of lymph nodes, organized follicular structures and follicular dendritic cell networks fail to form. These results show that in contrast to LTalphabeta signaling, TNF signaling through the p55TNF-R is not essential for lymphoid organogenesis but rather for interactions that determine the cellular and structural organization of B cell follicles in all secondary lymphoid tissues.

Lymphotoxin alpha (LT-alpha) may form secreted homotrimers binding to p55 and p75 tumor necrosis factor (TNF) receptors or cell surface-bound heterotrimers with LT-beta that interact with the LT-beta receptor. Genetic ablation of LT-alpha revealed that mutant mice have no detectable lymph nodes or Peyer's patches and that the organization of the splenic white pulp in T and B cell areas is disturbed. In this report we describe a novel function for the p55 TNF receptor during ontogeny and demonstrate that mice deficient for p55 completely lack organized Peyer's patches. In contrast, lymph nodes and spleen are present in p55-deficient mice and lymphocytes segregate normally into B and T cell areas in these organs. Lamina propria and intraepithelial lymphocytes of the small intestine were detected in normal number and distribution in p55 mutant mice. Lymphocytes and endothelial cells from p55-deficient mice express normal levels of adhesion molecules considered important for lymphocyte migration to mucosal organs; this indicates that the lack of Peyer's patches does not result from a defect in lymphocyte homing. In summary, the p55 receptor for TNF selectively mediates organogenesis of Peyer's patches throughout ontogeny, suggesting that the effects of LT-alpha on the development of lymphoid organs may be mediated by distinct receptors, each functioning in an organ-specific context.

Experimental allergic encephalomyelitis (EAE) was generated in SJL and B10.PL mice by using the synthetic myelin basic protein peptides. Inflammation in brain and spinal cord preceded clinical signs of disease. Infiltrating lymphocytes were predominantly Lyt1+ (CD5+), L3T4+ (CD4+) T cells, until day 18. After that, F4/80+ monocyte/macrophages outnumbered T cells. Ia+ cells were microglia, macrophages, and endothelial cells, but Ia was not detectable on astrocytes in this EAE model. Ia+ endothelial cells appeared later in the disease than Ia+ microglia and macrophages, suggesting that antigen presentation at the blood-brain barrier is not initially responsible for inflammation. Cells staining for interferon gamma, interleukin 2 (IL-2), and IL-2 receptors were more prominent than IL-4, IL-5, lymphotoxin (LT), and tumor necrosis factor alpha (TNF-alpha), which occurred transiently in the second week and were associated with fewer cells. TNF-alpha and LT were never seen in spinal cord, suggesting that these cytokines are not responsible for initiation of clinical disease. Few or no cells stained for IL-6, IL-1, or transforming growth factor beta. Control animals injected with complete Freund's adjuvant in saline or control antigen demonstrated no inflammatory cell infiltration or cytokine production. Thus, our findings suggest a peptide-induced EAE model in which Th1 T-cell-macrophage interactions result in the disease process.