To control and prevent the current COVID-19 pandemic, the development of novel vaccines is an emergent issue. In addition, we need to develop tools that can measure/monitor T-cell and B-cell responses to know how our immune system is responding to this deleterious virus. However, little information is currently available about the immune target epitopes of novel coronavirus (SARS-CoV-2) to induce host immune responses. Through a comprehensive bioinformatic screening of potential epitopes derived from the SARS-CoV-2 sequences for HLAs commonly present in the Japanese population, we identified 2013 and 1399 possible peptide epitopes that are likely to have the high affinity (<0.5%- and 2%-rank, respectively) to HLA class I and II molecules, respectively, that may induce CD8⁺ and CD4⁺ T-cell responses. These epitopes distributed across the structural (spike, envelope, membrane, and nucleocapsid proteins) and the nonstructural proteins (proteins corresponding to six open reading frames); however, we found several regions where high-affinity epitopes were significantly enriched. By comparing the sequences of these predicted T cell epitopes to the other coronaviruses, we identified 781 HLA-class I and 418 HLA-class II epitopes that have high homologies to SARS-CoV. To further select commonly-available epitopes that would be applicable to larger populations, we calculated population coverages based on the allele frequencies of HLA molecules, and found 2 HLA-class I epitopes covering 83.8% of the Japanese population. The findings in the current study provide us valuable information to design widely-available vaccine epitopes against SARS-CoV-2 and also provide the useful information for monitoring T-cell responses.

This work aimed to investigate tumor-infiltrating immune cells (TIICs) and immune-associated genes in the tumor microenvironment of osteosarcoma. An algorithm known as ESTIMATE was applied for immune score assessment, and osteosarcoma cases were assigned to the high and low immune score groups. Immune-associated genes between these groups were compared, and an optimal immune-related risk model was built by Cox regression analyses. The deconvolution algorithm (referred to as CIBERSORT) was applied to assess 22 TIICs for their amounts in the osteosarcoma microenvironment. Osteosarcoma cases with high immune score had significantly improved outcome (P<0.01). The proportions of naive B cells and M0 macrophages were significantly lower in high immune score tissues compared with the low immune score group (P<0.05), while the amounts of M1 macrophages, M2 macrophages, and resting dendritic cells were significantly higher (P<0.05). Important immune-associated genes were determined to generate a prognostic model by Cox regression analysis. Interestingly, cases with high risk score had poor outcome (P<0.01). The areas under the curve (AUC) for the risk model in predicting 1, 3 and 5-year survival were 0.634, 0.781, and 0.809, respectively. Gene set enrichment analysis suggested immunosuppression in high-risk osteosarcoma patients, in association with poor outcome.

OBJECTIVE

The role of liver resection in advanced hepatocellular carcinoma (multinodular or with macroscopic vascular involvement) is still controversial. The aim of this study is to evaluate the role of surgical resection compared to other therapeutic modalities in patients with advanced hepatocellular carcinoma (HCC).

METHODS

Four hundred sixty four patients with HCC observed from 1991 to 2007 were included in the study. All the patients were evaluated for the treatment of HCC in relation to the severity of liver impairment and tumor stage. All the patients included in the study had no evidence of distant metastases.

RESULTS

Median follow up time for surviving patients was 25 months (range 1-155). Two-hundred and eighty-three patients were in Child-Pugh class A, 161 in class B, and 20 in class C. Two-hundred and seventy-one patients had single HCC, 121 patients had two or three HCCs, and 72 more than three HCCs. One-hundred and thirty-six patients (29.3%) were submitted to liver resection (LR), 232 (50.0%) to local ablative therapies (LAT) (ethanol injection, radiofrequency ablation, chemoembolization), eight (1.7%) to liver transplantation (LT), and 88 (19%) to supportive therapy (ST). Median survival time for all patients was 36 months (95% CI 24-36). Median survival time was 57 months for LR, 30 months for LAT, and 8 months for ST, with a 5-year survival of 47%, 20%, and 2.5%, respectively (p = 0.001). Actuarial 5-year survival for patients submitted to LT was 75%. Overall survival was significantly shorter in patients with multiple HCCs compared to single HCC, with median survival times of 39, 16, and 11 months for patients with a single HCC, with two to three HCCs, and with more than three HCCs, respectively (p = 0.01). Survival for patients with single HCC was significantly longer in patients submitted to LR compared to LAT and ST with median survival times of 57, 37, and 14 months, respectively (p = 0.02). Also, in patients with multinodular HCCs (2-3 HCCs) LR showed the best results with a median survival time of 58 months compared to 22 and 8 months for LAT and ST (p = 0.01). In patients with more than three HCCs, LR did not show different results compared to LAT and ST. Seventy-three patients had evidence of macroscopic vascular involvement; median survival in this subgroup of patients was significantly shorter compared to patients without vascular involvement, 10 and 36 months, respectively. Survival for patients with macroscopic vascular involvement submitted to LR or LAT was significant longer compared to ST, with mean survivals of 27, 30, and 12 months, respectively (p = 0.01).

CONCLUSIONS

The present study shows that the surgery can achieve good results in patients with single HCC and good liver function. Also, patients with multinodular HCCs (two to three nodules) could benefit from LR where survival is longer than after LAT or ST. In patients with more than three HCCs, LR have similar results of LAT. Macroscopic vascular invasion is a major prognostic factor, and the LR is justified in selected patients, where it can allow good long-term results compared to ST.

Background: Social distancing measures are used to reduce the spreading of infection. Our aim was to assess the immediate effects of national lockdown orders due to coronavirus disease 2019 (COVID-19) on pediatric emergency room (ER) visits and respiratory tract infections in hospitals and nationwide in Finland.

Methods: This register-based study used hospital patient information systems and the Finnish national infectious disease register. The participants were all patients visiting pediatric ER in 2 Finnish hospitals (Kuopio University Hospital, Mikkeli Central Hospital) covering 1/5th of the Finnish children population, 4 weeks before and 4 weeks after the start of the nationwide lockdown on March 16, 2020. Nationwide weekly numbers of influenza (A + B) and respiratory syncytial virus (RSV) in children were assessed from the infectious disease register from 2015 to 2020.

Results: A major decrease in the rate of daily median pediatric ER visits was detected in both hospitals in the study during the nationwide lockdown compared with the study period before the lockdown (Mikkeli, 19 vs. 7, P < 0.001; Kuopio, 9 vs. 2,5, P < 0.001). The influenza season was shorter (8 weeks from peak to no cases), and the weekly rate of new cases decreased faster compared with the previous 4 influenza seasons (previously 15-20 weeks from peak to no cases). A similar decrease was also seen in RSV cases. No pediatric cases of COVID-19 were found in participating hospitals during the study period.

Conclusion: These results strongly suggest that social distancing and other lockdown strategies are effective to slow down the spreading of common respiratory viral diseases and decreasing the need for hospitalization among children.

<A<em>b</em>stractText>To evaluate the efficacy and safety of eptinezuma<em>b</em>, a humanized anti-calcitonin gene-related peptide monoclonal anti<em>b</em>ody, in the preventive treatment of chronic migraine (CM).</A<em>b</em>stractText><A<em>b</em>stractText>The Prevention of Migraine via Intravenous ALD403 Safety and Efficacy-2 (PROMISE-2) study was a phase 3, mu<em>lt</em>icenter, randomized, dou<em>b</em>le-<em>b</em>lind, place<em>b</em>o-controlled, parallel-group study. Adu<em>lt</em>s with CM were randomly assigned to receive IV eptinezuma<em>b</em> 100 mg, eptinezuma<em>b</em> 300 mg, or place<em>b</em>o administered on day 0 and week 12. The primary endpoint was change from <em>b</em>aseline in mean monthly migraine days (MMDs) over weeks 1 to 12.</A<em>b</em>stractText><p><div>(<em>b</em>)RESULTS</<em>b</em>)</div>Among treated participants (n = 1,072), <em>b</em>aseline mean num<em>b</em>er of MMDs was ≈16.1 across groups. Treatment with eptinezuma<em>b</em> 100 and 300 mg was associated with significant reductions in MMDs across weeks 1 to 12 compared with place<em>b</em>o (place<em>b</em>o -5.6, 100 mg -7.7, <i>p</i> &<em>lt</em>; 0.0001 vs place<em>b</em>o; 300 mg -8.2, <i>p</i> &<em>lt</em>; 0.0001 vs place<em>b</em>o). Treatment-emergent adverse events (TEAEs) were reported <em>b</em>y 43.5% (100 mg), 52.0% (300 mg), and 46.7% (place<em>b</em>o) of patients. Nasopharyngitis was the only TEAE reported for >2% of eptinezuma<em>b</em>-treated patients at an incidence of >2% over place<em>b</em>o; it occurred in the 300 mg eptinezuma<em>b</em> arm (eptinezuma<em>b</em> 9.4%, place<em>b</em>o 6.0%).</p><A<em>b</em>stractText>In patients with CM, eptinezuma<em>b</em> 100 and 300 mg was associated with a significant reduction in MMDs from the day after IV administration through week 12, was well tolerated, and demonstrated an accepta<em>b</em>le safety profile.</A<em>b</em>stractText><A<em>b</em>stractText>This study provides Class I evidence that for patients with CM, a single dose of eptinezuma<em>b</em> reduces MMDs over 12 weeks of treatment.</A<em>b</em>stractText><A<em>b</em>stractText>NCT02974153.</A<em>b</em>stractText>

<p><div>(<em>b</em>)Background</<em>b</em>)</div>The threat of drug-resistant <i>Pseudomonas aeruginosa</i> requires great efforts to develop highly effective and safe <em>b</em>actericide.</p><p><div>(<em>b</em>)O<em>b</em>jective</<em>b</em>)</div>This study aimed to investigate the anti<em>b</em>acterial activity and mechanism of silver nanoparticles (AgNPs) against mu<em>lt</em>idrug-resistant <i>P. aeruginosa</i>.</p><p><div>(<em>b</em>)Methods</<em>b</em>)</div>The antimicro<em>b</em>ial effect of AgNPs on clinical isolates of resistant <i>P. aeruginosa</i> was assessed <em>b</em>y minimal inhi<em>b</em>itory concentration (MIC) and minimal <em>b</em>actericidal concentration (MBC). In mu<em>lt</em>idrug-resistant <i>P. aeruginosa</i>, the a<em>lt</em>erations of morphology and structure were o<em>b</em>served <em>b</em>y the transmission electron microscopy (TEM); the differentially expressed proteins were analyzed <em>b</em>y quantitative proteomics; the production of reactive oxygen species (ROS) was assayed <em>b</em>y H<su<em>b</em>)2</su<em>b</em>)DCF-DA staining; the activity of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) was chemically measured and the apoptosis-like effect was determined <em>b</em>y flow cytometry.</p><p><div>(<em>b</em>)Resu<em>lt</em>s</<em>b</em>)</div>Antimicro<em>b</em>ial tests revealed that AgNPs had highly <em>b</em>actericidal effect on the drug-resistant or mu<em>lt</em>idrug-resistant <i>P. aeruginosa</i> with the MIC range of 1.406-5.625 µg/mL and the MBC range of 2.813-5.625 µg/mL. TEM showed that AgNPs could enter the mu<em>lt</em>idrug-resistant <em>b</em>acteria and impair their morphology and structure. The proteomics quantified that, in the AgNP-treated <em>b</em>acteria, the levels of SOD, CAT, and POD, such as alkyl hydroperoxide reductase and organic hydroperoxide resistance protein, were o<em>b</em>viously high, as well as the significant upregulation of low oxygen regulatory oxidases, including c<em>b</em><em>b</em>3-type cytochrome c oxidase su<em>b</em>unit P2, N2, and O2. Further resu<em>lt</em>s confirmed the excessive production of ROS. The antioxidants, reduced glutathione and ascor<em>b</em>ic acid, partially antagonized the anti<em>b</em>acterial action of AgNPs. The apoptosis-like rate of AgNP-treated <em>b</em>acteria was remarka<em>b</em>ly higher than that of the untreated <em>b</em>acteria (<i>P</i>&<em>lt</em>;0.01).</p><p><div>(<em>b</em>)Conclusion</<em>b</em>)</div>This study proved that AgNPs could play antimicro<em>b</em>ial roles on the mu<em>lt</em>idrug-resistant <i>P. aeruginosa</i> in a concentration- and time-dependent manner. The main mechanism involves the disequili<em>b</em>rium of oxidation and antioxidation processes and the failure to eliminate the excessive ROS.</p>

Background: Scaling SARS-CoV-2 testing to meet demands of safe reopenings continues to be plagued by assay costs and supply chain shortages. In response, we developed SalivaDirect, which received Emergency Use Authorization (EUA) from the U.S. Food and Drug Administration (FDA).

Methods: We simplified our saliva-based diagnostic test by (1) not requiring collection tubes with preservatives, (2) replacing nucleic acid extraction with a simple enzymatic and heating step, and (3) testing specimens with a dualplex qRT-PCR assay. Moreover, we validated SalivaDirect with reagents and instruments from multiple vendors to minimize supply chain issues.

Findings: From our hospital cohort, we show a high positive agreement (94%) between saliva tested with SalivaDirect and nasopharyngeal swabs tested with a commercial qRT-PCR kit. In partnership with the National Basketball Association (NBA) and National Basketball Players Association (NBPA), we tested 3,779 saliva specimens from healthy individuals and detected low rates of invalid (0.3%) and false-positive (&lt;0.05%) results.

Conclusions: We demonstrate that saliva is a valid alternative to swabs for SARS-CoV-2 screening and that SalivaDirect can make large-scale testing more accessible and affordable. Uniquely, we can designate other laboratories to use our sensitive, flexible, and simplified platform under our EUA (https://publichealth.yale.edu/salivadirect/).

Funding: This study was funded by the NBA and NBPA (N.D.G.), the Huffman Family Donor Advised Fund (N.D.G.), a Fast Grant from Emergent Ventures at the Mercatus Center at George Mason University (N.D.G.), the Yale Institute for Global Health (N.D.G.), and the Beatrice Kleinberg Neuwirth Fund (A.I.K.). C.B.F.V. is supported by NWO Rubicon 019.181EN.004.

Keywords: COVID-19; SARS-CoV-2; molecular testing; population screening; saliva.

BACKGROUND

It is recognized that airway inflammation has a central role in the pathogenesis of asthma, but how it relates to exercise-induced bronchoconstriction (EIB) is not completely understood.

OBJECTIVE

The aim of our study was to investigate the relationship between EIB and baseline concentrations of cysteinyl leukotrienes (Cys-LTs) and other inflammatory markers in exhaled breath condensate (EBC).

METHODS

EBC was collected, and the fraction of exhaled nitric oxide (FE NO ) was measured in a group of 19 asthmatic children, after which they performed a treadmill exercise test. Fourteen healthy children were enrolled as control subjects.

RESULTS

The asthmatic children were divided into the EIB group (decrease in FEV 1 ,>> or =12%) and the non-EIB group. The EBC was analyzed for the presence of Cys-LTs, leukotriene B 4 , and ammonia. Asthmatic patients with EIB (mean FEV 1 decrease, 23% +/- 3%) had higher Cys-LT concentrations than either asthmatic patients without EIB or control subjects (42.2 pg/mL [median] vs 11.7 pg/mL and 5.8 pg/mL; P < .05 and P < .001, respectively). Ammonia concentrations were lower in both the EIB and non-EIB groups than in control subjects (253.2 microM and 334.6 microM vs 798.4 microM; P < .01 and P < .05, respectively). No difference in EBC leukotriene B 4 levels was found among the 3 groups. Both asthmatic groups had higher FE NO levels than control subjects ( P < .001). EBC Cys-LT ( P < .01; r = 0.7) and FE NO ( P < .05; r = 0.5) values both correlated significantly with the postexercise FEV 1 decrease.

CONCLUSIONS

this study shows that EBC Cys-LT values are higher in asthmatic children with EIB and correlate with the decrease in FEV 1 after exercise. These findings suggest that the pathways of both Cys-LT and nitric oxide are involved in the pathogenesis of EIB.

Escherichia coli SA53 produces a new enterotoxin that has a biological activity similar to that of E. coli heat-labile enterotoxin (<em>LT</em>) but is not neutralized by antiserum against <em>LT</em> or cholera enterotoxin. Strain SA53 contained two plasmids, pR<em>B</em>1 (69.2 +/- 4.3 megadaltons) and pR<em>B</em>2 (57.6 +/- 5.3 megadaltons). Studies were undertaken to determine whether either plasmid was required for production of the <em>LT</em>-like toxin. We isolated a derivative of SA53 lacking both plasmids and confirmed that radioactively labeled pR<em>B</em>1 and pR<em>B</em>2 DNAs failed to hybridize to total DNA digests of the cured strain. The new enterotoxin was still produced by the cured strain, demonstrating that the gene(s) encoding the toxin was not located on pR<em>B</em>1 or pR<em>B</em>2 and was most likely on the bacterial chromosome. Although sonic extracts from SA53 contained no detectable <em>LT</em> antigen, plasmid pR<em>B</em>1 DNA did contain sequences with partial homology to the <em>LT</em>-A and <em>LT</em>-<em>B</em> genes. No sequence homology with <em>LT</em> genes was detected with pR<em>B</em>2 DNA. When the enterotoxin plasmid pCG86 was introduced into a rifampin-resistant derivative of SA53, <em>LT</em> was produced. Thus, plasmid-coded <em>LT</em> could be produced in the E. coli SA53 host, and the sequences homologous to <em>LT</em> in pR<em>B</em>1 were cryptic.

To improve its immunogenicity for children and adults and to make it suitable for routine immunization of infants against typhoid fever, the capsular polysaccharide of Salmonella typhi (Vi) was bound to the B subunit of the heat-labile toxin (LT-B) of Escherichia coli or the recombinant exoprotein A (rEPA) of Pseudomonas aeruginosa. The conjugates elicited higher levels of antibodies (micrograms per milliliter of serum) in mice and in guinea pigs than did Vi and, unlike Vi alone, elicited booster antibody responses in both species. In adult volunteers, Vi-LT-B and Vi-rEPA, respectively, elicited higher levels of antibodies than Vi alone after the first injection (4.74 versus 1.77 and 4.91 versus 1.77; P < 0.005) and 26 weeks later (2.32 and 2.69 versus 0.54; P < 0.04); a second injection of the conjugates did not elicit a booster response of Vi antibodies. None of the 51 vaccinees had fever or significant local reactions. Vi-rEPA elicited slightly higher levels of Vi antibodies than did Vi-LT-B at all intervals after injection, but these differences were not significant. Each conjugate elicited antibodies to its carrier protein. The antibody responses elicited in adults by Vi bound to LT-B and rEPA are similar to those of other polysaccharide-protein conjugates. These conjugates promise to be an improved Vi vaccine. Studies of Vi conjugates with adults and infants in areas where typhoid is endemic are planned.

Mice orally immunized with Salmonella dublin EL23, a nonreverting, aromatic-dependent, histidine-requiring mutant transformed with a plasmid which carries a gene that codes for production of the B subunit of the heat-labile toxin (LT-B) of enterotoxigenic Escherichia coli, or with purified LT-B alone were compared for their ability to initiate expression of interleukin-12 (IL-12) mRNAs at mucosal sites. At 6 or 20 h following oral immunization, the Peyer's patches and mesenteric lymph nodes were removed, and polyadenylated mRNA was prepared from each tissue. Constitutive expression of an mRNA encoding the p35 subunit of IL-12 was observed in control as well as immunized mice. Conversely, expression of an mRNA encoding the p40 subunit of IL-12 was not detected in control animals but was dramatically upregulated in immunized mice. By using semiquantitative reverse transcription-PCR (RT-PCR) followed by competitive RT-PCR, differences in the magnitude of IL-12 p40 mRNA expression were quantified. Six hours after oral immunization with the Salmonella construct, mice had 12.1- and 8.4-fold increases in expression of IL-12 p40 mRNA in the Peyer's patches and mesenteric lymph nodes, respectively, compared with control mice receiving only saline. By 20 h, the pattern of increased mRNA expression was reversed, showing 2.5- and 17.6-fold increases in the Peyer's patches and mesenteric lymph nodes, respectively. Oral immunization with LT-B alone also stimulated IL-12 p40 mRNA expression, but to a lesser extent. The constitutive expression of IL-12 p35 mRNA at these mucosal sites coupled with a rapid and dramatic induction of IL-12 p40 mRNA following immunization with wild-type or attenuated strains of S. dublin is consistent with other investigations which support a role for IL-12 in modulating cell-mediated immune responses against intracellular pathogens.

The expression of the mouse mutation, diabetes (db), was examined on eight different inbred genetic backgrounds. The influence of H-2 haplotype and sex was examined. Mice of both sexes in two diabetes (db) strains (C57BL/6J, 129/J) having the H-2b haplotype were resistant to the diabetogenic action of the mutant gene. On the contrary, two H-2d congenic diabetes stocks (C57BL/KsJ, DBA/2J) exhibited severe diabetes associated with beta-cell necrosis. However, diabetes resistance was not restricted to mice with H-2b haplotype since the congenic diabetes MA/J stock (H-2k) was also resistant. Similarly, diabetes susceptibility was not restricted to mice with the H-2d haplotype, since males, but generally not females, in the congenic CBA/Lt-db/db and C3HeB/FeJ-db/db stocks (both H-2k) also exhibited a severe diabetes. Males of the congenic SWR/J-db stock (H-2q) had a diabetes of intermediate severity. Female diabetes mice with H-2k and H-2q haplotypes exhibited a sustained hypertrophy and hyperplasia of beta-cells and were able to control hyperglycemia better than males. Thus, while the H-2b haplotype remains associated with resistance, and the H-2d haplotype with susceptibility to induction of genetic diabetes, the diabetes stocks with H-2k haplotype clearly illustrate the importance of non-H-2, but sex-associated, genetic modifiers.

Salmonella typhimurium strain LT-2 was found to utilize phosphoenolpyruvate, 2-phosphoglycerate, and 3-phosphoglycerate as sole sources of carbon and energy for growth, but Escherichia coli strains did not. The following evidence suggests that this growth difference was due to the presence in Salmonella cells of an inducible phosphoglycerate permease distinct from previously studied transport systems: (a) The ability of cells to take up 3-phospho[14-C]glycerate was induced by growth in the presence of phosphoenolpyruvate, 2-phosphoglycerate, or 3-phosphoglycerate, but not glycerate, alpha-glycerophosphate, or other carbon sources tested. (b) Uptake of 3-phospho[14-C]glycerate was strongly inhibited by the three nonradioactive inducers of 3-phosphoglycerate uptake, but not by glycerate or alpha-glycerophosphate. (c) Mutants which lost the ability to utilize and take up 3-phosphoglycerate simultaneously lost the ability to utilize 2-phosphoglycerate and phosphoenolpyruvate, but not other compounds tested. (d) Mutant strains which constitutively synthesized the phosphoglycerate transport system could use both phosphoglycerates and phosphoenolpyruvate as sole sources of phosphate at low substrate concentrations. (e) A strain lacking alkaline and acid phosphatases could still grow with 3-phosphoglycerate as sole carbon source. Maximal rates of 3-phospho[14-C]glycerate uptake occurred at pH 6 in the presence of an exogenous energy source. The apparent Km for 3-phosphoglycerate uptake under these conditions was about 10-minus 4 M. The maximal uptake rate (but not the Km) was dependent on potassium ions. Although synthesis of the phosphoglycerate transport system appeared to be under adenosine 3:5-monophosphate control, glucose repressed induction only slightly. The genes controlling synthesis of the phosphoglycerate transport system (pgt genes) appeared to map at about 74 min on the Salmonella chromosome.

Heat-labile enterotoxin (LT) was purified from cells of enterotoxigenic Escherichia coli isolated from a patient with traveller's diarrhea. Purified LT was separated into A and B subunits by treatment with 6 M urea solution in 0.1 M propionic acid (pH 4.0). Biologically active toxin was reconstituted from isolated A and B subunits of LT. Hybrid toxins with biological activity were obtained in vitro from the A subunit of cholera enterotoxin and B subunit of LT, and from the A subunit of LT and B subunit of cholera enterotoxin. The hybrid toxins show a similar toxicity to that of the parent toxins from which the A subunits were derived. The in vitro formations of the hybrid toxins were confirmed by polyacrylamide gel disk electrophoresis.

Alloxan (AL), a potent generator of superoxide and hydroxyl radicals, selectively destroys rodent pancreatic beta-cells. Alloxan-susceptible (ALS/Lt) and AL-resistant (ALR/Lt) are inbred mouse strains derived in Japan by inbreeding CD-1 (ICR) mice with concomitant selection for high or low sensitivity to a relatively low AL dose. The present study was undertaken to examine whether resistance was mediated by differences in either systemic or beta-cell antioxidant defense status. Superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), and glutathione peroxidase (GPX) activities were determined in tissues of AL-untreated ALR/Lt and ALS/Lt male mice at 7 weeks of age. Specific activities of pancreatic SOD1, GR, and GPX were significantly increased in ALR/Lt mice compared with ALS/Lt mice. ALR/Lt mice further exhibited higher levels of glutathione in plasma, blood, pancreas, and liver combined with lower constitutive lipid peroxides in serum, liver, and pancreas. These results support the hypothesis that the selection process leading to the development of an AL-resistant mouse strain entailed accumulation of a gene or genes contributing to upregulated antioxidant status.

The cause of acute liver failure (ALF) is a major determinant of its outcome. Acetaminophen (paracetamol) overdose is a leading cause of ALF in some developed countries, whereas in others, such as Spain, it is extremely rare. To analyze the etiology, characteristics, and outcome of ALF in Spain, we performed a retrospective analysis of 267 patients whom we observed from 1992 to 2000. Seventeen tertiary-care hospitals with active liver transplantation (LT) programs contributed data. Causes of ALF were viral hepatitis in 98 (37%; hepatitis B virus in 75 patients), unknown in 86 (32%), drug or toxic reactions in 52 (19.5%; acetaminophen overdose in 6), and miscellaneous in 31 (11.6%). Overall survival was 58%. LT was performed in 150 patients, with a survival of 69%. Despite fulfilling criteria, 51 patients were not transplanted because of contraindications; their survival was only 7.8%. Forty-seven (85.5%) of 55 patients without transplant criteria survived. Hepatitis B virus is the most common cause of ALF in Spain, although the origin of 30% of cases remains undetermined. Acetaminophen overdose represents a very rare cause of ALF. LT was performed in >50% of cases. Patients without transplant criteria had a very good prognosis; those who fulfilled these criteria but who had contraindications for transplantation had a high mortality rate.

(<em>b</em>)Background:</<em>b</em>) The risk factors for adverse events of Coronavirus Disease-19 (COVID-19) have not <em>b</em>een well descri<em>b</em>ed. We aimed to explore the predictive value of clinical, la<em>b</em>oratory and CT imaging characteristics on admission for short-term outcomes of COVID-19 patients. (<em>b</em>)Methods:</<em>b</em>) This mu<em>lt</em>icenter, retrospective, o<em>b</em>servation study enrolled 703 la<em>b</em>oratory-confirmed COVID-19 patients admitted to 16 tertiary hospitals from 8 provinces in China <em>b</em>etween January 10, 2020 and March 13, 2020. Demographic, clinical, la<em>b</em>oratory data, CT imaging findings on admission and clinical outcomes were collected and compared. The primary endpoint was in-hospital death, the secondary endpoints were composite clinical adverse outcomes including in-hospital death, admission to intensive care unit (ICU) and requiring invasive mechanical ventilation support (IMV). Mu<em>lt</em>ivaria<em>b</em>le Cox regression, Kaplan-Meier plots and log-rank test were used to explore risk factors related to in-hospital death and in-hospital adverse outcomes. (<em>b</em>)Resu<em>lt</em>s:</<em>b</em>) Of 703 patients, 55 (8%) developed adverse outcomes (including 33 deceased), 648 (92%) discharged without any adverse outcome. Mu<em>lt</em>ivaria<em>b</em>le regression analysis showed risk factors associated with in-hospital death included ≥ 2 comor<em>b</em>idities (hazard ratio [HR], 6.734; 95% CI; 3.239-14.003, p &<em>lt</em>; 0.001), leukocytosis (HR, 9.639; 95% CI, 4.572-20.321, p &<em>lt</em>; 0.001), lymphopenia (HR, 4.579; 95% CI, 1.334-15.715, p = 0.016) and CT severity score > 14 (HR, 2.915; 95% CI, 1.376-6.177, p = 0.005) on admission, while older age (HR, 2.231; 95% CI, 1.124-4.427, p = 0.022), ≥ 2 comor<em>b</em>idities (HR, 4.778; 95% CI; 2.451-9.315, p &<em>lt</em>; 0.001), leukocytosis (HR, 6.349; 95% CI; 3.330-12.108, p &<em>lt</em>; 0.001), lymphopenia (HR, 3.014; 95% CI; 1.356-6.697, p = 0.007) and CT severity score > 14 (HR, 1.946; 95% CI; 1.095-3.459, p = 0.023) were associated with increased odds of composite adverse outcomes. (<em>b</em>)Conclusion:</<em>b</em>) The risk factors of older age, mu<em>lt</em>iple comor<em>b</em>idities, leukocytosis, lymphopenia and higher CT severity score could help clinicians identify patients with potential adverse events.

Keywords: COVID-19; Coronavirus; Mortality; Pneumonia; Risk factor.

In an attempt to analyze the cellular and molecular basis of the capacity of bone marrow stromal cells to support hematopoiesis in culture, we developed a series of murine stromal cell lines from a single long-term bone marrow culture (BMC). The cytokines produced by these cells were analyzed using immunohistochemical techniques, ribonuclease protection assays (RPA) and RT-PCR. We examined the capacity of these cloned cell lines to replace primary bone marrow-derived stromal cells in long-term bone marrow cultures (<em>LT</em>-BMC) and sought correlations between the capacity to support hematopoiesis in culture with the production of known cytokines. These immortalized lines replicate many of the functions of the hematopoietic microenvironment. They express cytokines known to play a role in hematopoiesis. All of the lines constitutively express mRNA for PBSF (SDF-1), macrophage colony-stimulating factor (M-CSF), stem cell factor (SCF), F<em>LT</em>-3, thrombopoietin (TPO), interleukin 7 (IL-7), leukemia inhibitory factor (LIF), tumor necrosis factor-<em>beta</em> (TNF-<em>beta</em>), and interferon-gamma (IFN-gamma). Most lines also express granulocyte-macrophage colony-stimulating factor (GM-CSF) and G-CSF. They vary in their expression of IL-6, tumor growth factor-<em>beta</em>1 (TGF-<em>beta</em>1), TGF-<em>beta</em>2, and TNF-alpha. Growing these lines in the presence of cytokines that influence hematopoiesis alters the levels of cytokine message. The most striking effects were produced by TNF-alpha. In addition to the cytokine mRNAs, the cell lines express factors associated with bone formation such as osteoblast-specific factor-2 (OSF-2) and bone morphogenetic protein-1 (BMP-1). They also express the neural cell-adhesion molecule neuropilin and neurotrophic factors including nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). Several of the lines can maintain hematopoiesis in culture, as measured by the continuous production of myeloid colony-forming cells (CFU-c), for months. This capacity to support hematopoiesis does not correlate with any pattern of cytokine expression. Several of these lines also support the growth of human hematopoietic cells, and human CFU-c can be detected in the cultures in which CD34(+) bone marrow cells (BMC) are cultured on murine stromal cells. No correlation between the production of any of the known cytokines and the ability to support murine hematopoiesis was detected. In addition, there was no correlation between the capacity to support murine hematopoiesis and the capacity to maintain human HSC. Despite repeated cloning, the lines remain heterogeneous and are capable of producing cells with the properties of fibroblasts, osteoblasts, adipocytes, and myoblasts. In addition to the cytokine mRNAs, the cell lines express factors associated with bone formation such as OSF-2 and BMP-1. They also express the neural cell-adhesion molecule neuropilin and neurotrophic factors including NGF and BDNF.

Cholera toxin (CT) is a heterohexameric bacterial protein toxin belonging to a larger family of A/B ADP-ribosylating toxins. Each of these toxins undergoes limited proteolysis and/or disulfide bond reduction to form the enzymatically active toxic fragment. Nicking and reduction render both CT and the closely related heat-labile enterotoxin from Escherichia coli (LT) unstable in solution, thus far preventing a full structural understanding of the conformational changes resulting from toxin activation. We present the first structural glimpse of an active CT in structures from three crystal forms of a single-site A-subunit CT variant, Y30S, which requires no activational modifications for full activity. We also redetermined the structure of the wild-type, proenzyme CT from two crystal forms, both of which exhibit (i) better geometry and (ii) a different A2 "tail" conformation than the previously determined structure [Zhang et al. (1995) J. Mol. Biol. 251, 563-573]. Differences between wild-type CT and active CTY30S are observed in A-subunit loop regions that had been previously implicated in activation by analysis of the structure of an LT A-subunit R7K variant [van den Akker et al. (1995) Biochemistry 34, 10996-11004]. The 25-36 activation loop is disordered in CTY30S, while the 47-56 active site loop displays varying degrees of order in the three CTY30S structures, suggesting that disorder in the activation loop predisposes the active site loop to a greater degree of flexibility than that found in unactivated wild-type CT. On the basis of these six new views of the CT holotoxin, we propose a model for how the activational modifications experienced by wild-type CT are communicated to the active site.

The Escherichia coli heat-labile enterotoxin (LT) is a very potent mucosal immunogen. LT also has strong adjuvant activity towards coadministered unrelated antigens and is therefore of potential interest for development of mucosal vaccines. However, despite the great demand for such mucosal vaccines, the use of LT holotoxin as an adjuvant is essentially precluded by its toxicity. LT is composed of an A subunit, carrying the toxic ADP-ribosylation activity, and a pentamer of identical B subunits, which mediates binding to ganglioside GM1, the cellular receptor for the toxin. In this paper, we demonstrate that recombinant enzymatically inactive variants of LT, including the LTB pentamer by itself, retain the immunoadjuvant activity of LT holotoxin in a murine influenza model. Mice were immunized intranasally (i.n.) with influenza virus subunit antigen, consisting mostly of the isolated surface glycoprotein hemagglutinin (HA), supplemented with either recombinant LTB (rLTB), a nontoxic LT mutant (E112K, with a Glu112->>Lys substitution in the A subunit), or LT holotoxin, and the induction of systemic IgG and local S-IgA responses was evaluated by direct enzyme-linked immunosorbent assay (ELISA). Immunization with subunit antigen alone resulted in a poor systemic IgG response and no detectable S-IgA. However, supplementation of the antigen with E112K or rLTB resulted in a substantial stimulation of the serum IgG level and in induction of a strong S-IgA response in the nasal cavity. The adjuvant activity of E112K or rLTB under these conditions was essentially the same as that of the LT holotoxin. The present results demonstrate that nontoxic variants of LT, rLTB in particular, represent promising immunoadjuvants for potential application in an i.n. influenza virus subunit vaccine. Nontoxic LT variants may also be used in i.n. vaccine formulations directed against other mucosal pathogens. In this respect, it is of interest that LT(B)-stimulated antibody responses after i.n. immunization were also observed at distant mucosal sites, including the urogenital system. This, in principle, opens the possibility to develop i.n. vaccines against sexually transmitted infectious diseases.

Eosinophilia is a feature of airway inflammation associated with asthma. Leukotriene antagonists provide therapeutic benefit in asthma, but their potential antiinflammatory actions have not been fully explored. We have examined the role of eosinophil-derived cysteinyl leukotrienes in the maintenance of eosinophil survival, and the involvement of leukotrienes in the paracrine stimulation of eosinophil survival by mast cells and lymphocytes. We obtained eosinophils and autologous lymphocytes from peripheral blood of asthmatic subjects. Leukotriene (LT)-B(4), LTC(4) and LTD(4), granulocyte-macrophage colony-stimulating factor (GM-CSF), and fibronectin promoted eosinophil survival. LTD(4) (10(-)(6) M) was as effective as GM-CSF (5 ng/ml) and fibronectin (400 ng/ml) in promoting survival. Lymphocytes and conditioned medium from a human mast cell line (HMC-1) induced eosinophil survival. Blockade of cysteinyl leukotriene receptors with SKF 104353 (pobilukast, 3 nM), and inhibition of 5-lipoxygenase (5-LO) with BW A4C (1 microM) and of 5-LO activating protein with MK 886 (1 microM), all increased basal rates of eosinophil apoptosis and reversed GM-CSF-induced eosinophil survival. Fifty percent reversal of GM-CSF- induced survival was achieved with SKF 104353 at 0.3 nM. The potency of SKF 104353 was two orders of magnitude greater than that of the LTB(4) receptor antagonist SB 201146. Mast cell- and lymphocyte-induced eosinophil survival were completely reversed by SB 201146, SKF 104353, BW A4C, and MK 886. These findings provide evidence for the involvement of an autocrine cysteinyl leukotriene pathway that supports eosinophil survival in response to a range of survival stimuli. They also suggest that LTB(4) could act as a paracrine stimulus of eosinophil survival.

Bacillus anthracis secretes 2 toxins: lethal toxin (LT) and edema toxin (ET). We investigated their role in the physiopathologic mechanisms of inhalational anthrax by evaluating murine lung dendritic cell (LDC) functions after infection with B. anthracis strains secreting LT, ET, or both or with a nontoxinogenic strain. Three lung cell populations gated on CD11c/CD11b expression were obtained after lung digestion: (1) CD11c(high)/CD11b(low) (alveolar macrophages), (2) CD11c(intermediate (int))/CD11b(int) (LDCs), and (3) CD11c(low)/CD11b(high) (interstitial macrophages or monocytes). After infection with LT-secreting strains, a decrease in costimulatory molecule expression on LDCs was observed. All CD11c+ cells infected with a nontoxinogenic strain secreted tumor necrosis factor (TNF)- alpha , interleukin (IL)-10, and IL-6. LT-secreting strains inhibited overall cytokine secretion, whereas the ET-secreting strain inhibited only TNF- alpha secretion and increased IL-6 secretion. Similar results were obtained after preincubation with purified toxins. Our results suggest that anthrax toxins secreted during infection impair LDC function and suppress the innate immune response.