The type II heat-labile enterotoxins, LT-IIa and LT-IIb, exhibit potent adjuvant properties. However, little is known about their immunomodulatory activities upon interaction with innate immune cells, unlike the widely studied type I enterotoxins that include cholera toxin (CT). We therefore investigated interactions of LT-IIa and LT-IIb with human monocytic THP-1 cells. We found that LT-II enterotoxins were inactive in stimulating cytokine release, whereas CT induced low levels of interleukin-1beta (IL-1beta) and IL-8. However, all three enterotoxins potently regulated cytokine induction in cells activated by bacterial lipopolysaccharide or fimbriae. Induction of proinflammatory (tumor necrosis factor alpha [TNF-alpha]) or chemotactic (IL-8) cytokines was downregulated, whereas induction of cytokines with anti-inflammatory (IL-10) or mucosal adjuvant properties (IL-1beta) was upregulated by the enterotoxins. These effects appeared to depend on their A subunits, because isolated B-pentameric subunits lacked regulatory activity. Enterotoxin-mediated inhibition of proinflammatory cytokine induction in activated cells was partially attributable to synergism for endogenous production of IL-10 and to an IL-10-independent inhibition of nuclear factor kappaB (NF-kappaB) activation. In sharp contrast to the holotoxins, the B pentamers (LT-IIaB and, to a greater extent, LT-IIbB) stimulated cytokine production, suggesting a link between the absence of the A subunit and increased proinflammatory properties. In this regard, the ability of LT-IIbB to activate NF-kappaB and induce TNF-alpha and IL-8 was antagonized by the LT-IIb holotoxin. These findings support distinct immunomodulatory roles for the LT-II holotoxins and their respective B pentamers. Moreover, the anti-inflammatory properties of the holotoxins may serve to suppress innate immunity and promote the survival of the pathogen.

Lymphotoxin and tumor necrosis factor provide essential signals for the formation of secondary lymphoid tissue structures. Lymphotoxin in its membrane form (LT alpha 1 beta 2 heterotrimer) is required for the development of lymph nodes and Peyer's patches and supports the development of normal spleen structure. In the spleen, lymphotoxin acts during embryonic development to support the formation of distinct B and T cell zones. Lymphotoxin also acts in a tonic fashion-supporting the formation and maintenance of the follicular dendritic cell network and of primary B cell follicle structure. The cells that deliver the tonic lymphotoxin signal supporting follicular dendritic cell structure are B cells; thus, B cells participate fundamentally in the development of the lymphoid tissue structure in which they subsequently mature.

Although Fas (APO-1/CD95) is expressed ubiquitously and induces cell death, it is also known to mediate other responses such as inflammation and angiogenesis in vivo. Previously, we have reported that Fas ligation induces selective expression of chemokines (IL-8 and MCP-1) in human astroglioma cells in vitro. In this study, we investigated whether Fas ligation can induce expression of other cytokines. Expression of IL-1alpha, IL-1beta, IL-6, IL-10, IL-12, IFN-beta, IFN-gamma, LT-beta, TGF-beta, TNF-a and TNF-beta mRNA levels in CRT-MG human astroglioma cells upon Fas ligation was investigated using RNase protection assay (RPA). We found that IL-6 mRNA is selectively induced upon Fas ligation, and IL-6 mRNA and protein expression was further investigated using single probe RPA and ELISA. To investigate the in vivo expression of IL-6, human brain specimens were homogenized and ELISA was performed for IL-6 expression. Herein, we demonstrate that: (1) Among these cytokines, only IL-6 was induced upon Fas ligation in a dose- and time-dependent manner; (2) A selective p38 MAP kinase inhibitor, SB202190, and a MEK inhibitor, U0126, suppressed induction of IL-6 mRNA and protein expression by Fas ligation; and (3) Glioblastoma multiforme samples (n = 11) contain significantly higher levels of IL-6 compared to those of control brains (n = 5), which correlate with increased levels of Fas. These results suggest that the Fas-FasL system may play a role in the regulation of tumor growth and survival by inducing the pleiotropic cytokine IL-6.

The functional properties, regarding parasite growth inhibition in vitro, the cytotoxic potential and cytokine profiles of human gammadelta+ and alphabeta+ T cells, T-cell lines and clones stimulated with Plasmodium falciparum-antigen-or T-cell mitogen in vitro were investigated. Using reverse transcriptase-polymerase chain reaction (RT-PCR) and specific primers, mRNA for the cytolytic molecules perforin, granzyme A and B, Fas and Fas ligand (FasL) were detected in both the gammadelta- and the alphabetaT cells. Despite this fact, only gammadeltaT cells inhibited, both Vdelta1+ and Vdelta2+, the in vitro growth of the asexual blood stages in a dose dependent manner. The inhibition required cell-to-cell contact and was not observed until the second parasite replication implied that the likely gammadeltaT-cell target was the extracellular merozoite or schizont. The failure of alphabetaT cells to inhibit the growth of the parasite suggests requirement of additional cytolytic molecules/signals or different receptor specificities exhibited by the gammadeltaT cells. Both the gammadelta- and alphabetaT cells expressed mRNA for a large number of cytokines. Interferon (IFN)-gamma, interleukin (IL) IL-5, IL-6, IL-8, tumour necrosis factor alpha (TNFalpha), tumour necrosis factor beta (TNF-beta)/lymphotoxin (LT) and T-cell growth factor beta-1 (TGF-betabeta, IL-2, IL-4, IL-10 and GM-CSF were variably expressed. In conclusion, our data show that gammadeltaT cells in malaria nonimmune individuals inhibit the asexual blood stages of P. falciparum malaria, while similarly activated alphabetaT cells do not. Thus, it is likely that the gammadeltaT cells could play a mandatory role in the elimination of parasites and/or the regulation of the early immune response to malaria infection.

We investigated the immunogenicity of recombinant double-layered rotavirus-like particle (2/6-VLPs) vaccines derived from simian SA11 or human (VP6) Wa and bovine RF (VP2) rotavirus strains. The 2/6-VLPs were administered to gnotobiotic pigs intranasally (i.n.) with a mutant Escherichia coli heat-labile toxin, LT-R192G (mLT), as mucosal adjuvant. Pigs were challenged with virulent Wa (P1A[8],G1) human rotavirus at postinoculation day (PID) 21 (two-dose VLP regimen) or 28 (three-dose VLP regimen). In vivo antigen-activated antibody-secreting cells (ASC) (effector B cells) and in vitro antigen-reactivated ASC (derived from memory B cells) from intestinal and systemic lymphoid tissues (duodenum, ileum, mesenteric lymph nodes [MLN], spleen, peripheral blood lymphocytes [PBL], and bone marrow lymphocytes) collected at selected times were quantitated by enzyme-linked immunospot assays. Rotavirus-specific immunoglobulin M (IgM), IgA, and IgG ASC and memory B-cell responses were detected by PID 21 or 28 in intestinal and systemic lymphoid tissues after i.n. inoculation with two or three doses of 2/6-VLPs with or without mLT. Greater mean numbers of virus-specific ASC and memory B cells in all tissues prechallenge were induced in pigs inoculated with two doses of SA11 2/6-VLPs plus mLT compared to SA11 2/6-VLPs without mLT. After challenge, anamnestic IgA and IgG ASC and memory B-cell responses were detected in intestinal lymphoid tissues of all VLP-inoculated groups, but serum virus-neutralizing antibody titers were not significantly enhanced compared to the challenged controls. Pigs inoculated with Wa-RF 2/6-VLPs (with or without mLT) developed higher anamnestic IgA and IgG ASC responses in ileum after challenge compared to pigs inoculated with SA11 2/6-VLPs (with or without mLT). Three doses of SA 11 2/6-VLP plus mLT induced the highest mean numbers of IgG memory B cells in MLN, spleen, and PBL among all groups postchallenge. However, no significant protection against diarrhea or virus shedding was evident in any of the 2/6-VLP (with or without mLT)-inoculated pigs after challenge with virulent Wa human rotavirus. These results indicate that 2/6-VLP vaccines are immunogenic in gnotobiotic pigs when inoculated i.n. and that the adjuvant mLT enhanced their immunogenicity. However, i.n. inoculation of gnotobiotic pigs with 2/6-VLPs did not confer protection against human rotavirus challenge.

A sizeable subset of patients with the two most common organ-specific rheumatic autoimmune diseases, rheumatoid arthritis (RA) and Sjögren's syndrome (SS) develop ectopic lymphoid structures (ELS) in the synovial tissue and salivary glands, respectively. These structures are characterized by perivascular (RA) and periductal (SS) clusters of T and B lymphocytes, differentiation of high endothelial venules and networks of stromal follicular dendritic cells (FDC). Accumulated evidence from other and our group demonstrated that the formation and maintenance of ELS in these chronic inflammatory conditions is critically dependent on the ectopic expression of lymphotoxins (LT) and lymphoid chemokines CXCL13, CCL19, CCL21 and CXCL12. In this review we discuss recent advances highlighting the cellular and molecular mechanisms, which regulate the formation of ELS in RA and SS, with particular emphasis on the role of lymphoid chemokines. In particular, we shall focus on the evidence that in the inflammatory microenvironment of the RA synovium and SS salivary glands, several cell types, including resident epithelial, stromal and endothelial cells as well as different subsets of infiltrating immune cells, have been shown to be capable of producing lymphoid chemokines. Finally, we summarize accumulating data supporting the conclusion that ELS in RA and SS represent functional niches for B cells to undergo affinity maturation, clonal selection and differentiation into plasma cells autoreactive against disease-specific antigens, thus contributing to humoral autoimmunity over and above that of secondary lymphoid organs.

Spontaneous ovarian teratomas develop in a large proportion of female LT strain mice. These tumors display a large neuroectodermal component with morphologic differentiation ranging from primitive neuroepithelium (medulloepithelial and ependymoblastic rosettes) to mature neurons, and provide a useful system for the study of various asynchronous stages of neuroepithelial differentiation. The aim of this study was to assess the expression of various cytoskeletal proteins in conjunction with other differentiation-related antigens in these tumors. We found that the medulloepithelial rosettes reacted with only two anti-beta-tubulin monoclonal antibodies. One of these (TU27) recognizes an epitope common to all of the mammalian beta-tubulin isotypes. The other monoclonal antibody (TUJ1) recognizes an epitope unique to class III beta-tubulin isotypes (neuronal-associated). Whereas immunoreactivity in the ependymoblastic rosettes was limited to TU27, differentiating polar neuroblasts reacted with both TU27 and TUJ1 and expressed neuron-specific enolase, synaptophysin, and the 68 kilodalton subunit of neurofilament protein. In well-differentiated foci, mature neurons were positive for all three neurofilament protein subunits (68, 168 and 200 kilodaltons), microtubule-associated-protein 2, synaptophysin, and neuron-specific-enolase, and reacted with both TU27 and TUJ1. By contrast, glial elements expressed glial fibrillary acidic and S-100 proteins, Leu-7 and TU27 but not TUJ1. Myelin basic protein and myelin-associated glycoprotein reactivity was found in the neuropile of these mature areas. The neuroepithelial components were negative for retinal S-antigen and cytokeratin. The expression of the class III beta-tubulin isotype by medulloepithelial rosettes suggests that this isotype may be one of the earliest markers to signal neuronal commitment in primitive neuroepithelium.

Tumor necrosis factor (TNF)-alpha and lymphotoxin (LT) alpha/beta play multiple roles in the development and function of the immune system. This article focuses on three important aspects of the effects of these cytokines on the immune response and on autoimmunity. In several experimental systems (Jurkat T cells, murine T-cell hybridomas), TNF-alpha appears to cause a downregulation of signaling through the TCR, revealed by changes in calcium flux, activation of p21, p23 and ZAP70, and a decrease in nuclear activation of NF-kappaB. Previous and present results suggest that TNF-alpha interferes in some manner with signaling through the TCR, at a locus yet to be delineated. Transgenic expression of LTbetaR-Fc in nonobese diabetic (NOD) transgenic mice results in prevention of type 1 diabetes in NOD mice as long as the level of expression of the fusion protein (under the control of the cytomegalovirus promoter) remains above a level of 2-3 microg/ml. Once the expression levels of the fusion protein have dropped below this critical level, the diabetic process resumes and the animals become diabetic at 40-50 weeks of age, whereas nontransgenic littermates develop diabetes by 25-30 weeks of age. The paradoxical effects of neonatal TNF-alpha administration in NOD mice in increasing incidence of and hastening onset of type 1 diabetes, while neonatal anti-TNF administration completely prevents all signs of islet cell autoimmunity, are due partly to the low levels of CD4+CD25+ T cells in NOD mice. These low levels are reduced by a further 50% on neonatal administration of nontoxic levels of TNF-alpha. In contrast, neonatal administration of anti-TNF-alpha results in a dramatic increase in the levels of CD4+CD25+ regulatory T cells, to levels beyond those seen in wild-type untreated NOD mice. TNF-alpha and LTalpha/beta thus have pleomorphic regulatory effects on the development and expression of autoimmunity.

Molecular dynamics simulations coupled with functional analyses of the major yeast phosphatidylinositol/phosphatidylcholine transfer protein Sec14p identify structural elements involved in regulating the ability of Sec14p to execute phospholipid exchange. The molecular dynamics simulations suggest large rigid body motions within the Sec14p molecule accompany closing and opening of an A(10)/T(4)/A(11) helical gate, and that "state-of-closure" of this helical gate determines access to the Sec14p phospholipid binding cavity. The data also project that conformational dynamics of the helical gate are controlled by a hinge unit (residues F(212), Y(213), K(239), I(240), and I(242)) that links to the N- and C-terminal ends of the helical gate, and by a novel gating module (composed of the B(1)LB(2) and A(12)LT(5) substructures) through which conformational information is transduced to the hinge. The (114)TDKDGR(119) motif of B(1)LB(2) plays an important role in that transduction process. These simulations offer new mechanistic possibilities for an important half-reaction of the Sec14p phospholipid exchange cycle that occurs on membrane surfaces after Sec14p has ejected bound ligand, and is reloading with another phospholipid molecule. These conformational transitions further suggest structural rationales for known disease missense mutations that functionally compromise mammalian members of the Sec14-protein superfamily.

Innate recognition and signaling by Toll-like receptors (TLRs) is facilitated by functionally associated coreceptors, although the cooperativity mechanisms involved are poorly understood. As a model we investigated TLR2 interactions with the GD1a ganglioside binding subunit of type IIb Escherichia coli enterotoxin (LT-IIb-B(5)). Both LT-IIb-B(5) and a GD1a binding-defective mutant (LT-IIb-B(5)(T13I)) could modestly bind to TLR2, but only the wild-type molecule displayed a dramatic increase in TLR2 binding activity in the presence of GD1a (although not in the presence of irrelevant gangliosides). Moreover, fluorescence resonance energy transfer experiments indicated that LT-IIb-B(5) induces lipid raft recruitment of TLR2 and TLR1 and their clustering with GD1a, in contrast to the GD1a binding-defective mutant, which moreover fails to activate TLR2 signaling. LT-IIb-B(5)-induced cell activation was critically dependent upon the Toll/IL-1 receptor domain-containing adaptor protein, which was induced to colocalize with TLR2 and GD1a, as shown by confocal imaging. Therefore, GD1a provides TLR2 coreceptor function by enabling the ligand to recruit, bind, and activate TLR2. These findings establish a model of TLR2 coreceptor function and, moreover, suggest novel mechanisms of adjuvanticity by non-toxic derivatives of type II enterotoxins dependent upon GD1a/TLR2 cooperative activity.

We tested the efficacy of a killed oral vaccine for enterotoxigenic Escherichia coli (ETEC) diarrhoea to determine if two doses of vaccine with colonization factor antigens (CF) and cholera B subunit would protect against ETEC diarrhoea of travellers. Six hundred seventy-two healthy travellers going to Mexico or Guatemala were studied in a prospective, randomised, placebo-controlled trial. The primary outcome was a vaccine preventable outcome (VPO), defined as an episode of ETEC diarrhoea with an ETEC organism producing heat labile toxin (LT) or CF homologous with the vaccine, without other known causes. The vaccine was safe and stimulated anti-heat labile toxin antibodies. There was a significant decrease in more severe VPO episodes (PE=77%, p=0.039) as defined by symptoms that interfered with daily activities or more than five loose stools in a day, although the total number of VPO events did not differ significantly in the vaccine and placebo groups. We conclude that the new oral ETEC vaccine reduces the rate of more severe episodes of traveller's diarrhoea (TD) due to VPO-ETEC, but it did not reduce the overall rate of ETEC diarrhoea or of travellers' diarrhoea due to other causes.

(<em>b</em>)Background:</<em>b</em>) The spread of the COVID-19 pandemic, the partial lockdown, the disease intensity, weak governance in the hea<em>lt</em>hcare system, insufficient medical facilities, unawareness, and the sharing of misinformation in the mass media has led to people experiencing fear and anxiety. The present study intended to conduct a perception-<em>b</em>ased analysis to get an idea of people's psychosocial and socio-economic crisis, and the possi<em>b</em>le environmental crisis, amidst the COVID-19 pandemic in Bangladesh. (<em>b</em>)Methods:</<em>b</em>) A perception-<em>b</em>ased questionnaire was put online for Bangladeshi citizens of 18 years and/or older. The sample size was 1,066 respondents. Datasets were analyzed through a set of statistical techniques including principal component and hierarchical cluster analysis. (<em>b</em>)Resu<em>lt</em>s:</<em>b</em>) There was a positive significant association <em>b</em>etween fear of the COVID-19 out<em>b</em>reak with the struggling hea<em>lt</em>hcare system (<i>p</i> &<em>lt</em>; 0.05) of the country. Also, there was a negative association <em>b</em>etween the fragile hea<em>lt</em>h system of Bangladesh and the government's a<em>b</em>ility to deal with the pandemic (<i>p</i> &<em>lt</em>; 0.05), revealing the poor governance in the hea<em>lt</em>hcare system. A positive association of shutdown and social distancing with the fear of losing one's own or a family mem<em>b</em>ers' life, influenced <em>b</em>y a lack of hea<em>lt</em>hcare treatment (<i>p</i> &<em>lt</em>; 0.05), reveals that, due to the decision of shutting down normal activities, people may <em>b</em>e experiencing mental and economic stress. However, a positive association of the socio-economic impact of the shutdown with poor people's suffering, the price hike of <em>b</em>asic essentials, the hindering of formal education (<i>p</i> &<em>lt</em>; 0.05), and the possi<em>b</em>ility of a severe socio-economic and hea<em>lt</em>h crisis will <em>b</em>e aggravated. Moreover, there is a possi<em>b</em>ility of a climate change-induced disaster and infectious diseases like dengue during/after the COVID-19 situation, which will create severe food insecurity (<i>p</i> &<em>lt</em>; 0.01) and a further hea<em>lt</em>hcare crisis. (<em>b</em>)Conclusions:</<em>b</em>) The partial lockdown in Bangladesh due to the COVID-19 pandemic increased community transmission and worsened the hea<em>lt</em>hcare crisis, economic <em>b</em>urden, and loss of GDP despite the resuming of industrial operations. In society, it has created psychosocial and socio-economic insecurity among people due to the loss of lives and livelihoods. The government should take proper inclusive steps for risk assessment, communications, and financial stimulus toward the pu<em>b</em>lic to alleviate their fear and anxiety, and to take proper action to <em>b</em>oost mental hea<em>lt</em>h and well-<em>b</em>eing.

Keywords: COVID-19; fear; linear regression model; perception-based questionnaire; principal component analysis (PCA); social conflict.

<p><div>(<em>b</em>)BACKGROUND</<em>b</em>)</div>It is unknown whether patients undergoing primary percutaneous coronary intervention (PCI) <em>b</em>enefit from genotype-guided selection of oral P2Y<su<em>b</em>)12</su<em>b</em>) inhi<em>b</em>itors.</p><p><div>(<em>b</em>)METHODS</<em>b</em>)</div>We conducted a randomized, open-la<em>b</em>el, assessor-<em>b</em>linded trial in which patients undergoing primary PCI with stent implantation were assigned in a 1:1 ratio to receive either a P2Y<su<em>b</em>)12</su<em>b</em>) inhi<em>b</em>itor on the <em>b</em>asis of early <i>CYP2C19</i> genetic testing (genotype-guided group) or standard treatment with either ticagrelor or prasugrel (standard-treatment group) for 12 months. In the genotype-guided group, carriers of <i>CYP2C19</i>*2 or <i>CYP2C19</i>*3 loss-of-function alleles received ticagrelor or prasugrel, and noncarriers received clopidogrel. The two primary outcomes were net adverse clinical events - defined as death from any cause, myocardial infarction, definite stent throm<em>b</em>osis, stroke, or major <em>b</em>leeding defined according to Platelet Inhi<em>b</em>ition and Patient Outcomes (PLATO) criteria - at 12 months (primary com<em>b</em>ined outcome; tested for noninferiority, with a noninferiority margin of 2 percentage points for the a<em>b</em>solute difference) and PLATO major or minor <em>b</em>leeding at 12 months (primary <em>b</em>leeding outcome).</p><A<em>b</em>stractText>For the primary analysis, 2488 patients were included: 1242 in the genotype-guided group and 1246 in the standard-treatment group. The primary com<em>b</em>ined outcome occurred in 63 patients (5.1%) in the genotype-guided group and in 73 patients (5.9%) in the standard-treatment group (a<em>b</em>solute difference, -0.7 percentage points; 95% confidence interval [CI], -2.0 to 0.7; P&<em>lt</em>;0.001 for noninferiority). The primary <em>b</em>leeding outcome occurred in 122 patients (9.8%) in the genotype-guided group and in 156 patients (12.5%) in the standard-treatment group (hazard ratio, 0.78; 95% CI, 0.61 to 0.98; P = 0.04).</A<em>b</em>stractText><p><div>(<em>b</em>)CONCLUSIONS</<em>b</em>)</div>In patients undergoing primary PCI, a <i>CYP2C19</i> genotype-guided strategy for selection of oral P2Y<su<em>b</em>)12</su<em>b</em>) inhi<em>b</em>itor therapy was noninferior to standard treatment with ticagrelor or prasugrel at 12 months with respect to throm<em>b</em>otic events and resu<em>lt</em>ed in a lower incidence of <em>b</em>leeding. (Funded <em>b</em>y the Netherlands Organization for Hea<em>lt</em>h Research and Development; POPular Genetics ClinicalTrials.gov num<em>b</em>er, NCT01761786; Netherlands Trial Register num<em>b</em>er, NL2872.).</p>

(<em>b</em>)O<em>b</em>jectives</<em>b</em>) Severe or critical COVID-19 is associated with intensive care unit admission, increased secondary infection rate, and would lead to significant worsened prognosis. Risks and characteristics relating to secondary infections in severe COVID-19 has not <em>b</em>een descri<em>b</em>ed. (<em>b</em>)Methods</<em>b</em>) Severe and critical COVID-19 patients from Shanghai were included. We collected lower respiratory, urine, catheters and <em>b</em>lood samples according to clinical necessity and cu<em>lt</em>ure and mNGS were performed. Clinical and la<em>b</em>oratory data were archived. (<em>b</em>)Resu<em>lt</em>s</<em>b</em>) We found 57.89% (22/38) patients developed secondary infections. The patient receiving invasive mechanical ventilation or in critical state has a higher chance of secondary infections(P&<em>lt</em>;0.0001). The most common infections were respiratory, <em>b</em>lood-stream and urinary infections, and in respiratory infections, the most detected pathogens were gram-negative <em>b</em>acteria (26, 50.00%), following <em>b</em>y gram-positive <em>b</em>acteria (14, 26.92%), virus (6, 11.54%), fungi (4, 7.69%) and others (2, 3.85%). Respiratory Infection rate post high flow, tracheal intu<em>b</em>ation and tracheotomy were 12.90% (4/31), 30.43% (7/23), and 92.31% (12/13) respectively. Secondary infections would lead to lower discharge rate and higher mortality rate. (<em>b</em>)Conclusion</<em>b</em>) Our study originally illustrated secondary infection proportion in severe and critical COVID-19 patients. Cu<em>lt</em>ure accompanied with metagenomics sequencing increased pathogen diagnostic rate. Secondary infections risks increased after receiving invasive respiratory ventilations and intravascular devices, and would lead to a lower discharge rate and a higher mortality rate.

Keywords: COVID-19; SARS-CoV-2; intensive care; secondary infection.

CSF biomarkers, including total-tau, neurofilament light chain (NfL) and amyloid-β, are increasingly being used to define and stage Alzheimer's disease. These biomarkers can be measured more quickly and less invasively in plasma and may provide important information for early diagnosis of Alzheimer's disease. We used stored plasma samples and clinical data obtained from 4444 non-demented participants in the Rotterdam study at baseline (between 2002 and 2005) and during follow-up until January 2016. Plasma concentrations of total-tau, NfL, amyloid-βββlt; 0.0001] and 0.59 (95% CI, 0.43-0.79; P = 0.0006), respectively. Conversely, a log2 higher baseline plasma NfL level was associated with a higher risk of all-cause dementia [adjusted HR 1.59 (95% CI, 1.38-1.83); P &lt; 0.0001] or Alzheimer's disease [adjusted HR 1.50 (95% CI, 1.26-1.78); P &lt; 0.0001]. Combining the lowest quartile group of amyloid-βlted in a stronger association with all-cause dementia [adjusted HR 9.5 (95% CI, 2.3-40.4); P &lt; 0.002] and with Alzheimer's disease [adjusted HR 15.7 (95% CI, 2.1-117.4); P &lt; 0.0001], compared to the highest quartile group of amyloid-ββlt; 0.0001), plasma values for cases diverged from controls 9.6 years before Alzheimer's disease diagnosis. Amyloid-βlthough the decline did not reach significance compared to dementia-free participants. In conclusion, our study shows that low amyloid-ββlthough not specific, may also be useful in monitoring progression of Alzheimer's disease dementia.

<A<em>b</em>stractText>Efficacy and safety of the GLP-1 analog, oral semaglutide, and the SGLT-2 inhi<em>b</em>itor, empagliflozin, were compared in patients with type 2 dia<em>b</em>etes uncontrolled on metformin.</A<em>b</em>stractText><p><div>(<em>b</em>)RESEARCH DESIGN AND METHODS</<em>b</em>)</div>Patients were randomized to once-daily open-la<em>b</em>el treatment with oral semaglutide 14 mg (<i>n</i> = 412) or empagliflozin 25 mg (<i>n</i> = 410) in a 52-week trial. Key endpoints were change from <em>b</em>aseline to week 26 in H<em>b</em>A<su<em>b</em>)1c</su<em>b</em>) (primary) and <em>b</em>ody weight (confirmatory secondary). Two estimands addressed efficacy-related questions: treatment policy (regardless of trial product discontinuation or rescue medication) and trial product (on trial product without rescue medication) in all randomized patients.</p><p><div>(<em>b</em>)RESULTS</<em>b</em>)</div>Four-hundred patients (97.1%) in the oral semaglutide group and 387 (94.4%) in the empagliflozin group completed the trial. Oral semaglutide provided superior reductions in H<em>b</em>A<su<em>b</em>)1c</su<em>b</em>) versus empagliflozin at week 26 (treatment policy: -1.3% vs. -0.9% [-14 vs. -9 mmol/mol]; estimated treatment difference [ETD]: -0.4%, 95% CI -0.6, -0.3 [-5 mmol/mol, 95% CI -6, -3]; <i>P</i>&<em>lt</em>; 0.0001). The treatment difference in H<em>b</em>A<su<em>b</em>)1c</su<em>b</em>) significantly favored oral semaglutide at week 26 for the trial product estimand (-1.4% vs. -0.9% [-15 vs. -9 mmol/mol]; ETD: -0.5%, 95% CI -0.7, -0.4 [-6 mmol/mol, 95% CI -7, -5]; <i>P</i>&<em>lt</em>; 0.0001), and at week 52 for <em>b</em>oth estimands (<i>P</i>&<em>lt</em>; 0.0001). Superior weight loss was not confirmed at week 26 (treatment policy), <em>b</em>ut oral semaglutide was significantly <em>b</em>etter than empagliflozin at week 52 (trial product: -4.7 vs. -3.8 kg; <i>P</i> = 0.0114). Gastrointestinal adverse events were more common with oral semaglutide.</p><p><div>(<em>b</em>)CONCLUSIONS</<em>b</em>)</div>Oral semaglutide was superior to empagliflozin in reducing H<em>b</em>A<su<em>b</em>)1c</su<em>b</em>) <em>b</em>ut not <em>b</em>ody weight at 26 weeks in patients with type 2 dia<em>b</em>etes uncontrolled on metformin. At week 52, H<em>b</em>A<su<em>b</em>)1c</su<em>b</em>) and <em>b</em>ody weight (trial product estimand) were significantly reduced versus empagliflozin. Oral semaglutide was well tolerated within the esta<em>b</em>lished safety profile of GLP-1 receptor agonists.</p>

DA, GDVII and H101 are neurovirulent strains of Theiler's murine encephalomyelitis virus that cause very different neuropathology and CNS disease when inoculated into SJL/J mice. DA virus causes a chronic demyelinating disease, GDVII virus causes an acute fatal polioencephalomyelitis, and H101 virus causes an acute pachymeningitis with hydrocephalus. Performing RNase protection assays, we detected the same pattern of chemokine (RANTES, MCP-1, IP-10, MIP-1beta, MIP-1alpha and MIP-2) mRNA expression in brain and spinal cord during all three infections. In contrast, IFN-beta and IL-6 mRNA were highly expressed only in GDVII virus infection, whereas high levels of LT-alpha mRNA were only found during DA virus infection. Our study demonstrates that proinflammatory cytokines are involved in the neuropathogenesis of CNS disease and modulate the acute and chronic process underlying different pathologic features of disease.

BACKGROUND

We have developed a new oral vaccine against enterotoxigenic Escherichia coli (ETEC), which is the most common cause of bacterial diarrhea in children in developing countries and in travelers.

METHODS

The vaccine was tested for safety and immunogenicity alone and together with double-mutant heat-labile toxin (dmLT) adjuvant in a double-blind, placebo-controlled Phase I study in 129 Swedish adults. The vaccine consists of four inactivated recombinant E. coli strains overexpressing the major ETEC colonization factors (CFs) CFA/I, CS3, CS5, and CS6 mixed with an LT B-subunit related toxoid, LCTBA. Volunteers received two oral doses of vaccine alone, vaccine plus 10 μg or 25 μg dmLT or placebo. Secretory IgA antibody responses in fecal samples and IgA responses in secretions from circulating intestine-derived antibody secreting cells were assessed as primary measures of vaccine immunogenicity.

RESULTS

The vaccine was safe and well tolerated; adverse events were few and generally mild with no significant differences between subjects receiving placebo or vaccine with or without adjuvant. As many as 74% of subjects receiving vaccine alone and 83% receiving vaccine plus 10 μg dmLT showed significant mucosal IgA responses to all five primary vaccine antigens and about 90% of all vaccinees responded to at least four of the antigens. Subjects receiving vaccine plus 10 μg dmLT responded with significantly increased intestine-derived anti-CS6 responses compared to subjects receiving vaccine alone.

CONCLUSIONS

The vaccine was safe and broadly immunogenic. dmLT further enhanced mucosal immune responses to CF antigens present in low amounts in the vaccine. Based on these encouraging results, the vaccine will be tested for safety and immunogenicity in different age groups including infants in Bangladesh and for protective efficacy in travelers.

<A<em>b</em>stractText>In patients with heart failure and reduced ejection fraction (HFrEF), treatment with sacu<em>b</em>itril-valsartan reduces N-terminal pro-<em>b</em>-type natriuretic peptide (NT-proBNP) concentrations. The effect of sacu<em>b</em>itril-valsartan on cardiac remodeling is uncertain.</A<em>b</em>stractText><A<em>b</em>stractText>To determine whether NT-proBNP changes in patients with HFrEF treated with sacu<em>b</em>itril-valsartan correlate with changes in measures of cardiac volume and function.</A<em>b</em>stractText><A<em>b</em>stractText>Prospective, 12-month, single-group, open-la<em>b</em>el study of patients with HFrEF enrolled in 78 outpatient sites in the United States. Sacu<em>b</em>itril-valsartan was initiated and the dose adjusted. Enrollment commenced on Octo<em>b</em>er 25, 2016, and follow-up was completed on Octo<em>b</em>er 22, 2018.</A<em>b</em>stractText><A<em>b</em>stractText>NT-proBNP concentrations among patients treated with sacu<em>b</em>itril-valsartan.</A<em>b</em>stractText><A<em>b</em>stractText>The primary outcome was the correlation <em>b</em>etween changes in log2-NT-proBNP concentrations and left ventricular (LV) EF, LV end-diastolic volume index (LVEDVI), LV end-systolic volume index (LVESVI), left atrial volume index (LAVI), and ratio of early transmitral Doppler velocity/early diastolic annular velocity (E/e') at 12 months.</A<em>b</em>stractText><A<em>b</em>stractText>Among 794 patients (mean age, 65.1 years; 226 women [28.5%]; mean LVEF = 28.2%), 654 (82.4%) completed the study. The median NT-proBNP concentration at <em>b</em>aseline was 816 pg/mL (interquartile range [IQR], 332-1822) and 455 pg/mL (IQR, 153-1090) at 12 months (difference, P &<em>lt</em>; .001). At 12 months, the change in log2-NT-proBNP concentration was correlated with changes in LVEF (r = -0.381 [IQR, -0.448 to -0.310]; P &<em>lt</em>; .001), LVEDVI (r = 0.320 [IQR, 0.246 to 0.391]; P &<em>lt</em>; .001), LVESVI (r = 0.405 [IQR, 0.335 to 0.470]; P &<em>lt</em>; .001), LAVI (r = 0.263 [IQR, 0.186 to 0.338]; P &<em>lt</em>; .001), and E/e' (r = 0.269 [IQR, 0.182 to 0.353]; P &<em>lt</em>; .001). At 12 months, LVEF increased from 28.2% to 37.8% (difference, 9.4% [95% CI, 8.8% to 9.9%]; P &<em>lt</em>; .001), while LVEDVI decreased from 86.93 to 74.15 mL/m2 (difference, -12.25 mL/m2 [IQR, -12.92 to -11.58]; P &<em>lt</em>; .001) and LVESVI decreased from 61.68 to 45.46 mL/m2 (difference, -15.29 mL/m2 [95% CI, -16.03 to -14.55]; P &<em>lt</em>; .001). LAVI and E/e' ratio also decreased significantly. The most frequent adverse events were hypotension (17.6%), dizziness (16.8%), hyperkalemia (13.2%), and worsening kidney function (12.3%).</A<em>b</em>stractText><A<em>b</em>stractText>In this exploratory study of patients with HFrEF treated with sacu<em>b</em>itril-valsartan, reduction in NT-proBNP concentration was weakly yet significantly correlated with improvements in markers of cardiac volume and function at 12 months. The o<em>b</em>served reverse cardiac remodeling may provide a mechanistic explanation for the effects of sacu<em>b</em>itril-valsartan in patients with HFrEF.</A<em>b</em>stractText><A<em>b</em>stractText>ClinicalTrials.gov Identifier: NCT02887183.</A<em>b</em>stractText>

BACKGROUND

From initiation to plaque rupture, immune system components contribute to atherosclerosis. We investigated variation in inflammation-related genes - interleukin (IL)-1beta, IL-6, C-reactive protein (CRP), IL-10, IL-18, and the tumor necrosis factor (TNF) superfamily [lymphotoxin(LT)-alpha, TNF-alpha, LT-beta] - with respect to nonfatal incident myocardial infarction (MI) or ischemic stroke risk.

RESULTS

A population-based case-control study recruited postmenopausal and/or hypertensive Group Health members aged 30-79 years. We chose a subset of single nucleotide polymorphisms (SNPs) to describe common gene-wide variation on the basis of linkage disequilibrium. 36 SNPs, describing 38 common haplotypes for 5 genes and a 3-gene cluster, were genotyped among 856 MI cases, 368 stroke cases, and 2688 controls. Associations of SNPs or PHASE-inferred haplotypes and risk were estimated using logistic regression; significance of gene-level associations was assessed with global Wald tests and permutation tests. Gene-wide IL-18 variation was associated with higher MI risk and an IL-1B haplotype was associated with lower stroke risk. In secondary analyses of SNPs, we observed associations of several IL-1B polymorphisms with risk of MI or stroke. IL-6, CRP, IL-10, and TNF superfamily gene variation was not associated with MI or stroke risk.

CONCLUSIONS

Our results support prior reports associating an IL-18 gene variant and MI risk, contribute additional evidence to reports of IL-1B and cardiovascular risk, and fail to confirm risk differences previously observed for CRP, IL-6, and TNF-alpha promoter variants.

<A<em>b</em>stractText>We aimed to evaluate the value of deep learning on positron emission tomography with computed tomography (PET/CT)-<em>b</em>ased radiomics for individual induction chemotherapy (IC) in advanced nasopharyngeal carcinoma (NPC).</A<em>b</em>stractText><p><div>(<em>b</em>)EXPERIMENTAL DESIGN</<em>b</em>)</div>We constructed radiomics signatures and nomogram for predicting disease-free survival (DFS) <em>b</em>ased on the extracted features from PET and CT images in a training set (<i>n</i> = 470), and then validated it on a test set (<i>n</i> = 237). Harrell's concordance indices (C-index) and time-independent receiver operating characteristic (ROC) analysis were applied to evaluate the discriminatory a<em>b</em>ility of radiomics nomogram, and compare radiomics signatures with plasma Epstein-Barr virus (EBV) DNA.</p><p><div>(<em>b</em>)RESULTS</<em>b</em>)</div>A total of 18 features were selected to construct CT-<em>b</em>ased and PET-<em>b</em>ased signatures, which were significantly associated with DFS (<i>P</i> &<em>lt</em>; 0.001). Using these signatures, we proposed a radiomics nomogram with a C-index of 0.754 [95% confidence interval (95% CI), 0.709-0.800] in the training set and 0.722 (95% CI, 0.652-0.792) in the test set. Consequently, 206 (29.1%) patients were stratified as high-risk group and the other 501 (70.9%) as low-risk group <em>b</em>y the radiomics nomogram, and the corresponding 5-year DFS rates were 50.1% and 87.6%, respectively (<i>P</i> &<em>lt</em>; 0.0001). High-risk patients could <em>b</em>enefit from IC while the low-risk could not. Moreover, radiomics nomogram performed significantly <em>b</em>etter than the EBV DNA-<em>b</em>ased model (C-index: 0.754 vs. 0.675 in the training set and 0.722 vs. 0.671 in the test set) in risk stratification and guiding IC.</p><A<em>b</em>stractText>Deep learning PET/CT-<em>b</em>ased radiomics could serve as a relia<em>b</em>le and powerful tool for prognosis prediction and may act as a potential indicator for individual IC in advanced NPC.</A<em>b</em>stractText>

<A<em>b</em>stractText>Standard-of-care treatment for patients with newly diagnosed mu<em>lt</em>iple myeloma includes com<em>b</em>ination therapies for patients who are not eligi<em>b</em>le for autologous stem-cell transplantation. At the primary analysis for progression-free survival of the phase 3 ALCYONE trial, progression-free survival was significantly longer with daratumuma<em>b</em> in com<em>b</em>ination with <em>b</em>ortezomi<em>b</em>, melphalan, and prednisone (D-VMP) versus <em>b</em>ortezomi<em>b</em>, melphalan, and prednisone (VMP) alone in patients with transplant-ineligi<em>b</em>le, newly diagnosed mu<em>lt</em>iple myeloma. Here we report updated efficacy and safety resu<em>lt</em>s from a prespecified, interim, overall survival analysis of ALCYONE with more than 36 months of follow-up.</A<em>b</em>stractText><p><div>(<em>b</em>)METHODS</<em>b</em>)</div>ALCYONE was a mu<em>lt</em>icentre, randomised, open-la<em>b</em>el, active-controlled, phase 3 trial that enrolled patients <em>b</em>etween Fe<em>b</em> 9, 2015, and July 14, 2016, at 162 sites in 25 countries across North America, South America, Europe, and the Asia-Pacific region. Patients were eligi<em>b</em>le for inclusion if they had newly diagnosed mu<em>lt</em>iple myeloma and were ineligi<em>b</em>le for high-dose chemotherapy with autologous stem-cell transplantation, <em>b</em>ecause of their age (≥65 years) or <em>b</em>ecause of su<em>b</em>stantial comor<em>b</em>idities. Patients were randomly assigned in a 1:1 ratio and <em>b</em>y permuted <em>b</em>lock randomisation to receive D-VMP or VMP. An interactive we<em>b</em>-<em>b</em>ased randomisation system was used. Randomisation was stratified <em>b</em>y International Staging System disease stage, geographical region, and age. There was no masking to treatment assignments. All patients received up to nine 6-week cycles of su<em>b</em>cutaneous <em>b</em>ortezomi<em>b</em> (1·3 mg/m² of <em>b</em>ody surface area on days 1, 4, 8, 11, 22, 25, 29, and 32 of cycle one and on days 1, 8, 22, and 29 of cycles two through nine), oral melphalan (9 mg/m² once daily on days 1 through 4 of each cycle), and oral prednisone (60 mg/m² once daily on days 1 through 4 of each cycle). Patients in the D-VMP group also received intravenous daratumuma<em>b</em> (16 mg/kg of <em>b</em>odyweight, once weekly during cycle one, once every 3 weeks in cycles two through nine, and once every 4 weeks thereafter as maintenance therapy until disease progression or unaccepta<em>b</em>le toxicity). The primary endpoint was progression-free survival, which has <em>b</em>een reported previously. Resu<em>lt</em>s presented are from a prespecified interim analysis for overall survival. The primary analysis population (including for overall survival) was the intention-to-treat population of all patients who were randomly assigned to treatment. The safety population included patients who received any dose of study treatment. This trial is registered with ClinicalTrials.gov, NCT02195479.</p><A<em>b</em>stractText>706 patients were randomly assigned to treatment groups (350 to the D-VMP group, 356 to the VMP group). At a median follow-up of 40·1 months (IQR 37·4-43·1), a significant <em>b</em>enefit in overall survival was o<em>b</em>served for the D-VMP group. The hazard ratio (HR) for death in the D-VMP group compared with the VMP group was 0·60 (95% CI 0·46-0·80; p=0·0003). The Kaplan-Meier estimate of the 36-month rate of overall survival was 78·0% (95% CI 73·2-82·0) in the D-VMP group and 67·9% (62·6-72·6) in the VMP group. Progression-free survival, the primary endpoint, remained significantly improved for the D-VMP group (HR 0·42 [0·34-0·51]; p&<em>lt</em>;0·0001). The most frequent adverse events during maintenance daratumuma<em>b</em> monotherapy in patients in the D-VMP group were respiratory infections (54 [19%] of 278 patients had upper respiratory tract infections; 42 [15%] had <em>b</em>ronchitis, 34 [12%] had viral upper respiratory tract infections), cough (34 [12%]), and diarrhoea (28 [10%]).</A<em>b</em>stractText><A<em>b</em>stractText>D-VMP prolonged overall survival in patients with newly diagnosed mu<em>lt</em>iple myeloma who were ineligi<em>b</em>le for stem-cell transplantation. With more than 3 years of follow-up, the D-VMP group continued to show significant improvement in progression-free survival, with no new safety concerns.</A<em>b</em>stractText><A<em>b</em>stractText>Janssen Research & Development.</A<em>b</em>stractText>