When activated by specific antigen, complement, or other transmembrane stimuli, mast cells (MCs) generate three eicosanoids: prostaglandin (PG)D(2), leukotriene (LT)B(4), and LTC(4), the parent molecule of the cysteinyl leukotrienes (cysLTs). These diverse lipid mediators, which are generated from a single cell membrane-associated precursor, arachidonic acid, can initiate, amplify, or dampen inflammatory responses and influence the magnitude, duration, and nature of subsequent immune responses. PGD(2) and cysLTs, which were originally recognized for their bronchoconstricting and vasoactive properties, also serve diverse and pivotal functions in effector cell trafficking, antigen presentation, leukocyte activation, matrix deposition, and fibrosis. LTB(4) is a powerful chemoattractant for neutrophils and certain lymphocyte subsets. Thus, MCs can contribute to each of these processes through eicosanoid generation. Additionally, MCs express G-protein-coupled receptors specific for cysLTs, LTB(4), and another eicosanoid, PGE(2). Each of these receptors can regulate MC functions in vivo by autocrine and paracrine mechanisms. This review focuses on the biologic functions for MC-associated eicosanoids, the regulation of their production, and the mechanisms by which eicosanoids may regulate MC function in host defense and disease.

Background: No effective oral therapy exists for early coronavirus disease 2019 (COVID-19).

Objective: To investigate whether hydroxychloroquine could reduce COVID-19 severity in adult outpatients.

Design: Randomized, double-blind, placebo-controlled trial conducted from 22 March through 20 May 2020. (ClinicalTrials.gov: NCT04308668).

Setting: Internet-based trial across the United States and Canada (40 states and 3 provinces).

Participants: Symptomatic, nonhospitalized adults with laboratory-confirmed COVID-19 or probable COVID-19 and high-risk exposure within 4 days of symptom onset.

<strong class="su<em>b</em>-title"> Intervention: </strong> Oral hydroxychloroquine (800 mg once, followed <em>b</em>y 600 mg in 6 to 8 hours, then 600 mg daily for 4 more days) or masked place<em>b</em>o. (<em>b</em>)Measures</<em>b</em>): Symptoms and severity at <em>b</em>aseline and then at days 3, 5, 10, and 14 using a 10-point visual analogue scale. The primary end point was change in overall symptom severity over 14 days.

Results: Of 491 patients randomly assigned to a group, 423 contributed primary end point data. Of these, 341 (81%) had laboratory-confirmed infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or epidemiologically linked exposure to a person with laboratory-confirmed infection; 56% (236 of 423) were enrolled within 1 day of symptoms starting. Change in symptom severity over 14 days did not differ between the hydroxychloroquine and placebo groups (difference in symptom severity: relative, 12%; absolute, -0.27 points [95% CI, -0.61 to 0.07 points]; P = 0.117). At 14 days, 24% (49 of 201) of participants receiving hydroxychloroquine had ongoing symptoms compared with 30% (59 of 194) receiving placebo (P = 0.21). Medication adverse effects occurred in 43% (92 of 212) of participants receiving hydroxychloroquine versus 22% (46 of 211) receiving placebo (P < 0.001). With placebo, 10 hospitalizations occurred (2 non-COVID-19-related), including 1 hospitalized death. With hydroxychloroquine, 4 hospitalizations occurred plus 1 nonhospitalized death (P = 0.29).

Limitations: Only 58% of participants received SARS-CoV-2 testing because of severe U.S. testing shortages.

Conclusion: Hydroxychloroquine did not substantially reduce symptom severity in outpatients with early, mild COVID-19.

Primary funding source: Private donors.

We present evidence that toxic shock syndrome toxin 1 (TSST-1) induces the production of high levels of TNF by human blood monocytes. Enriched lymphocyte preparations incubated with the staphylococcal toxin produced significant levels of TNF-like activity that is not neutralized by anti-rHuTNF antibodies and is likely to be lymphotoxin (LT or TNF-beta). We demonstrate also that TSST-1 is a potent inducer of IFN-gamma. When lymphocyte preparations were costimulated with PMA, the TSST-1 effect was strongly potentiated and the levels of cytotoxic factors, IFN-gamma, and IL-2 present in supernatant fluids were comparable to those observed after treatment with PMA and PHA. Thus, TSST-1, which is also known as an inducer of IL-1 and IL-2, stimulates the production of endogenous mediators that could play a role in the physiopathological processes of toxic shock syndrome (TSS). The described results suggest that the discrepancies in the clinical features between TSS and endotoxin shock may be related to qualitative differences in cytokine production.

Affinity maturation by somatic hypermutation is thought to occur within germinal centres. Mice deficient in lymphotoxin-alpha (LT alpha-/- mice) have no lymph nodes or Peyer's patches, and fail to form germinal centres in the spleen. We tested whether germinal centres are essential for maturation of antibody responses to T-cell-dependent antigens. LT alpha-/- mice immunized with low doses of (4-hydroxy-3-nitrophenyl)acetyl-ovalbumin (NP-OVA) showed dramatically impaired production of high-affinity anti-NP IgG1. However, LT alpha-/- mice immunized with high doses of NP-OVA, even though they failed to produce germinal centres, manifested a high-affinity anti-NP IgG1 response similar to wild-type mice. Furthermore, when LT alpha-/- mice were multiply immunized with high doses of NP-OVA, the predominantly expressed anti-NP VH gene segment VH186.2 showed somatic mutations typical of affinity maturation. Thus, B-cell memory and affinity maturation are not absolutely dependent on the presence of germinal centres.

OBJECTIVE

LIGHT (lymphotoxin-like inducible protein that competes with glycoprotein D for herpes virus entry on T cells) is a tumor necrosis factor core family member that regulates T-cell activation and causes experimental inflammatory bowel disease. Additional data suggest that LIGHT may be involved in the pathogenesis of human inflammatory bowel disease. The aim of this study was to determine if LIGHT is capable of signaling directly to intestinal epithelia and to define the mechanisms and consequences of such signaling.

METHODS

The effects of LIGHT and interferon-gamma on barrier function, cytoskeletal regulation, and tight junction structure were assessed in mice and intestinal epithelial monolayers.

RESULTS

LIGHT induced barrier loss in cultured epithelia via myosin II regulatory light chain (MLC) phosphorylation; both barrier loss and MLC phosphorylation were reversed by MLC kinase (MLCK) inhibition. Pretreatment with interferon-gamma, which induced lymphotoxin beta receptor (LT beta R) expression, was required for these effects, and neither barrier dysfunction nor intestinal epithelial MLC phosphorylation occurred in LT beta R knockout mice. In cultured monolayers, endocytosis of the tight junction protein occludin correlated with barrier loss. Internalized occludin colocalized with caveolin-1. LIGHT-induced occludin endocytosis and barrier loss were both prevented by inhibition of caveolar endocytosis.

CONCLUSIONS

T cell-derived LIGHT activates intestinal epithelial LT beta R to disrupt barrier function. This requires MLCK activation and caveolar endocytosis. These data suggest a novel role for LIGHT in disease pathogenesis and suggest that inhibition of MLCK-dependent caveolar endocytosis may represent an approach to restoring barrier function in inflammatory bowel disease.

<A<em>b</em>stractText>The aim of this study was to further clarify clinical characteristics and predict mortality risk among patients with viral pneumonia.</A<em>b</em>stractText><A<em>b</em>stractText>A total of 528 patients with viral pneumonia at RuiJin hospital in Shanghai from May 2015 to May 2019 were recruited. Mu<em>lt</em>iplex real-time RT-PCR was used to detect respiratory viruses. Demographic information, comor<em>b</em>idities, routine la<em>b</em>oratory examinations, immunological indexes, etiological detections, radiological images and treatment were collected on admission.</A<em>b</em>stractText><p><div>(<em>b</em>)Resu<em>lt</em>s</<em>b</em>)</div>76 (14.4%) patients died within 90 days in hospital. A predictive MuLBSTA score was calculated on the <em>b</em>asis of a mu<em>lt</em>ivariate logistic regression model in order to predict mortality with a weighted score that included mu<em>lt</em>ilo<em>b</em>ular infi<em>lt</em>rates (OR = 5.20, 95% CI 1.41-12.52, <i>p</i> = 0.010; 5 points), lymphocyte ≤ 0.8^∗10⁹/L (OR = 4.53, 95% CI 2.55-8.05, <i>p</i> &<em>lt</em>; 0.001; 4 points), <em>b</em>acterial coinfection (OR = 3.71, 95% CI 2.11-6.51, <i>p</i> &<em>lt</em>; 0.001; 4 points), acute-smoker (OR = 3.19, 95% CI 1.34-6.26, <i>p</i> = 0.001; 3 points), quit-smoker (OR = 2.18, 95% CI 0.99-4.82, <i>p</i> = 0.054; 2 points), hypertension (OR = 2.39, 95% CI 1.55-4.26, <i>p</i> = 0.003; 2 points) and age ≥60 years (OR = 2.14, 95% CI 1.04-4.39, <i>p</i> = 0.038; 2 points). 12 points was used as a cut-off value for mortality risk stratification. This model showed sensitivity of 0.776, specificity of 0.778 and a <em>b</em>etter predictive a<em>b</em>ility than CURB-65 (AUROC = 0.773 vs. 0.717, <i>p</i> &<em>lt</em>; 0.001).</p><A<em>b</em>stractText>Here, we designed an easy-to-use clinically predictive tool for assessing 90-day mortality risk of viral pneumonia. It can accurately stratify hospitalized patients with viral pneumonia into relevant risk categories and could provide guidance to make further clinical decisions.</A<em>b</em>stractText>

<A<em>b</em>stractText>The aim of the current study was to conduct a pooled analysis of studies that have investigated the prognostic value of tumor-infi<em>lt</em>rating lymphocytes (TILs) in early-stage triple negative <em>b</em>reast cancer (TNBC).</A<em>b</em>stractText><A<em>b</em>stractText>Participating studies had evaluated the percentage infi<em>lt</em>ration of stromally located TILs (sTILs) that were quantified in the same manner in patient diagnostic samples of early-stage TNBC treated with anthracycline-<em>b</em>ased chemotherapy with or without taxanes. Cox proportional hazards regression models stratified <em>b</em>y trial were used for invasive disease-free survival (iDFS; primary end point), distant disease-free survival (D-DFS), and overall survival (OS), fitting sTILs as a continuous varia<em>b</em>le adjusted for clinicopathologic factors.</A<em>b</em>stractText><p><div>(<em>b</em>)RESULTS</<em>b</em>)</div>We collected individual data from 2,148 patients from nine studies. Average age was 50 years (range, 22 to 85 years), and 33% of patients were node negative. The average value of sTILs was 23% (standard deviation, 20%), and 77% of patients had 1% or more sTILs. sTILs were significantly lower with older age ( P = .001), larger tumor size ( P = .01), more nodal involvement ( P = .02), and lower histologic grade ( P = .001). A total of 736 iDFS and 548 D-DFS events and 533 deaths were o<em>b</em>served. In the mu<em>lt</em>ivaria<em>b</em>le model, sTILs added significant independent prognostic information for all end points (likelihood ratio χ², 48.9 iDFS; P &<em>lt</em>; .001; χ², 55.8 D-DFS; P &<em>lt</em>; .001; χ², 48.5 OS; P &<em>lt</em>; .001). Each 10% increment in sTILs corresponded to an iDFS hazard ratio of 0.87 (95% CI, 0.83 to 0.91) for iDFS, 0.83 (95% CI, 0.79 to 0.88) for D-DFS, and 0.84 (95% CI, 0.79 to 0.89) for OS. In node-negative patients with sTILs ≥ 30%, 3-year iDFS was 92% (95% CI, 89% to 98%), D-DFS was 97% (95% CI, 95% to 99%), and OS was 99% (95% CI, 97% to 100%).</p><A<em>b</em>stractText>This pooled data analysis confirms the strong prognostic role of sTILs in early-stage TNBC and excellent survival of patients with high sTILs after adjuvant chemotherapy and supports the integration of sTILs in a clinicopathologic prognostic model for patients with TNBC. This model can <em>b</em>e found at www.tilsin<em>b</em>reastcancer.org .</A<em>b</em>stractText>

Toll-like receptor (TLR)-dependent pathways control the production of IFNalphabeta, a key cytokine in innate immune control of viruses including mouse cytomegalovirus (MCMV). The lymphotoxin (LT) alphabeta-LTbeta receptor signaling pathway is also critical for defense against MCMV and thought to aid in the IFNbeta response. We find that upon MCMV infection, mice deficient for lymphotoxin (LT)alphabeta signaling cannot mount the initial part of a biphasic IFNalphabeta response, but show normal levels of IFNalphabeta during the sustained phase of infection. Significantly, the LTalphabeta-dependent, IFNalphabeta response is independent of TLR signaling. B, but not T, cells expressing LTbeta are essential for promoting the initial IFNalphabeta response. LTbetaR expression is required strictly in splenic stromal cells for initial IFNalphabeta production to MCMV and is dependent upon the NF-kappaB-inducing kinase (NIK). These results reveal a TLR-independent innate host defense strategy directed by B cells in communication with stromal cells via the LTalphabeta cytokine system.

Homozygosity for the severe combined immunodeficiency (scid) mutation results in a block in T- and B-lymphocyte development. An unusually high incidence of spontaneous thymic lymphoma development was observed after transfer of this mutation from the C.B-17 congenic strain background onto the diabetes-susceptible nonobese diabetic (NOD) background. Thymomagenesis in the NOD-scid/scid mouse was associated with expression of an NOD mouse-unique endogenous ecotropic murine leukemia provirus locus (Emv-30, mapped to proximal region of chromosome 11) not expressed in the standard substrain NOD/Lt thymus. All tumors exhibited insertions of ecotropic proviruses, whereas only a subset also exhibited proviral integrations of mink cell focus-forming retrovirus. Neither class of retrovirus was associated with consistent integration into genes previously associated with activation of oncogenesis. We propose that the unusual features of T-cell ontogeny characteristic of the NOD inbred strain synergize with the scid-imparted block in thymocyte development, leading to activation of the NOD-unique Emv-30 to initiate thymomagenesis.

Cytokines with bone-resorbing activity include IL 1 beta (pI 7), IL 1 alpha (pI 5), tumor necrosis factor (TNF), and lymphotoxin (LT). Possible interaction between IL 1 beta, the major mediator with osteoclast-activating factor (OAF) activity, and other cytokines was studied. By itself, IL 1 beta was 13-fold more potent than IL 1 alpha and 1000-fold more potent than either TNF or LT in stimulating bone resorption. Suboptimal concentrations of IL 1 beta or IL 1 alpha in combination with suboptimal concentrations of TNF or LT resulted in synergistic bone-resorptive responses (1.5 to 10 times the expected responses if their effects were additive). Synergy between either form of IL 1 and TNF or LT resulted in a twofold increase in activity of IL 1, and a 100-fold increase in activity of TNF or LT. However, even with optimal synergy, IL 1 beta remained 20-fold more potent in inducing bone resorption than TNF or LT. Because IL 1 beta is considerably more potent than TNF and LT in stimulating bone resorption either alone or under synergistic conditions, it is unlikely that TNF and LT are responsible for more than a minor proportion of the total bone-resorbing activity formerly referred to as OAF.

(<em>b</em>)Background:</<em>b</em>) To investigate deep vein throm<em>b</em>osis (DVT) in hospitalized patients with coronavirus disease 2019 (COVID-19), we performed a single institutional study to evaluate its prevalence, risk factors, prognosis, and potential throm<em>b</em>oprophylaxis strategies in a large referral and treatment center. (<em>b</em>)Methods:</<em>b</em>) We studied a total of 143 patients with COVID-19 from January 29 to Fe<em>b</em>ruary 29, 2020. Demographic and clinical data, la<em>b</em>oratory data, including u<em>lt</em>rasound scans of the lower extremities, and outcome varia<em>b</em>les were o<em>b</em>tained, comparisons were made <em>b</em>etween DVT and non-DVT groups. (<em>b</em>)Resu<em>lt</em>s:</<em>b</em>) Of the 143 patients hospitalized with COVID-19 (aged 63 ± 14 years; 74 [51.7%] man), 66 patients developed lower extremity DVT (46.1%, included 23 [34.8%] with proximal DVT and 43 [65.2%] with distal DVT).Compared with patients who with no DVT, patients with DVT were older and had a lower oxygenation index, a higher rate of cardiac injury, and worse prognosis including an increased proportion of deaths (23 [34.8%] vs 9 [11.7%], <i>P</i> = 0.001) and a decreased proportion of patients discharged (32 [48.5%] vs 60 [77.9%], <i>P</i> &<em>lt</em>; 0.001). Mu<em>lt</em>ivariant analysis only showed an association <em>b</em>etween CURB-65 score 3-5 (OR = 6.122, <i>P</i> = 0.031), Padua prediction score ≥ 4 (OR = 4.016, <i>P</i> = 0.04), and D-dimer >1.0 (μg/ml) (OR = 5.818, <i>P</i> &<em>lt</em>; 0.014) and DVT in this cohort, respectively. The com<em>b</em>ination of a CURB-65 score 3-5, a Padua prediction score ≥ 4, and D-dimer > 1.0 (μg/ml) has a sensitivity of 88.52% and a specificity of 61.43% for screening for DVT. In the su<em>b</em>group of patients with a Padua prediction score ≥ 4 and whose u<em>lt</em>rasound scans were performed ˃72 hours after admission, DVT was present in 18 (34.0%) of the su<em>b</em>group receiving venous throm<em>b</em>oem<em>b</em>olism prophylaxis vs 35 (63.3%) in the nonprophylaxis group (<i>P</i> = 0.010). (<em>b</em>)Conclusions:</<em>b</em>) The prevalence of DVT is high and is associated with adverse outcomes in hospitalized patients with COVID-19. Prophylaxis for venous throm<em>b</em>oem<em>b</em>olism may <em>b</em>e protective in patients with a Padua protection score ≥ 4 after admission. Our data seem to suggest that COVID-19 is pro<em>b</em>a<em>b</em>ly an additional risk factor for DVT in the hospitalized patients.

Keywords: COVID-19; novel coronavirus.

The ability of Escherichia coli heat-labile enterotoxin (LT) to influence the induction and maintenance of tolerance was examined in animals primed orally with a soluble protein antigen, ovalbumin (OVA), or in animals primed orally with two unrelated protein antigens administered simultaneously, OVA and bovine serum albumin (BSA). LT is immunologically and structurally related to the cholera enterotoxin (CT), which has been shown to be capable of abrogating oral tolerance to protein antigens when delivered simultaneously with the antigens. In this study, simultaneous administration of LT with OVA was shown to prevent the induction of tolerance to OVA and to increase the serum anti-OVA IgG response 30- to 90-fold over OVA-primed and PBS-primed animals, respectively. This effect was determined to be a function of the enzymatically active A subunit of the toxin since the B (binding) subunit alone was unable to influence tolerance induction. Animals fed LT with OVA after the initial OVA prime developed a significantly lower serum IgG and mucosal IgA anti-OVA response than those fed LT with OVA in the initial immunization, indicating that prior exposure to the antigen reduces the effectiveness of LT to influence tolerance and its ability to act as an adjuvant. LT was not able to abrogate tolerance once it had been established. Serum IgG and mucosal IgA responses in animals receiving LT on only a single occasion, that being upon first exposure to antigen, were equivalent to responses after three OVA/LT primes, indicating that commitment to responsiveness occurs early and upon first exposure to antigen.(ABSTRACT TRUNCATED AT 250 WORDS)

<A<em>b</em>stractText>To identify markers associated with in-hospital death in patients with coronavirus disease 2019 (COVID-19)-associated pneumonia.</A<em>b</em>stractText><p><div>(<em>b</em>)PATIENTS AND METHODS</<em>b</em>)</div>A retrospective cohort study was conducted of 140 patients with moderate to critical COVID-19-associated pneumonia requiring oxygen supplementation admitted to the hospital from January 28, 2020, through Fe<em>b</em>ruary 28, 2020, and followed up through March 13, 2020, in Union Hospital, Wuhan, China. Oxygen saturation (SpO<su<em>b</em>)2</su<em>b</em>)) and other measures were tested as predictors of in-hospital mortality in survival analysis.</p><p><div>(<em>b</em>)RESULTS</<em>b</em>)</div>Of 140 patients with COVID-19-associated pneumonia, 72 (51.4%) were men, with a median age of 60 years. Patients with SpO<su<em>b</em>)2</su<em>b</em>) values of 90% or less were older and were more likely to <em>b</em>e men, to have hypertension, and to present with dyspnea than those with SpO<su<em>b</em>)2</su<em>b</em>) values greater than 90%. Overall, 36 patients (25.7%) died during hospitalization after median 14-day follow-up. Higher SpO<su<em>b</em>)2</su<em>b</em>) levels after oxygen supplementation were associated with reduced mortality independently of age and sex (hazard ratio per 1-U SpO<su<em>b</em>)2</su<em>b</em>), 0.93; 95% CI, 0.91 to 0.95; P&<em>lt</em>;.001). The SpO<su<em>b</em>)2</su<em>b</em>) cutoff value of 90.5% yielded 84.6% sensitivity and 97.2% specificity for prediction of survival. Dyspnea was also independently associated with death in mu<em>lt</em>ivaria<em>b</em>le analysis (hazard ratio, 2.60; 95% CI, 1.24 to 5.43; P=.01).</p><A<em>b</em>stractText>In this cohort of patients with COVID-19, hypoxemia was independently associated with in-hospital mortality. These resu<em>lt</em>s may help guide the clinical management of patients with severe COVID-19, particularly in settings requiring strategic allocation of limited critical care resources.</A<em>b</em>stractText><A<em>b</em>stractText>Chictr.org.cn Identifier: ChiCTR2000030852.</A<em>b</em>stractText>

NOD/Lt mice harboring a hybrid rat insulin-promoter/SV40 large T-antigen gene spontaneously develop beta-cell adenomas. NIT-1 is a pancreatic beta-cell line established from one of these transgenic mice. Immunocytochemical staining of passage 18 cells showed most contained insulin, with less than 5% containing glucagon, and none containing pancreatic polypeptide or somatostatin. Glucagon content radioimmunoassayed in cell extracts was only 0.27% of the insulin content. Two-hour insulin secretion at 16.5 mM glucose was 638 ng/10(6) cells (41% of intracellular content) compared to only 1.3 ng glucagon (32% of intracellular content). Stimulated insulin secretion was consistently observed in response to 11 and 16.5 mM glucose between passages 11 and 19. At passage 19, both theophylline and tolbutamide stimulated insulin secretion at 5.5 mM glucose. Northern-blot analysis confirmed high levels of insulin mRNA but only trace glucagon mRNA and undetectable somatostatin mRNA. Interferon-gamma (IFN-gamma)-induced MHC class I RNA expression was correlated with markedly increased antigen expression at the cell surface. Similarly, a MHC-linked "occult" class I-like antigen detected by Cr release assay only after exposure of standard NOD/Lt islet cells to IFN-gamma was strongly induced by IFN-gamma in NIT-1 cells. Cell surface MHC class II antigen was not constitutively expressed on NIT-1 cells and could not be detected after IFN-gamma incubation, despite demonstration of IFN-gamma-induced Aa, Ab, and Li invariant-chain RNA transcripts. Similarly IFN-gamma induction of intercellular adhesion molecule 1 (Icam-1) transcripts was not accompanied by demonstrable cell surface expression of ICAM-1 antigen.(ABSTRACT TRUNCATED AT 250 WORDS)

Cholera toxin (CT) and the analogous heat-labile enterotoxin (LT) from Escherichia coli have several immunomodulating effects which alone or in combination might explain their strong adjuvant action in stimulating mucosal IgA and other immune responses to admixed unrelated antigens after oral immunization. These effects include, depending on animal species and experimental systems, enhanced antigen presentation by a variety of cell types; promotion of isotype differentiation in B cells leading to increased IgA formation; and complex stimulatory as well as inhibitory effects on T-cell proliferation and lymphokine production. This adjuvant activity appears to be closely linked to the ADP-ribosylating action of CT and LT associated with enhanced cyclic AMP formation in the affected cells, and thus it may prove difficult to eliminate the enterotoxic activity without loss of adjuvanticity. However, through a separate mechanism, as an antigen-carrier system providing specific binding to epithelium including the M cells of intestinal Peyer's patches, both CT and its non-toxic binding subunit moiety (CTB) have been shown to markedly enhance the mucosal immune response to various foreign antigens or epitopes covalently linked to these molecules. This gives promise for the future use of CTB or related non-toxic binding derivatives as vehicles to facilitate induction of mucosal immune responses to a broad range of antigens for human vaccination purposes.

Synovial inflammation in rheumatoid arthritis is closely related to the formation of ectopic lymphoid microstructures. In synovial tissue from some patients, one finds seemingly diffuse infiltrates; in others, T cells and B cells cluster in aggregates with interdigitating dendritic cells (DCs) but no follicular DCs (FDCs). In a third group, T cell/B cell follicles with germinal center (GC) reactions are generated. Within a given patient, aggregates and GCs are mutually exclusive and stable over time. Because antigen storage capacity, lymphoid density, and three-dimensional topography of GCs optimize immune responses, synovial GCs should play a crucial role in the breakdown of self-tolerance. We have identified factors critical for ectopic GCs, thereby transforming the synovial inflammatory process. Tissues with GCs produced 10- to 20-fold higher amounts of the chemokines CXCL13 and CCL21. CXCL13 derived from three sources, endothelial cells, synovial fibroblasts, and FDC networks. The level of CXCL13 transcripts strongly predicted GCs; however, some tissues had high levels of CXCL13 but lacked GCs. Tissue expression of LT-beta emerged as a second key factor. LT-beta protein was detected on follicular center and mantle zone B cells. Multivariate regression analysis identified CXCL13 and LT-beta as the only cytokines predicting GCs. Remarkably, LT-alpha did not contribute independently. The contribution of B cells to ectopic lymphoid organogenesis was not limited to LT-beta production. Rather, synovial tissue B cells were critical in regulating T cell activation. In adoptive transfer experiments in human synovium-SCID mouse chimeras, activation of synovium-derived CD4 T cells was strictly dependent on T cell/B cell follicles. Depletion of synovial tissue B cells abrogated T cell function, and non-B cell antigen-presenting cells could not maintain T cell stimulation. Unexpectedly, GC function in the rheumatoid lesion was also dependent on CD8 T cells. The majority of T cell receptors derived from CD8 T cells were shared between distinct GCs. Depletion of CD8 T cells disrupted synovial GCs, FDC networks disappeared, and transcription of LT-beta, IgG, and Igkappa declined. Follicle-sustaining CD8 T cells were located at the edge of or within the mantle zone. Cell-cell communication in the mantle zone, including CD8 T cells, appears to be critical for ectopic GC formation in rheumatoid synovitis.

Lymphotoxins (LT alpha and LT beta), LIGHT [homologous to LT, inducible expression, competes with herpes simplex virus (HSV) glycoprotein D for HSV entry mediator (HVEM), a receptor expressed on T lymphocytes], tumor necrosis factor (TNF), and their specific receptors LT beta R, HVEM, and TNF receptor 1 (TNFR1) and TNFR2, form the immediate family of the larger TNF superfamily. These cytokines establish a critical communication system required for the development of secondary lymphoid tissues; however, knowledge of the target genes activated by these signaling pathways is limited. Target genes regulated by the LT alpha beta-LT beta R pathway include the tissue-organizing chemokines, CXCL13, CCL19, and CCL21, which establish cytokine circuits that regulate LT expression on lymphocytes, leading to organized lymphoid tissue. Infectious disease models have revealed that LT alpha beta pathways are also important for innate and adaptive immune responses involved in host defense. Here, regulation of interferon-beta by LT beta R and TNFR signaling may play a crucial role in certain viral infections. Regulation of autoimmune regulator in the thymus via LT beta R implicates LT/LIGHT involvement in central tolerance. Dysregulated expression of LIGHT overrides peripheral tolerance leading to T-cell-driven autoimmune disease. Blockade of TNF/LT/LIGHT pathways as an intervention in controlling autoimmune diseases is attractive, but such therapy may have risks. Thus, identifying and understanding the target genes may offer an opportunity to fine-tune inhibitory interventions.

The Escherichia coli heat-labile enterotoxin (LT) was found to have the same subunit structure as cholera toxin, namely, one A subunit and five B subunits. Reaction with a bisimidate generated all the possible cross-linked derivatives of A5B: B,2B ... 5B and A, AB ... A5B. The isolated B component, coligenoid, contained five B subunits and showed some tendency of polymerize: with a bisimidate it became covalently connected into the set B ... 5B with lesser amounts of 6B ... 10B, etc. The subunit formulas of two independently prepared samples of LT were both proved to be A5B by cross-linking, but their B pentamers migrated at different rates on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, indicating that they have different conformations. The faster (R) form could be converted to a diffuse slower (C) form by incubating it at 50 degrees C or at 37 degrees C with 0.2 M galactose, which is the terminal sugar of ganglioside GM1, the natural receptor for LT. Cholera toxin resembled the R form more than the C form of LT.

We have previously reported on a universal human influenza A vaccine, based on the external domain of the transmembrane viral M2-protein (M2e) [Nature Medicine 5 (1999) 1119]. M2-protein is scarcely present on the virus but is abundantly expressed on virus-infected cells. The external domain, M2e, is 23-amino acids long and as such weakly immunogenic. But when presented on an appropriate carrier, such as hepatitis B virus core (HBc) particles, it induces a high titer antibody response that in mice effectively protects against a potentially lethal influenza infection. The advantage of M2e as an antigen is the conservation of its sequence that has hardly changed since the first influenza virus was isolated in 1933, despite numerous epidemics and several pandemics. Various constructs, e.g. M2e fused at the N-terminus of the HBc subunit or inserted in the immuno-dominant loop, were evaluated as a vaccine. They conferred full protection when administered together with an adjuvant. Several adjuvants were tested in conjunction with intraperitoneal vaccine administration, while the non-toxic enterotoxin mutant LT(R192G) was used for intranasal vaccination. Appropriate combinations of vaccine construct and adjuvant allowed to obtain anti-M2e IgG2a serum titers above 10,000, and this provided complete protection.

Three-hundred and twenty-four patients were enrolled in an open-label, multicenter, international study in which pre- and post-liver transplantation (LT) patients with recurrent chronic hepatitis B (CHB) and evidence of lamivudine-resistant HBV were treated with adefovir dipivoxil 10 mg once daily. In the pre- and post-LT cohorts, 128 and 196 patients were treated for a median duration of 18.7 and 56.1 weeks, respectively. In patients who received 48 weeks of treatment, 81% of the pre-LT and 34% of the post-LT cohort achieved undetectable serum hepatitis B virus (HBV) DNA (Roche Amplicor Monitor polymerase chain reaction [PCR] assay lower limit of quantification [LLQ] < 400 copies/mL) with a median change in serum HBV DNA from baseline of -4.1 log(10) and -4.3 log(10) copies/mL, respectively. Serum alanine aminotransferase (ALT), albumin, bilirubin, and prothrombin time normalized in 76%, 81%, 50%, and 83% of pre-LT patients and 49%, 76%, 75%, and 20% of post-LT patients. The Child-Pugh-Turcotte (CPT) score improved in over 90% of patients in both cohorts. Genotypic analysis of 122 HBV baseline samples revealed that 98% of these patients had lamivudine-resistant mutant HBV. No adefovir resistance mutations were identified in patients after 48 weeks of therapy. One-year survival was 84% for pre-LT and 93% for post-LT patients (Kaplan-Meier analysis). Treatment-related adverse effects associated with adefovir dipivoxil in this setting were primarily mild to moderate in severity. In conclusion, 48 weeks of adefovir dipivoxil resulted in significant improvements in virologic, biochemical, and clinical parameters in CHB patients pre- and post-LT with lamivudine-resistant HBV.

Tumor necrosis factor (TNF) receptor-associated factors (TRAFs) are signal transducers for several members of the TNF receptor superfamily. We have identified a novel member of the TRAF family by degenerate oligonucleotide polymerase chain reaction amplification that contains a zinc RING finger and zinc finger motifs, a coiled-coil region, and a C-terminal "TRAF" homology domain. In vitro translated TRAF5 binds to the cytoplasmic region of the lymphotoxin-beta receptor (LT-betaR) but not to several other related receptors including CD40, both TNF receptors, Fas, and nerve growth factor receptor. TRAF5 and LT-betaR coimmunoprecipitate when overexpressed in COS7 cells. TRAF5 mRNA expression is found in all visceral organs and overlaps with LT-betaR. These features distinguish TRAF5 from the other members of the TRAF family. The transcription factor NF-kappaB is activated in HEK293 cells by overexpression of full-length TRAF5 but not a truncated form lacking the zinc binding region. Furthermore, overexpression of LT-betaR in HEK293 cells also results in activation of NF-kappaB, which is partially inhibited by the truncated TRAF5 mutant. These results show TRAF5 is functionally similar to TRAF2 in that both mediate activation NF-kappaB and implicate TRAF5 as a signal transducer for LT-betaR.

The Brief Repeatable Battery of Neuropsychological Tests (BRB) is by far the most widely used instrument to estimate cognitive dysfunction in multiple sclerosis (MS) patients. However, the paucity of normative data currently limits its applicability. We administered the BRB to 200 healthy subjects to obtain normative values. Moreover, we assessed the influence of demographic factors on the test scores and calculated corrections for these relevant factors. To test executive functions not explored by the BRB, we also included the Stroop word-color task (ST). Higher educational level was associated with better performance on all the tests, except for the world list generation (WLG) and the ST, considering version A, and on Symbol Digit Modalities Test (SDMT), Paced Auditory Serial Addition Test (PASAT) and Selective Reminding Test-Delayed (SRT-D), considering version B. Females performed better than males on the WLG considering version A, and on the SRT-Long-Term Storage (SRT-LTS) and SRT-Consistent Long-Term Retrieval (SRT-CLTR) considering version B. Increasing age was associated with worse performance on the ST in version A, and on the SRT-LTS, SRT-CLTR and WLG in version B. Our data can improve the applicability of the BRB for both clinical and research purposes.

<A<em>b</em>stractText>CheckMate 568 is an open-la<em>b</em>el phase II trial that evaluated the efficacy and safety of nivoluma<em>b</em> plus low-dose ipilimuma<em>b</em> as first-line treatment of advanced/metastatic non-small-cell lung cancer (NSCLC). We assessed the association of efficacy with programmed death ligand 1 (PD-L1) expression and tumor mutational <em>b</em>urden (TMB).</A<em>b</em>stractText><A<em>b</em>stractText>Two hundred eighty-eight patients with previously untreated, recurrent stage IIIB/IV NSCLC received nivoluma<em>b</em> 3 mg/kg every 2 weeks plus ipilimuma<em>b</em> 1 mg/kg every 6 weeks. The primary end point was o<em>b</em>jective response rate (ORR) in patients with 1% or more and less than 1% tumor PD-L1 expression. Efficacy on the <em>b</em>asis of TMB (FoundationOne CDx assay) was a secondary end point.</A<em>b</em>stractText><p><div>(<em>b</em>)RESULTS</<em>b</em>)</div>Of treated patients with tumor availa<em>b</em>le for testing, 252 patients (88%) of 288 were evalua<em>b</em>le for PD-L1 expression and 98 patients (82%) of 120 for TMB. ORR was 30% overall and 41% and 15% in patients with 1% or greater and less than 1% tumor PD-L1 expression, respectively. ORR increased with higher TMB, plateauing at 10 or more mutations/mega<em>b</em>ase (mut/M<em>b</em>). Regardless of PD-L1 expression, ORRs were higher in patients with TMB of 10 or more mut/M<em>b</em> (n = 48: PD-L1, ≥ 1%, 48%; PD-L1, &<em>lt</em>; 1%, 47%) versus TMB of fewer than 10 mut/M<em>b</em> (n = 50: PD-L1, ≥ 1%, 18%; PD-L1, &<em>lt</em>; 1%, 5%), and progression-free survival was longer in patients with TMB of 10 or more mut/M<em>b</em> versus TMB of fewer than 10 mut/M<em>b</em> (median, 7.1 <i>v</i> 2.6 months). Grade 3 to 4 treatment-related adverse events occurred in 29% of patients.</p><A<em>b</em>stractText>Nivoluma<em>b</em> plus low-dose ipilimuma<em>b</em> was effective and tolera<em>b</em>le as a first-line treatment of advanced/metastatic NSCLC. TMB of 10 or more mut/M<em>b</em> was associated with improved response and prolonged progression-free survival in <em>b</em>oth tumor PD-L1 expression 1% or greater and less than 1% su<em>b</em>groups and was thus identified as a potentially relevant cutoff in the assessment of TMB as a <em>b</em>iomarker for first-line nivoluma<em>b</em> plus ipilimuma<em>b</em>.</A<em>b</em>stractText>