Despite their widespread expression, the in vivo recruitment activities of CCL19 (EBV-induced molecule 1 ligand chemokine) and CXCL12 (stromal cell-derived factor 1) have not been established. Furthermore, although CXCL13 (B lymphocyte chemoattractant) has been shown to induce lymphoid neogenesis through induction of lymphotoxin (LT)alpha1beta2, it is unclear whether other homeostatic chemokines have this property. In this work we show that ectopic expression in pancreatic islets of CCL19 leads to small infiltrates composed of lymphocytes and dendritic cells and containing high endothelial venules and stromal cells. Ectopic CXCL12 induced small infiltrates containing few T cells but enriched in dendritic cells, B cells, and plasma cells. Comparison of CCL19 transgenic mice with mice expressing CCL21 (secondary lymphoid tissue chemokine) revealed that CCL21 induced larger and more organized infiltrates. A more significant role for CCL21 is also suggested in lymphoid tissues, as CCL21 protein was found to be present in lymph nodes and spleen at much higher concentrations than CCL19. CCL19 and CCL21 but not CXCL12 induced LTalpha1beta2 expression on naive CD4 T cells, and treatment of CCL21 transgenic mice with LTbetaR-Fc antagonized development of organized lymphoid structures. LTalpha1beta2 was also induced on naive T cells by the cytokines IL-4 and IL-7. These studies establish that CCL19 and CXCL12 are sufficient to mediate cell recruitment in vivo and they indicate that LTalpha1beta2 may function downstream of CCL21, CCL19, and IL-2 family cytokines in normal and pathological lymphoid tissue development.

The role of bacteria in the initiation of periodontitis is well-documented and the end result, destruction of the alveolar bone and periodontal connective tissue, is readily observed; but the events occurring between these two points in time remain obscure and are the focus of this paper. Bacteria induce tissue destruction indirectly by activating host defense cells, which in turn produce and release mediators that stimulate the effectors of connective tissue breakdown. Components of microbial plaque have the capacity to induce the initial infiltrate of inflammatory cells including lymphocytes, macrophages, and PMNs. Microbial components, especially lipopolysaccharide (LPS), have the capacity to activate macrophages to synthesize and secrete a wide array of molecules including the cytokines interleukin-1 (IL-1) and tumor-necrosis factor-alpha (TNF-alpha), prostaglandins, especially PGE2, and hydrolytic enzymes. Likewise, bacterial substances activate T lymphocytes and they produce IL-1 and lymphotoxin (LT), a molecule having properties very similar to TNF-alpha. These cytokines manifest potent proinflammatory and catabolic activities, and play key roles in periodontal tissue breakdown. They induce fibroblasts and macrophages to produce neutral metalloproteinases such as procollagenase and prostromelysin, the serine proteinase urokinase-type plasminogen activator (u-PA), tissue inhibitor of metalloproteinase (TIMP), and prostaglandins, u-PA converts plasminogen into plasmin, which can activate neutral metalloproteinase proenzymes, and these enzymes degrade the extracellular matrix components. TIMP inactivates the active enzymes and thereby blocks further tissue degradation. Several amplification and suppression mechanisms are involved in the process. While LPS activates macrophages to produce IL-1, IL-1 is autostimulatory and can therefore amplify and perpetuate its own production. Interferon-gamma (INF-gamma) suppresses autostimulation, but it enhances LPS-induced IL-1 production. PGE2 exerts a control over the whole process by suppressing production of both IL-1 and TNF-alpha. Furthermore, the activated cells produce an IL-1 receptor antagonist that binds to the IL-1 receptor but does not induce the biologic consequences of IL-1 binding. Other cytokines such as transforming growth factor-beta (TGF-beta) suppress production of metalloproteinases and u-PA. Thus the progression and extent of tissue degradation is likely to be determined in major part by relative concentrations and half-life of IL-1, TNF-alpha, and related cytokines, competing molecules such as the IL-1 receptor antagonist, and suppressive molecules such as TGF-beta and PGE2. These molecules control levels of latent and active metalloproteinase and u-PA, and the availability and concentration of TIMP determines the extent and duration of degradative activity.

Targeted mutagenesis in embryonic stem cells was used to generate mice deficient in lymphotoxin-alpha (LT-alpha). Mice lacking LT-alpha -/- (LT-alpha -/- mice) exhibit a phenotype dominated by defects in secondary lymphoid organ development. LT-alpha -/- mice lack lymph nodes and Peyer's patches, and possess spleens in which the usual architecture is disrupted. However, in a few of the mutants, abnormal lymph node-like structures were observed, mainly within the mesenteric fat. Abnormal clusters of lymphocytes were also found to accumulate in the periportal and perivascular regions of the liver and lung of LT-alpha -/- mice. Yet, lymphocytes from LT-alpha -/- mice appeared phenotypically normal, expressing the expected ratios of B and T cell surface markers as well as the lymphocyte homing marker, L-selectin. In addition, bone marrow cells from LT-alpha -/- mice were able to successfully reconstitute the lymphoid organs of severe combined immunodeficient mice. However, LT-alpha -/- mutant mice examined for humoral immune responsiveness were found to be impaired in their ability to respond to different Ag. These data illustrate the utility of this mouse model as a system for understanding lymphoid organ development and its effects on immune responsiveness.

mAb to murine TNF (MuTNF) were produced after immunization of Armenian hamsters with purified, Escherichia coli-derived rMuTNF-alpha. Antibody produced from clone TN3-19.12, was purified and was found to inhibit 100% of the lytic activity of either recombinant or natural MuTNF-alpha at an antibody input of 25 ng/U. TN3-19.12 also inhibited all the lytic activity in culture supernatants from a variety of T cell sources, including activated T cell clones and T cell hybridomas (all of which expressed high levels of TNF-alpha and TNF-beta (lymphotoxin, LT) mRNA). Western blot analysis was used to document the physical form(s) of MuTNF recognized by TN3-19.12. Recombinant and macrophage-derived TNF displayed identical patterns of a single band with Mr 17 kDa. In contrast, T cell culture supernatants exhibited patterns consisting of two bands with Mr 17 and 24.7 kDa. The higher m.w. form was glycosylated based on its sensitivity to n-glycanase and displayed a m.w. consistent with that of TNF-beta (LT). These data suggest that TN3-19.12 recognizes both MuTNF-alpha and MuTNF-beta (LT). Monoclonal TN3-19.12 and polyvalent rabbit anti-rTNF were used to establish a MuTNF-specific ELISA capable of detecting picogram quantities of recombinant or natural TNF. This assay was used to detect TNF in the sera of mice challenged with a lethal dose of LPS. Peak TNF serum levels of 11 ng/ml were observed in these animals 90 min after i.p. LPS administration and then rapidly declined to near base line levels by 3 h. These values were confirmed by quantitating levels of TNF functional activity in the same samples. TN3-19.12 injected into mice subsequently treated with LPS prevented the detection of TNF in the circulation by either assay and protected mice from the lethal effects of endotoxin shock. Thus, TN3-19.12 effectively neutralizes endogenously produced TNF in vivo.

Uncertainty regarding pathogenic mechanisms has been a major impediment to effective prevention and treatment for human neurologic diseases such as multiple sclerosis, tropical spastic paraparesis, and AIDS demyelinating disease. Here, we implicate lymphotoxin (LT) (tumor necrosis factor beta [TNF-beta]) and TNF-alpha in experimental allergic encephalomyelitis (EAE), a murine model of an autoimmune demyelinating disease. In this communication, we report that treatment of recipient mice with an antibody that neutralizes LT and TNF-alpha prevents transfer of clone-mediated EAE. LNC-8, a myelin basic protein-specific T cell line, produces high levels of LT and TNF-alpha after activation by concanavalin A, antibody to the CD-3 epsilon component of the T cell receptor, or myelin basic protein presented in the context of syngeneic spleen cells. LNC-8 cells transfer clinical signs of EAE. When LNC-8 recipient mice were also treated with TN3.19.12, a monoclonal antibody that neutralizes LT and TNF-alpha, the severity of the transferred EAE was reduced, while control antibodies did not alter the disease. The effect of anti-LT/TNF-alpha treatment was long lived and has been sustained for 5 mo. These findings suggest that LT and TNF-alpha and the T cells that produce them play an important role in EAE.

Fracture healing is a unique biological process regulated by a complex array of signaling molecules and proinflammatory cytokines. Recent evidence for the role of tumor necrosis family members in the coupling of cellular functions during skeletal homeostasis suggests that they also may be involved in the regulation of skeletal repair. The expression of a number of cytokines and receptors that are of functional importance to bone remodeling (osteoprotegerin [OPG], macrophage colony-stimulating factor [M-CSF], and osteoprotegerin ligand [receptor activator of NF-kappaB ligand (RANKL)]), as well as inflammation (tumor necrosis factor alpha [TNF-alpha] and its receptors, and interleukin-1alpha [IL-1alpha] and -beta and their receptors) were analyzed over a 28-day period after the generation of simple transverse fractures in mouse tibias. OPG was expressed constitutively in unfractured bones and elevated levels of expression were detected throughout the repair process. It showed two distinct peaks of expression: the first occurring within 24 h after fracture and the second at the time of peak cartilage formation on day 7. In contrast, the expression of RANKL was nearly undetectable in unfractured bones but strongly induced throughout the period of fracture healing. The peak in expression of RANKL did not correlate with that of OPG, because maximal levels of expression were seen on day 3 and day 14, when OPG levels were decreasing. M-CSF expression followed the temporal profile of RANKL but was expressed at relatively high basal levels in unfractured bones. TNF-alpha, lymphotoxin-beta (LT-beta), IL-1alpha, and IL-1beta showed peaks in expression within the first 24 h after fracture, depressed levels during the period of cartilage formation, and increased levels of expression on day 21 and day 28 when bone remodeling was initiated. Both TNF-alpha receptors (p55 and p75) and the IL-1RII receptor showed identical patterns of expression to their ligands, while the IL-1R1 was expressed only during the initial period of inflammation on day 1 and day 3 postfracture. Both TNF-alpha and IL-1alpha expression were localized primarily in macrophages and inflammatory cells during the early periods of inflammation and seen in mesenchymal and osteoblastic cells later during healing. TNF-alpha expression also was detected at very high levels in hypertrophic chondrocytes. These data imply that the expression profiles for OPG, RANKL, and M-CSF are tightly coupled during fracture healing and involved in the regulation of both endochondral resorption and bone remodeling. TNF-alpha and IL-1 are expressed at both very early and late phases in the repair process, which suggests that these cytokines are important in the initiation of the repair process and play important functional roles in intramembraneous bone formation and trabecular bone remodeling.

Hepatitis B and C viruses (HBV and HCV) cause chronic hepatitis and hepatocellular carcinoma (HCC) by poorly understood mechanisms. We show that cytokines lymphotoxin (LT) alpha and beta and their receptor (LTbetaR) are upregulated in HBV- or HCV-induced hepatitis and HCC. Liver-specific LTalphabeta expression in mice induces liver inflammation and HCC, causally linking hepatic LT overexpression to hepatitis and HCC. Development of HCC, composed in part of A6(+) oval cells, depends on lymphocytes and IKappa B kinase beta expressed by hepatocytes but is independent of TNFR1. In vivo LTbetaR stimulation implicates hepatocytes as the major LT-responsive liver cells, and LTbetaR inhibition in LTalphabeta-transgenic mice with hepatitis suppresses HCC formation. Thus, sustained LT signaling represents a pathway involved in hepatitis-induced HCC.

<A<em>b</em>stractText>There is no proven specific pharmacological treatment for patients with the acute respiratory distress syndrome (ARDS). The efficacy of corticosteroids in ARDS remains controversial. We aimed to assess the effects of dexamethasone in ARDS, which might change pulmonary and systemic inflammation and resu<em>lt</em> in a decrease in duration of mechanical ventilation and mortality.</A<em>b</em>stractText><p><div>(<em>b</em>)METHODS</<em>b</em>)</div>We did a mu<em>lt</em>icentre, randomised controlled trial in a network of 17 intensive care units (ICUs) in teaching hospitals across Spain in patients with esta<em>b</em>lished moderate-to-severe ARDS (defined <em>b</em>y a ratio of partial pressure of arterial oxygen to the fraction of inspired oxygen of 200 mm Hg or less assessed with a positive end-expiratory pressure of 10 cm H<su<em>b</em>)2</su<em>b</em>)O or more and FiO<su<em>b</em>)2</su<em>b</em>) of 0·5 or more at 24 h after ARDS onset). Patients with <em>b</em>rain death, terminal-stage disease, or receiving corticosteroids or immunosuppressive drugs were excluded. Eligi<em>b</em>le patients were randomly assigned <em>b</em>ased on <em>b</em>alanced treatment assignments with a computerised randomisation allocation sequence using <em>b</em>locks of 10 opaque, sealed envelopes to receive immediate treatment with dexamethasone or continued routine intensive care (control group). Patients in the dexamethasone group received an intravenous dose of 20 mg once daily from day 1 to day 5, which was reduced to 10 mg once daily from day 6 to day 10. Patients in <em>b</em>oth groups were ventilated with lung-protective mechanical ventilation. Allocation concealment was maintained at all sites during the trial. Primary outcome was the num<em>b</em>er of ventilator-free days at 28 days, defined as the num<em>b</em>er of days alive and free from mechanical ventilation from day of randomisation to day 28. Secondary outcome was all-cause mortality 60 days after randomisation. All analyses were done according to the intention-to-treat principle. This study is registered with ClinicalTrials.gov, NCT01731795.</p><A<em>b</em>stractText>Between March 28, 2013, and Dec 31, 2018, we enrolled 277 patients and randomly assigned 139 patients to the dexamethasone group and 138 to the control group. The trial was stopped <em>b</em>y the data safety monitoring <em>b</em>oard due to low enrolment rate after enrolling more than 88% (277/314) of the planned sample size. The mean num<em>b</em>er of ventilator-free days was higher in the dexamethasone group than in the control group (<em>b</em>etween-group difference 4·8 days [95% CI 2·57 to 7·03]; p&<em>lt</em>;0·0001). At 60 days, 29 (21%) patients in the dexamethasone group and 50 (36%) patients in the control group had died (<em>b</em>etween-group difference -15·3% [-25·9 to -4·9]; p=0·0047). The proportion of adverse events did not differ significantly <em>b</em>etween the dexamethasone group and control group. The most common adverse events were hyperglycaemia in the ICU (105 [76%] patients in the dexamethasone group vs 97 [70%] patients in the control group), new infections in the ICU (eg, pneumonia or sepsis; 33 [24%] vs 35 [25%]), and <em>b</em>arotrauma (14 [10%] vs 10 [7%]).</A<em>b</em>stractText><A<em>b</em>stractText>Early administration of dexamethasone could reduce duration of mechanical ventilation and overall mortality in patients with esta<em>b</em>lished moderate-to-severe ARDS.</A<em>b</em>stractText><A<em>b</em>stractText>Fundación Mutua Madrileña, Instituto de Salud Carlos III, The European Regional Development's Funds, Asociación Científica Pulmón y Ventilación Mecánica.</A<em>b</em>stractText>

The alymphoplasia (aly) mutation of mouse is autosomal recessive and characterized by the systemic absence of lymph nodes (LN) and Peyer's patches (PP) and disorganized splenic and thymic structures with immunodeficiency. Although recent reports have shown that the interaction between lymphotoxin (LT) and the LT beta-receptor (Ltbeta r, encoded by Ltbr) provides a critical signal for LN genesis in mice, the aly locus on chromosome 11 is distinct from those for LT and its receptor. We found that the aly allele carries a point mutation causing an amino acid substitution in the carboxy-terminal interaction domain of Nf-kappa b-inducing kinase (Nik, encoded by the gene Nik). Transgenic complementation with wild-type Nik restored the normal structures of LN, PP, spleen and thymus, and the normal immune response in aly/aly mice. In addition, the aly mutation in a kinase domain-truncated Nik abolished its dominant-negative effect on Nf-kappa b activation induced by an excess of Ltbeta r. Our observations agree with previous reports that Ltbeta r-deficient mice showed defects in LN genesis and that Nik is a common mediator of Nf-kappa b activation by the tumour necrosis factor (TNF) receptor family. Nik is able to interact with members of the TRAF family (Traf1, 2, 3, 5 and 6), suggesting it acts downstream of TRAF-associating receptor signalling pathways, including Tnfr, Cd40, Cd30 and Ltbeta r. The phenotypes of aly/aly mice are more severe than those of Ltbr-/- mice, however, indicating involvement of Nik in signal transduction mediated by other receptors.

OBJECTIVE

To study antibody-independent contributions of B cells to inflammatory disease activity, and the immune consequences of B-cell depletion with rituximab, in patients with multiple sclerosis (MS).

METHODS

B-Cell effector-cytokine responses were compared between MS patients and matched controls using a 3-signal model of activation. The effects of B-cell depletion on Th1/Th17 CD4 and CD8 T-cell responses in MS patients were assessed both ex vivo and in vivo, together with pharmacokinetic/pharmacodynamic studies as part of 2 rituximab clinical trials in relapsing-remitting MS.

RESULTS

B Cells of MS patients exhibited aberrant proinflammatory cytokine responses, including increased lymphotoxin (LT):interleukin-10 ratios and exaggerated LT and tumor necrosis factor (TNF)-alpha secretion, when activated in the context of the pathogen-associated TLR9-ligand CpG-DNA, or the Th1 cytokine interferon-gamma, respectively. B-Cell depletion, both ex vivo and in vivo, resulted in significantly diminished proinflammatory (Th1 and Th17) responses of both CD4 and CD8 T cells. Soluble products from activated B cells of untreated MS patients reconstituted the diminished T-cell responses observed following in vivo B-cell depletion in the same patients, and this effect appeared to be largely mediated by B-cell LT and TNFalpha.

CONCLUSIONS

We propose that episodic triggering of abnormal B-cell cytokine responses mediates 'bystander activation' of disease-relevant proinflammatory T cells, resulting in new relapsing MS disease activity. Our findings point to a plausible mechanism for the long-recognized association between infections and new MS relapses, and provide novel insights into B-cell roles in both health and disease, and into mechanisms contributing to therapeutic effects of B-cell depletion in human autoimmune diseases, including MS.

<p><div>(<em>b</em>)BACKGROUND</<em>b</em>)</div>Three different glucagon-like peptide-1 (GLP-1) receptor agonists reduce cardiovascular outcomes in people with type 2 dia<em>b</em>etes at high cardiovascular risk with high glycated haemoglo<em>b</em>in A<su<em>b</em>)1c</su<em>b</em>) (H<em>b</em>A<su<em>b</em>)1c</su<em>b</em>)) concentrations. We assessed the effect of the GLP-1 receptor agonist dulaglutide on major adverse cardiovascular events when added to the existing antihyperglycaemic regimens of individuals with type 2 dia<em>b</em>etes with and without previous cardiovascular disease and a wide range of glycaemic control.</p><A<em>b</em>stractText>This mu<em>lt</em>icentre, randomised, dou<em>b</em>le-<em>b</em>lind, place<em>b</em>o-controlled trial was done at 371 sites in 24 countries. Men and women aged at least 50 years with type 2 dia<em>b</em>etes who had either a previous cardiovascular event or cardiovascular risk factors were randomly assigned (1:1) to either weekly su<em>b</em>cutaneous injection of dulaglutide (1·5 mg) or place<em>b</em>o. Randomisation was done <em>b</em>y a computer-generated random code with stratification <em>b</em>y site. All investigators and participants were masked to treatment assignment. Participants were followed up at least every 6 months for incident cardiovascular and other serious clinical outcomes. The primary outcome was the first occurrence of the composite endpoint of non-fatal myocardial infarction, non-fatal stroke, or death from cardiovascular causes (including unknown causes), which was assessed in the intention-to-treat population. This study is registered with ClinicalTrials.gov, num<em>b</em>er NCT01394952.</A<em>b</em>stractText><p><div>(<em>b</em>)FINDINGS</<em>b</em>)</div>Between Aug 18, 2011, and Aug 14, 2013, 9901 participants (mean age 66·2 years [SD 6·5], median H<em>b</em>A<su<em>b</em>)1c</su<em>b</em>) 7·2% [IQR 6·6-8·1], 4589 [46·3%] women) were enrolled and randomly assigned to receive dulaglutide (n=4949) or place<em>b</em>o (n=4952). During a median follow-up of 5·4 years (IQR 5·1-5·9), the primary composite outcome occurred in 594 (12·0%) participants at an incidence rate of 2·4 per 100 person-years in the dulaglutide group and in 663 (13·4%) participants at an incidence rate of 2·7 per 100 person-years in the place<em>b</em>o group (hazard ratio [HR] 0·88, 95% CI 0·79-0·99; p=0·026). All-cause mortality did not differ <em>b</em>etween groups (536 [10·8%] in the dulaglutide group vs 592 [12·0%] in the place<em>b</em>o group; HR 0·90, 95% CI 0·80-1·01; p=0·067). 2347 (47·4%) participants assigned to dulaglutide reported a gastrointestinal adverse event during follow-up compared with 1687 (34·1%) participants assigned to place<em>b</em>o (p&<em>lt</em>;0·0001).</p><A<em>b</em>stractText>Dulaglutide could <em>b</em>e considered for the management of glycaemic control in middle-aged and older people with type 2 dia<em>b</em>etes with either previous cardiovascular disease or cardiovascular risk factors.</A<em>b</em>stractText><A<em>b</em>stractText>Eli Lilly and Company.</A<em>b</em>stractText>

The factors regulating dendritic cell (DC) development and homeostasis are incompletely understood. Here, we demonstrate that DCs express the lymphotoxin (LT)-beta receptor (LT beta R) and that in mice lacking the LT beta R in hematopoietic cells, spleen, and lymph node, CD8- DC numbers are reduced. B cells are a key source of LT alpha 1 beta 2 for splenic DC homeostasis, and transgenic overexpression of LT alpha 1 beta 2 on B cells leads to expansion of the CD8- DC compartment. Furthermore, we find that about 5% of splenic DCs are undergoing cell division, and the number of dividing CD8- DCs is disproportionately reduced in the absence of the LT beta R. In parabiosis experiments, splenic DCs were only partially replaced by circulating precursors over a 6 week period. We conclude that LT alpha 1 bet a2 acts on DCs or DC precursors to promote DC homeostasis, and we suggest that DC proliferation is an important pathway for locally maintaining these cells in the steady state.

We have previously shown that nonobese diabetic (NOD) mice are selectively deficient in alpha/beta-T cell receptor (TCR)+CD4-CD8- NKT cells, a defect that may contribute to their susceptibility to the spontaneous development of insulin-dependent diabetes mellitus (IDDM). The role of NKT cells in protection from IDDM in NOD mice was studied by the infusion of thymocyte subsets into young female NOD mice. A single intravenous injection of 10(6) CD4-/lowCD8- or CD4-CD8- thymocytes from female (BALB/c x NOD)F1 donors protected intact NOD mice from the spontaneous onset of clinical IDDM. Insulitis was still present in some recipient mice, although the cell infiltrates were principally periductal and periislet, rather than the intraislet pattern characteristic of insulitis in unmanipulated NOD mice. Protection was not associated with the induction of "allogenic tolerance" or systemic autoimmunity. Accelerated IDDM occurs after injection of splenocytes from NOD donors into irradiated adult NOD recipients. When alpha/beta-TCR+ and alpha/beta-TCR- subsets of CD4-CD8- thymocytes were transferred with diabetogenic splenocytes and compared for their ability to prevent the development of IDDM in irradiated adult recipients, only the alpha/beta-TCR+ population was protective, confirming that NKT cells were responsible for this activity. The protective effect in the induced model of IDDM was neutralized by anti-IL-4 and anti-IL-10 monoclonal antibodies in vivo, indicating a role for at least one of these cytokines in NKT cell-mediated protection. These results have significant implications for the pathogenesis and potential prevention of IDDM in humans.

For more than a decade, the biological roles and the apparent redundancy of the cytokines tumor necrosis factor (TNF) and lymphotoxin (LT) have been debated. LT alpha exists in its soluble form as a homotrimer, which like TNF only binds the TNF receptors, TNF-R55 or TNF-R75. The cell surface form of LT exists as a heteromer of LT alpha and LT beta subunits and this complex specifically binds the LT beta receptor (LT beta-R). To discriminate the functions of the LT and TNF systems, soluble LT beta-R-immunoglobulin (Ig) or TNF-R-Ig fusion proteins were introduced into embryonic circulation by injecting pregnant mice. Exposure to LT beta-R-Ig during gestation disrupted lymph node development and splenic architecture in the progeny indicating that both effects are mediated by the surface LT alpha/beta complex. These data are the first to identify a cell surface ligand involved in immune organ morphogenesis. Moreover, they unambiguously discriminate the functions of the various TNF/LT ligands, provide a unique model to study compartmentalization of immune responses and illustrate the generic utility of receptor-Ig fusion proteins for dissecting/ordering ontogenetic events in the absence of genetic modifications.

We examined the roles of cell- and antibody-mediated immunity in urease vaccine-induced protection against Helicobacter pylori infection. Normal and knockout mice deficient in major histocompatibility complex (MHC) class I, MHC class II, or B cell responses were mucosally immunized with urease plus Escherichia coli heat-labile enterotoxin (LT), or parenterally immunized with urease plus aluminum hydroxide or a glycolipid adjuvant, challenged with H. pylori strain X47-2AL, and H. pylori organisms and leukocyte infiltration in the gastric mucosa quantified. In an adjuvant/route study in normal mice, there was a direct correlation between the level of protection and the density of T cells recruited to the gastric mucosa. In knockout studies, oral immunization with urease plus LT protected MHC class I knockout mice [beta2-microglobulin (-/-)] but not MHC class II knockout mice [I-Ab (-/-)]. In B cell knockout mice [microMT (-/-)], vaccine-induced protection was equivalent to that observed in immunized wild-type (+/+) mice; no IgA+ cells were detected in the stomach, but levels of CD4(+) cells equivalent to those in the wild-type strain (+/+) were seen. These studies indicate that protection of mice against H. pylori infection by immunization with the urease antigen is dependent on MHC class II-restricted, cell-mediated mechanisms, and antibody responses to urease are not required for protection.

HLA class II molecules are surface glycoproteins which are essential in the initiation of immune responses. It has been postulated that induction of class II in epithelial cells such as endocrine cells, which are normally class II negative, may result in autoimmunity. In type I diabetes, islet beta cells, the target of the autoimmune process, selectively express class II antigens. But in contrast to most other cell types, islet beta cells are not stimulated to express class II by interferon-gamma (IFN-gamma) and thus the conditions under which this induction occurs have been particularly elusive. The cytotoxins tumour necrosis factor (TNF) and lymphotoxin (LT) synergize with IFN-gamma in a number of activities. We report here that IFN-gamma in combination with either TNF or LT induces islet cell class II expression. This finding has important implications for the pathogenesis of type I diabetes and the understanding of the differential control of class II expression.

Influenza vaccines that induce greater cross-reactive or heterosubtypic immunity (Het-I) may overcome limitations in vaccine efficacy imposed by the antigenic variability of influenza A viruses. We have compared mucosal versus traditional parenteral administration of inactivated influenza vaccine for the ability to induce Het-I in BALB/c mice and evaluated a modified Escherichia coli heat-labile enterotoxin adjuvant, LT(R192G), for augmentation of Het-I. Mice that received three intranasal (i.n.) immunizations of H3N2 vaccine in the presence of LT(R192G) were completely protected against lethal challenge with a highly pathogenic human H5N1 virus and had nasal and lung viral titers that were at least 2,500-fold lower than those of control mice receiving LT(R192G) alone. In contrast, mice that received three vaccinations of H3N2 vaccine subcutaneously in the presence or absence of LT(R192G) or incomplete Freund's adjuvant were not protected against lethal challenge and had no significant reductions in tissue virus titers observed on day 5 post-H5N1 virus challenge. Mice that were i.n. administered H3N2 vaccine alone, without LT(R192G), displayed partial protection against heterosubtypic challenge. The immune mediators of Het-I were investigated. The functional role of B and CD8+ T cells in Het-I were evaluated by using gene-targeted B-cell (IgH-6(-/-))- or beta2-microglobulin (beta2m(-/-))-deficient mice, respectively. beta2m(-/-) but not IgH-6(-/-) vaccinated mice were protected by Het-I and survived a lethal infection with H5N1, suggesting that B cells, but not CD8+ T cells, were vital for protection of mice against heterosubtypic challenge. Nevertheless, CD8+ T cells contributed to viral clearance in the lungs and brain tissues of heterotypically immune mice. Mucosal but not parenteral vaccination induced subtype cross-reactive lung immunoglobulin G (IgG), IgA, and serum IgG anti-hemagglutinin antibodies, suggesting the presence of a common cross-reactive epitope in the hemagglutinins of H3 and H5. These results suggest a strategy of mucosal vaccination that stimulates cross-protection against multiple influenza virus subtypes, including viruses with pandemic potential.

Cholera toxin (CT) and the Escherichia coli heat-labile toxin (LT) are functionally, structurally and immunologically similar enterotoxins. Both toxins cause the elevation of cyclic AMP levels in gut epithelial cells by catalysing the NAD-dependent ADP ribosylation of membrane proteins. Each toxin is composed of two dissimilar subunits. The A subunit has an enzymatic activity and is the adenylate cyclase-activating component of the enterotoxin. The B subunit recognizes membrane components and binds the holotoxin to the target call juxtaposing the A subunit with its substrates. Binding studies and competition experiments indicate that the membrane receptors for cholera toxin B subunit (CT-B) and LT-B are similar but not identical (these studies were performed before by LT was purified to homogeneity). The monosialosylganglioside GMI has been shown to be the receptor for the cholera toxin, and it probably composes part of the receptor for LT. Gyles and Barnum, first reported that LT and cholera toxin were immunologically related, and it has subsequently been shown that they share common antigenic determinants in both A and B subunits. The primary structure of CT-B has been determined. We report here a comparison between the amino acid sequences of LT-B and CT-B. The nucleotide sequence of the LT-B cistron (eltB) was determined using a recombinant plasmid encoding LT. Translation of this sequence revealed that LT-B and CT-B show significant amino acid sequence homology. In addition, several features of the eltB cistron were revealed by the sequence analysis.

Anthrax poses a clear and present danger as an agent of biological terrorism. Infection with Bacillus anthracis, the causative agent of anthrax, if untreated can result in rampant bacteraemia, multisystem dysfunction and death. Anthrax lethal toxin (LT) is a critical virulence factor of B. anthracis, which occurs as a complex of protective antigen and lethal factor. Here we demonstrate that LT severely impairs the function of dendritic cells--which are pivotal to the establishment of immunity against pathogens--and host immune responses by disrupting the mitogen-activated protein (MAP) kinase intracellular signalling network. Dendritic cells exposed to LT and then stimulated with lipopolysaccharide do not upregulate co-stimulatory molecules, secrete greatly diminished amounts of proinflammatory cytokines, and do not effectively stimulate antigen-specific T cells in vivo. Furthermore, injections of LT induce a profound impairment of antigen-specific T- and B-cell immunity. These data suggest a role for LT in suppressing host immunity during B. anthracis infections, and represent an immune evasion strategy, where a microbe targets MAP kinases in dendritic cells to disarm the immune response.

In presenting a unifying concept for chronic inflammation and lymphoid organogenesis, we suggest that lymphotoxin's (LT, LT-alpha, TNF-beta) crucial role in these processes is pivotal and similar. Chronic inflammatory lesions that developed in the kidney and pancreas at the sites of transgene expression in rat insulin promoter-LT (RIP-LT) mice resembled lymph nodes with regard to cellular composition (T cells, B cells, plasma cells, and antigen-presenting cells), delineated T and B cell areas, primary and secondary follicles, characteristic morphologic and antigenic (ICAM-1, VCAM-1, MAdCAM-1, and PNAd) features of high endothelial venules, and ability to respond to antigen and undergo Ig class switching when obtained from mice immunized with SRBC. The vascular changes, with the exception of PNAd, appear to be the direct consequence of transgene derived LT expression, as they were also observed in RIP-LT mice lacking mature T and B cells. These data show that LT-induced chronic inflammation has the characteristics of organized lymphoid tissue.

BACKGROUND

Nasal polyps (NPs) are characterized by eosinophilic inflammation and often coexist with asthma. However, the role of atopy and IgE in NP pathogenesis is unclear.

OBJECTIVE

We sought to determine whether there is an association between total and specific IgE to a variety of allergens in polyp and nonpolyp tissue and markers of eosinophilic inflammation or skin test results.

METHODS

Homogenates were prepared from nasal tissue of 20 patients with NPs and 20 patients without NPs and analyzed for concentrations of IL-5, IL-4, eotaxin, leukotriene (LT) C4/D4/E4, sCD23, and histamine (ELISA). Eosinophil cationic protein (ECP), tryptase, and total and specific IgE for inhalant allergens and Staphylococcus aureus enterotoxins were measured (ImmunoCAP).

RESULTS

The concentrations of total IgE, IL-5, eotaxin, ECP, LTC4/D4/E4, and sCD23 were significantly higher in NP tissue compared with nonpolyp tissue. Total IgE was significantly correlated to IL-5, ECP, LTC4/D4/E4, and sCD23 and to the number of eosinophils in NPs. On the basis of the presence of specific IgE antibodies in tissue, 3 NP groups were defined. NP group 1 demonstrated no measurable specific IgE, and NP group 2 selected specific IgE. The third group demonstrated a multiclonal specific IgE, including IgE to S aureus enterotoxins, a high total IgE level, and a high prevalence of asthma.

CONCLUSIONS

These studies suggest that there is an association between increased levels of total IgE, specific IgE, and eosinophilic inflammation in NPs, which may be of relevance in the pathophysiology of nasal polyposis. Similarly, the presence of specific IgE to staphylococcal enterotoxins A and B also points to a possible role of bacterial superantigens.

<A<em>b</em>stractText>Immunotherapy in com<em>b</em>ination with chemotherapy has shown promising efficacy across many different tumour types. We report the prespecified second interim overall survival analysis of the phase 3 IMpassion130 study assessing the efficacy and safety of atezolizuma<em>b</em> plus na<em>b</em>-paclitaxel in patients with unresecta<em>b</em>le, locally advanced or metastatic triple-negative <em>b</em>reast cancer.</A<em>b</em>stractText><p><div>(<em>b</em>)METHODS</<em>b</em>)</div>In this randomised, place<em>b</em>o-controlled, dou<em>b</em>le-<em>b</em>lind, phase 3 trial, done in 246 academic centres and community oncology practices in 41 countries, patients aged 18 years or older, with previously untreated, histologically documented, locally advanced or metastatic triple-negative <em>b</em>reast cancer, and Eastern Cooperative Oncology Group performance status of 0 or 1 were eligi<em>b</em>le. Patients were randomly assigned (1:1) using a permuted <em>b</em>lock method (<em>b</em>lock size of four) and an interactive voice-we<em>b</em> response system. Randomisation was stratified <em>b</em>y previous taxane use, liver metastases, and PD-L1 expression on tumour-infi<em>lt</em>rating immune cells. Patients received atezolizuma<em>b</em> 840 mg or matching place<em>b</em>o intravenously on day 1 and day 15 of every 28-day cycle and na<em>b</em>-paclitaxel 100 mg/m² of <em>b</em>ody surface area intravenously on days 1, 8, and 15 until progression or unaccepta<em>b</em>le toxicity. Investigators, patients, and the funder were masked to treatment assignment. Coprimary endpoints were investigator-assessed progression-free survival per Response Evaluation Criteria in Solid Tumors version 1.1 and overall survival, assessed in the intention-to-treat population and in patients with PD-L1 immune cell-positive tumours (tumours with ≥1% PD-L1 expression). The final progression-free survival resu<em>lt</em>s were previously reported at the first interim overall survival analysis. The prespecified statistical testing hierarchy meant that overall survival in the su<em>b</em>group of PD-L1 immune cell-positive patients could only <em>b</em>e formally tested if overall survival was significantly different <em>b</em>etween the treatment groups in the intention-to-treat population. This study is registered with ClinicalTrials.gov, NCT02425891.</p><A<em>b</em>stractText>Between June 23, 2015, and May 24, 2017, 902 patients were enrolled, of whom 451 were randomly assigned to receive atezolizuma<em>b</em> plus na<em>b</em>-paclitaxel and 451 were assigned to receive place<em>b</em>o plus na<em>b</em>-paclitaxel (the intention-to-treat population). Six patients from each group did not receive treatment. At the second interim analysis (data cutoff Jan 2, 2019), median follow-up was 18·5 months (IQR 9·6-22·8) in the atezolizuma<em>b</em> group and 17·5 months (8·4-22·4) in the place<em>b</em>o group. Median overall survival in the intention-to-treat patients was 21·0 months (95% CI 19·0-22·6) with atezolizuma<em>b</em> and 18·7 months (16·9-20·3) with place<em>b</em>o (stratified hazard ratio [HR] 0·86, 95% CI 0·72-1·02, p=0·078). In the exploratory overall survival analysis in patients with PD-L1 immune cell-positive tumours, median overall survival was 25·0 months (95% CI 19·6-30·7) with atezolizuma<em>b</em> versus 18·0 months (13·6-20·1) with place<em>b</em>o (stratified HR 0·71, 0·54-0·94]). As of Sept 3, 2018 (the date up to which updated safety data were availa<em>b</em>le), the most common grade 3-4 adverse events were neutropenia (38 [8%] of 453 patients in the atezolizuma<em>b</em> group vs 36 [8%] of 437 patients in the place<em>b</em>o group), peripheral neuropathy (25 [6%] vs 12 [3%]), decreased neutrophil count (22 [5%] vs 16 [4%]), and fatigue (17 [4%] vs 15 [3%]). Treatment-related deaths occurred in two (&<em>lt</em>;1%) patients in the atezolizuma<em>b</em> group (autoimmune hepatitis related to atezolizuma<em>b</em> [n=1] and septic shock related to na<em>b</em>-paclitaxel [n=1]) and one (&<em>lt</em>;1%) patient in the place<em>b</em>o group (hepatic failure). No new treatment-related deaths have <em>b</em>een reported since the primary clinical data cutoff date (April 17, 2018).</A<em>b</em>stractText><A<em>b</em>stractText>Consistent with the first interim analysis, this second interim overall survival analysis of IMpassion130 indicates no significant difference in overall survival <em>b</em>etween the treatment groups in the intention-to-treat population <em>b</em>ut suggests a clinically meaningful overall survival <em>b</em>enefit with atezolizuma<em>b</em> plus na<em>b</em>-paclitaxel in patients with PD-L1 immune cell-positive disease. However, this positive resu<em>lt</em> could not <em>b</em>e formally tested due to the prespecified statistical testing hierarchy. For patients with PD-L1 immune cell-positive metastatic triple-negative <em>b</em>reast cancer, atezolizuma<em>b</em> plus na<em>b</em>-paclitaxel is an important therapeutic option in a disease with high unmet need.</A<em>b</em>stractText><A<em>b</em>stractText>F Hoffmann-La Roche and Genentech.</A<em>b</em>stractText>

Tertiary lymphoid organs (TLOs) are organized aggregates of B and T cells formed in postembryonic life in response to chronic immune responses to infectious agents or self-antigens. Although CD11c+ dendritic cells (DCs) are consistently found in regions of TLO, their contribution to TLO organization has not been studied in detail. We found that CD11c(hi) DCs are essential for the maintenance of inducible bronchus-associated lymphoid tissue (iBALT), a form of TLO induced in the lungs after influenza virus infection. Elimination of DCs after the virus had been cleared from the lung resulted in iBALT disintegration and reduction in germinal center (GC) reactions, which led to significantly reduced numbers of class-switched plasma cells in the lung and bone marrow and reduction in protective antiviral serum immunoglobulins. Mechanistically, DCs isolated from the lungs of mice with iBALT no longer presented viral antigens to T cells but were a source of lymphotoxin (LT) beta and homeostatic chemokines (CXCL-12 and -13 and CCL-19 and -21) known to contribute to TLO organization. Like depletion of DCs, blockade of LTbeta receptor signaling after virus clearance led to disintegration of iBALT and GC reactions. Together, our data reveal a previously unappreciated function of lung DCs in iBALT homeostasis and humoral immunity to influenza virus.

<A<em>b</em>stractText>Atezolizuma<em>b</em> (a monoclonal anti<em>b</em>ody against PD-L1), which restores anticancer immunity, improved overall survival in patients with previously treated non-small-cell lung cancer and also showed clinical <em>b</em>enefit when com<em>b</em>ined with chemotherapy as first-line treatment of non-small-cell lung cancer. IMpower130 aimed to assess the efficacy and safety of atezolizuma<em>b</em> plus chemotherapy versus chemotherapy alone as first-line therapy for non-squamous non-small-cell lung cancer.</A<em>b</em>stractText><p><div>(<em>b</em>)METHODS</<em>b</em>)</div>IMpower130 was a mu<em>lt</em>icentre, randomised, open-la<em>b</em>el, phase 3 study done in 131 centres across eight countries (the USA, Canada, Belgium, France, Germany, Italy, Spain, and Israel). Eligi<em>b</em>le patients were aged 18 years or older, and had histologically or cytologically confirmed stage IV non-squamous non-small-cell lung cancer, an Eastern Cooperative Oncology Group performance status of 0 or 1, and received no previous chemotherapy for stage IV disease. Patients were randomly assigned (2:1; permuted <em>b</em>lock [<em>b</em>lock size of six] with an interactive voice or we<em>b</em> response system) to receive atezolizuma<em>b</em> (1200 mg intravenously every 3 weeks) plus chemotherapy (car<em>b</em>oplatin [area under the curve 6 mg/mL per min every 3 weeks] plus na<em>b</em>-paclitaxel [100 mg/m² intravenously every week]) or chemotherapy alone for four or six 21-day cycles followed <em>b</em>y maintenance therapy. Stratification factors were sex, <em>b</em>aseline liver metastases, and PD-L1 tumour expression. Co-primary endpoints were investigator-assessed progression-free survival and overall survival in the intention-to-treat wild-type (ie, EGFR^wt and ALK^wt) population. The safety population included patients who received at least one dose of the study drug. This study is registered with ClinicalTrials.gov, num<em>b</em>er NCT02367781.</p><A<em>b</em>stractText>Between April 16, 2015, and Fe<em>b</em> 13, 2017, 724 patients were randomly assigned and 723 were included in the intention-to-treat population (one patient died <em>b</em>efore randomisation, <em>b</em>ut was assigned to a treatment group; this patient was excluded from the intention-to-treat population) of the atezolizuma<em>b</em> plus chemotherapy group (483 patients in the intention-to-treat population and 451 patients in the intention-to-treat wild-type population) or the chemotherapy group (240 patients in the intention-to-treat population and 228 patients in the intention-to-treat wild-type population). Median follow-up in the intention-to-treat wild-type population was similar <em>b</em>etween groups (18·5 months [IQR 15·2-23·6] in the atezolizuma<em>b</em> plus chemotherapy group and 19·2 months [15·4-23·0] in the chemotherapy group). In the intention-to-treat wild-type population, there were significant improvements in median overall survival (18·6 months [95% CI 16·0-21·2] in the atezolizuma<em>b</em> plus chemotherapy group and 13·9 months [12·0-18·7] in the chemotherapy group; stratified hazard ratio [HR] 0·79 [95% CI 0·64-0·98]; p=0·033) and median progression-free survival (7·0 months [95% CI 6·2-7·3] in the atezolizuma<em>b</em> plus chemotherapy group and 5·5 months [4·4-5·9] in the chemotherapy group; stratified HR 0·64 [95% CI 0·54-0·77]; p&<em>lt</em>;0·0001]). The most common grade 3 or worse treatment-related adverse events were neutropenia (152 [32%] of 473 in the atezolizuma<em>b</em> plus chemotherapy group vs 65 [28%] of 232 in the chemotherapy group), anaemia (138 [29%] vs 47 [20%]), and decreased neutrophil count (57 [12%] vs 19 [8%]). Treatment-related serious adverse events were reported in 112 (24%) of 473 patients in the atezolizuma<em>b</em> plus chemotherapy group and 30 (13%) of 232 patients in the chemotherapy group. Treatment-related (any treatment) deaths occurred in eight (2%) of 473 patients in the atezolizuma<em>b</em> plus chemotherapy group and one (&<em>lt</em>;1%) of 232 patients in the chemotherapy group.</A<em>b</em>stractText><A<em>b</em>stractText>IMpower130 showed a significant and clinically meaningful improvement in overall survival and a significant improvement in progression-free survival with atezolizuma<em>b</em> plus chemotherapy versus chemotherapy as first-line treatment of patients with stage IV non-squamous non-small-cell lung cancer and no ALK or EGFR mutations. No new safety signals were identified. This study supports the <em>b</em>enefit of atezolizuma<em>b</em>, in com<em>b</em>ination with platinum-<em>b</em>ased chemotherapy, as first-line treatment of metastatic non-small-cell lung cancer.</A<em>b</em>stractText><A<em>b</em>stractText>F. Hoffmann-La Roche.</A<em>b</em>stractText>