Phenotypically nontoxinogenic mutants of Vibrio cholerae were isolated after infection with either of two mutagenic vibriophages, VcAI and VcA2ctsl. DNA isolated from these mutants was analyzed for toxin gene sequences by the Southern blotting method with 32P-labeled probes derived from the cloned A and B subunit genes for the heat-labile enterotoxin of Escherichia coli, designated LT. Several of the mutant isolates were shown by this method to have lost all sequences hybridizing to the LT probes, indicating that these clones contain deletion mutations that removed the structural gene(s) for cholera toxin. The mutants were prototrophic and grew normally, in vitro, demonstrating that the toxin is not essential for the growth and viability of V. cholerae. Moreover, the toxin gene deletion mutants multiplied well in vivo in ligated rabbit intestine. Because of these growth properties and the stability of deletion mutations, these strains are promising candidates for testing as live oral vaccine strains for protection against cholera.

The lymphotoxin beta receptor (LT beta R) was originally described as a transcribed sequence encoded on human chromosome 12p, with homology to the TNF receptor family. Subsequently, a recombinant LT beta R was shown to bind LT alpha LT beta heteromeric complexes. In this study, we have shown that LT beta R is expressed in a variety of tissues and cell lines of monocytic lineage, as well as in fibroblast and human melanoma cell lines. Unlike other members of the TNF receptor family, LT beta R is not expressed by peripheral blood T cells. A chimeric fusion protein consisting of the extracellular domain of LT beta R fused to the Fc region of human IgG1 was used to develop mAbs against LT beta R. Cross-linking LT beta R on A375 melanoma cells with these Abs generated an antiproliferative signal. In addition, the IL-8 and RANTES chemokines, early indicators of inflammation, were secreted by the A375 melanoma line and the WI38VA13 fibroblast line in response to cross-linking of LT beta R. These same activities could be induced by membrane-bound and soluble LT beta and LT alpha LT beta oligomers.

Mutagenesis of the B-subunit gene of Escherichia coli heat-labile enterotoxin LT-IIa was performed in vitro with sodium bisulfite. Mutants were screened initially by radial passive immune hemolysis assays for loss of binding to erythrocytes. Mutant B polypeptides were characterized for immunoreactivity; for binding to gangliosides GD1b, GD1a, and GM1; for formation of holotoxin; and for biological activity. Mutant alleles that determined altered binding specificities were sequenced. Three such mutant alleles encoded Thr-to-Ile substitutions at residues 13, 14, and 34 in the mature B polypeptide of LT-IIa. Each mutant protein failed to bind to ganglioside GD1b, although the Ile-14 mutant retained the ability to bind to ganglioside GM1. Site-specific mutagenesis was used to construct mutants with various amino acid substitutions at residue 13, 14, or 34. Only those mutant proteins with Ser substituted for Thr at position 13, 14, or 34 retained the ability to bind to ganglioside GD1b, thereby suggesting a role for the hydroxyl group of Thr or Ser in ganglioside GD1b binding.

The ADP-ribosylating enterotoxins, cholera toxin (CT) and the Escherichia coli heat-labile toxin (<em>LT</em>-IIa), have been shown to enhance mucosal and systemic antibody (Ab) responses to coadministered antigens. The purpose of the present study was to compare the ability of the nontoxic A2/<em>B</em> subunits of these toxins, which have distinct targeting properties, to augment the immunogenicity of a genetically coupled protein antigen. Structurally similar chimeric proteins were generated by genetically replacing the toxic A1 subunit of CT or <em>LT</em>-IIa with the saliva-binding region (S<em>B</em>R) from the streptococcal adhesin AgI/II. Intranasal immunization of <em>B</em>AL<em>B</em>/c mice with either chimeric protein induced significantly higher plasma and mucosal anti-S<em>B</em>R immunoglobulin A (IgA) and IgG Ab responses than S<em>B</em>R alone. Moreover, compared to S<em>B</em>R-<em>LT</em>-IIaA2/<em>B</em>, S<em>B</em>R-CTA2/<em>B</em> elicited significantly higher levels of plasma IgG1 and salivary IgA anti-S<em>B</em>R Ab responses. Ex vivo and in vitro experiments revealed that S<em>B</em>R-CTA2/<em>B</em> selectively up-regulated <em>B</em>7-2 expression on murine <em>B</em> cells isolated from both the nasal associated lymphoid tissue, cervical lymph nodes, and spleen. In contrast, S<em>B</em>R-<em>LT</em>-IIaA2/<em>B</em> had little effect on <em>B</em>7-1 or <em>B</em>7-2 expression on <em>B</em>220(+), CD11b(+), or CD11c(+) cells. Analysis of the functional costimulatory activity of S<em>B</em>R-CTA2/<em>B</em>-treated <em>B</em> cells revealed a significant enhancement in anti-CD3-stimulated CD4(+) T-cell proliferative responses, and this proliferation was significantly reduced by treatment with anti-<em>B</em>7-2 but not with anti-<em>B</em>7-1 or isotype control Abs. Thus, S<em>B</em>R-CTA2/<em>B</em> and S<em>B</em>R-<em>LT</em>-IIaA2/<em>B</em> exhibit distinct patterns of antibody responses associated with differential effects on <em>B</em>7-2 expression and subsequent costimulatory effects on CD4(+) T cells.

Cholera toxin (CT), LT-IIa, and LT-IIb are potent adjuvants which induce distinct T-helper (Th)-cell cytokine profiles and immunoglobulin G (IgG) subclass and IgA antibody responses. To determine if the distinct immune regulatory effects observed for LT-IIa, LT-IIb, and CT are elicited by binding of the enterotoxins to their cognate ganglioside receptors, the lineages of lymphoid cells that interact with the three enterotoxins and their effects on various lymphocyte responses in vitro were evaluated. Binding patterns of LT-IIa, LT-IIb, and CT to several lymphoid cell populations were distinctive for each enterotoxin. LT-IIa and CT, but not LT-IIb, induced apoptosis in CD8(+) T cells. LT-IIa(T34I), a mutant with no detectable binding to gangliosides, did not induce apoptosis. Blockade of GM(1) on the surface of CD8(+) T cells by LT-IIa(T14I), a mutant that binds only to GM(1) but does not induce apoptosis, did not inhibit induction of apoptosis by LT-IIa. Mitogen-induced proliferation of CD8(+) T cells was abrogated by treatment with CT, while resting CD8(+) T cells which were sensitive to LT-IIa-induced apoptosis became more resistant to apoptosis after mitogen activation. Exposure to CT, but not to LT-IIa or LT-IIb, inhibited mitogen-driven CD4(+) T-cell proliferation and expression of CD25 and CD69. In mitogen-stimulated B cells, CT, but not LT-IIa or LT-IIb, enhanced expression levels of CD86, while only CT induced B-cell differentiation into plasma cells. Thus, LT-IIa, LT-IIb, and CT exhibit distinguishable immunomodulatory properties which are likely dependent upon their capacities to recognize different ganglioside receptors on lymphocytes.

Various conditions affecting the release of heat-labile enterotoxin (LT) by enterotoxigenic Escherichia coli have been examined. The pH of a defined medium containing three amino acids, M-9 salts, and 0.5% glucose decreased to less than 7.0 in early log phase of growth, and no extracellular LT was detected. Adjustment of the pH at 8 h from 6.0 to 8.0 resulted in a concomitant increase in LT activity in culture supernatants. The release of cell-associated LT was significantly reduced by preincubation with protease inhibitors and increased by preincubation with trypsin. Cell-associated LT was not released by pH adjustment of cells grown at 21 degrees C; however, polymyxin B treatment released a toxin species active in only the pigeon erythrocyte lysate (PEL) assay system. As the growth temperature was increased, polymyxin B released toxin species which exhibited both PEL and Y-1 adrenal tumor cell activity. Polymyxin B extracts of enterotoxigenic E. coli in early log phase grown at 37 degrees C possessed only PEL activity, whereas extracts from cells in late-log and stationary phases had biological activity in both assay systems. Also, LT released by pH adjustment from mid-log to stationary phase was active in both PEL and Y-1 adrenal tumor cell assays. Gel electrophoresis of polymyxin B extracts revealed at least three molecular weight species active in either the PEL (22,000 daltons and 30,000 daltons) or both the PEL and the Y-1 adrenal tumor cell assay (72,000 daltons), depending on the growth temperature. These observations may help to explain the chemical and biological heterogeneity of most LT preparations and facilitate purification of LT by increasing the yield of enterotoxin.

Transmissible spongiform encephalopathy or prion diseases are fatal neurodegenerative disorders of humans and animals often initiated by oral intake of an infectious agent. Current evidence suggests that infection occurs initially in the lymphoid tissues and subsequently in the central nervous system (CNS). The identity of infected lymphoid cells remains controversial, but recent studies point to the involvement of both follicular dendritic cells (FDC) and CD11c(+) lymphoid dendritic cells. FDC generation and maintenance in germinal centers is dependent on lymphotoxin alpha (LT-alpha) and LT-beta signaling components. We report here that by the oral route, LT-alpha -/- mice developed scrapie while LT-beta -/- mice did not. Furthermore, LT-alpha -/- mice had a higher incidence and shorter incubation period for developing disease following inoculation than did LT-beta -/- mice. Transplantation of lymphoid tissues from LT-beta -/- mice, which have cervical and mesenteric lymph nodes, into LT-alpha -/- mice, which do not, did not alter the incidence of CNS scrapie. In other studies, a virus that is tropic for and alters functions of CD11c(+) cells did not alter the kinetics of neuroinvasion of scrapie. Our results suggest that neither FDC nor CD11c(+) cells are essential for neuroinvasion after high doses of RML scrapie. Further, it is possible that an as yet unidentified cell found more abundantly in LT-alpha -/- than in LT-beta -/- mice may assist in the amplification of scrapie infection in the periphery and favor susceptibility to CNS disease following peripheral routes of infection.

Two combined rotavirus vaccination regimens were evaluated in a gnotobiotic pig model of rotavirus infection and disease and were compared to previously tested rotavirus vaccination regimens. The first (AttHRV/VLP2x) involved oral inoculation with one dose of attenuated (Att) Wa human rotavirus (HRV), followed by two intranasal (i.n.) doses of a rotavirus-like particle (2/6-VLPs) vaccine derived from Wa (VP6) and bovine RF (VP2) rotavirus strains. The 2/6-VLPs were coadministered with a mutant Escherichia coli heat-labile toxin, LT-R192G (mLT) adjuvant. For the second regimen (VLP2x/AttHRV), two i.n. doses of 2/6-VLPs+mLT were given, followed by one oral dose of attenuated Wa HRV. To compare the protective efficacy and immune responses induced by the combined vaccine regimens with individual rotavirus vaccine regimens, we included in the experiments the following vaccine groups: one oral dose of attenuated Wa HRV (AttHRV1x and Mock2x/AttHRV, respectively), three oral doses of attenuated Wa HRV (AttHRV3x), three i.n. doses of 2/6-VLPs plus mLT (VLP3x), three i.n. doses of purified double-layered inactivated Wa HRV plus mLT (InactHRV3x), mLT alone, and mock-inoculated pigs. The isotype, magnitude, and tissue distribution of antibody-secreting cells (ASCs) in the intestinal and systemic lymphoid tissues were evaluated using an enzyme-linked immunospot assay. The AttHRV/VLP2x regimen stimulated the highest mean numbers of intestinal immunoglobulin A (IgA) ASCs prechallenge among all vaccine groups. This regimen induced partial protection against virus shedding (58%) and diarrhea (44%) upon challenge of pigs with virulent Wa HRV. The reverse VLP2x/AttHRV regimen was less efficacious than the AttHRV/VLP2x regimen in inducing IgA ASC responses and protection against diarrhea (25% protection rate) but was more efficacious than VLP3x or InactHRV3x (no protection). In conclusion, the AttHRV/VLP2x vaccination regimen stimulated the strongest B-cell responses in the intestinal mucosal immune system at challenge and conferred a moderately high protection rate against rotavirus disease, indicating that priming of the mucosal inductive site at the portal of natural infection with a replicating vaccine, followed by boosting with a nonreplicating vaccine at a second mucosal inductive site, may be a highly effective approach to stimulate the mucosal immune system and induce protective immunity against various mucosal pathogens.

OBJECTIVE

Inflammatory bowel conditions, particularly ulcerative colitis, are associated with an increased incidence of neoplastic transformation. High levels of proinflammatory leukotrienes (LTs) and up-regulated expression of cyclooxygenase (COX)-2 are characteristic of inflammation. Moreover, COX-2 has been implicated in cell survival and early colon carcinogenesis. Other aspects of interest for intestinal cell viability are the levels of beta-catenin and the antiapoptotic protein Bcl-2. We investigated the possibility that LTs participate in the regulation of these survival factors.

METHODS

We used the human intestinal epithelial cell line Int 407 and the rat intestinal epithelial cell line IEC-6. Immunoblotting was applied to ascertain protein expression and distribution, and enzyme immunoassay methodology was used to measure prostaglandin E(2) (PGE(2)) production. Apoptotic ability was assessed by trypan blue exclusion, Hoechst staining, DNA fragmentation, and a caspase-3 activity assay.

RESULTS

LTD(4) and LTB(4), but not LTC(4), caused a time- and dose-dependent increase in expression and/or membrane accumulation of COX-2, beta-catenin, and Bcl-2, as well as PGE(2) production. Apoptosis assays showed that the effects of LTs on these transformation-associated proteins correlated well with the ability of these LTs to reduce programmed cell death.

CONCLUSIONS

The results suggest that inflammatory conditions are associated with the expression and distribution of proteins that are characteristic of transformed cells; such conditions may involve a signaling mechanism comprising an altered rate of apoptosis.

Mononuclear cells may be important regulators of fibroblast glycosaminoglycan (GAG) biosynthesis. However, the soluble factors mediating these effects, the importance of intercytokine interactions in this regulation and the mechanisms of these alterations remain poorly understood. We analyzed the effect of recombinant (r) tumor necrosis factor (TNF), lymphotoxin (LT), and gamma, alpha, and beta 1 interferons (INF-gamma, -alpha and -beta 1), alone and in combination, on GAG production by normal human lung fibroblasts. rTNF, rLT, and rINF-gamma each stimulated fibroblast GAG production. In addition, rIFN-gamma synergized with rTNF and rLT to further augment GAG biosynthesis. In contrast, IFN-alpha A, -alpha D, and -beta 1 neither stimulated fibroblast GAG production nor interacted with rTNF or rLT to regulate GAG biosynthesis. The effects of the stimulatory cytokines and cytokine combinations were dose dependent and were abrogated by the respective monoclonal antibodies. In addition, these cytokines did not cause an alteration in the distribution of GAG between the fibroblast cell layer and supernatant. However, the stimulation was at least partially specific for particular GAG moieties with hyaluronic acid biosynthesis being markedly augmented without a comparable increase in the production of sulfated GAGs. Fibroblast prostaglandin production did not mediate these alterations since indomethacin did not decrease the stimulatory effects of the cytokines. In contrast, protein and mRNA synthesis appeared to play a role since the stimulatory effects of the cytokines were abrogated by cyclohexamide and actinomycin D, respectively. In addition, the cytokines and cytokine combinations increased cellular hyaluronate synthetase activity in proportion to their effects on hyaluronic acid suggesting that induction of this enzyme(s) is important in this stimulatory process. These studies demonstrate that IFN-gamma, TNF, and LT are important stimulators of fibroblast GAG biosynthesis, that interactions between these cytokines may be important in this regulatory process, that these cytokines predominantly stimulate hyaluronic acid production and that this effect may be mediated by stimulation of fibroblast hyaluronate synthetase activity.

Two homozygous lines of transgenic NOD/Lt mice expressing MHC class II I-E molecules at quantitatively different levels were utilized to study mechanisms of I-E-mediated diabetes prevention. In line 12, I-E expression on APC at levels comparable with that in BALB/cByJ controls conferred only partial diabetes resistance. In line 5, greater than normal I-E levels on APC correlated with nearly complete resistance. Levels of endogenously encoded I-Ag7 correlated inversely with transgene-induced I-E expression. T cell transfer experiments into NOD/severe combined immunodeficient mice demonstrated the presence of pathogenic T cells in I-E+ donors, and that continuous expression of I-E on hemopoietically derived APC was required to block their pathogenic function. T cells from transgenic and nontransgenic NOD/Lt mice primed in vivo against the beta cell autoantigen 65-kDa isoform of glutamic acid decarboxylase (GAD65) and two peptides derived from this protein proliferated when restimulated in vitro. However, reverse-transcription PCR and ELISA measurements of cytokine mRNA and protein levels showed that the GAD65-reactive T cells from both line 5 and line 12 mice produced higher levels of IL-4 and lower levels of IFN-gamma than similar T cells from standard NOD/Lt mice. Thus, the inverse relationship between I-E and I-Ag7 expression was associated with qualitative differences in T cell responses to putative beta cell autoantigens. Collectively, these data indicate quantitative increases in I-E expression on APC may block insulin-dependent diabetes mellitus by altering the balance of cytokines produced by beta cell autoreactive T cells.

BACKGROUND

It has been recently reported that major pathogens Staphylococcus aureus and Pseudomonas aeruginosa accelerate a normal process of cell surface syndecan-1 (Synd1) ectodomain shedding as a mechanism of host damage due to the production of shedding-inducing virulence factors. We tested if acceleration of Synd1 shedding takes place in vitro upon treatment of epithelial cells with B. anthracis hemolysins, as well as in vivo during anthrax infection in mice.

RESULTS

The isolated anthrax hemolytic proteins AnlB (sphingomyelinase) and AnlO (cholesterol-binding pore-forming factor), as well as ClnA (B. cereus homolog of B. anthracis phosphatidyl choline-preferring phospholipase C) cause accelerated shedding of Synd1 and E-cadherin from epithelial cells and compromise epithelial barrier integrity within a few hours. In comparison with hemolysins in a similar range of concentrations, anthrax lethal toxin (LT) also accelerates shedding albeit at slower rate. Individual components of LT, lethal factor and protective antigen are inactive with regard to shedding. Inhibition experiments favor a hypothesis that activities of tested bacterial shedding inducers converge on the stimulation of cytoplasmic tyrosine kinases of the Syk family, ultimately leading to activation of cellular sheddase. Both LT and AnlO modulate ERK1/2 and p38 MAPK signaling pathways, while JNK pathway seems to be irrelevant to accelerated shedding. Accelerated shedding of Synd1 also takes place in DBA/2 mice challenged with Bacillus anthracis (Sterne) spores. Elevated levels of shed ectodomain are readily detectable in circulation after 24 h.

CONCLUSIONS

The concerted acceleration of shedding by several virulence factors could represent a new pathogenic mechanism contributing to disruption of epithelial or endothelial integrity, hemorrhage, edema and abnormal cell signaling during anthrax infection.

Prior heavy exercise (above the lactate threshold, LT) reduces the amplitude of the pulmonary oxygen uptake (VO2) slow component during heavy exercise, yet the precise effect of prior heavy exercise on the phase II VO2 response remains to be established. This study was designed to test the hypotheses that (1) prior heavy exercise increases the amplitude of the phase II VO2 response independently of changes in the baseline VO2 value and (2) the effect of prior exercise depends on the amount of external work done during prior exercise, irrespective of the intensity of the prior exercise. Nine subjects performed two 6 min bouts of heavy cycling exercise separated by 6 min baseline pedalling recovery (A), two 6 min heavy exercise bouts separated by 12 min recovery (6 min rest and 6 min baseline pedalling, B), and a bout of moderate exercise (below the LT) in which the same amount of external work was performed as during the prior heavy exercise, followed by 6 min heavy exercise (C). In both tests A and B, prior heavy exercise significantly increased the absolute VO2 amplitude at the end of phase II (by approximately 150 ml x min(-1)), and reduced the amplitude of the VO2 slow component by a similar amount. Following 12 min of recovery (B), baseline VO2, but not blood [lactate], had returned to pre-exercise levels, indicating that these effects occurred independently of changes in baseline VO2. Prior moderate exercise (C) had no effect on either the VO2 or blood [lactate] responses to subsequent heavy exercise. The VO2 response to heavy exercise was therefore dependent on the intensity of prior exercise, and the effects on the amplitudes of the phase II and slow VO2 components persisted for at least 12 min following prior heavy exercise.

TNF/LTalpha/LTbeta (tumor necrosis factor/lymphotoxin-alpha/lymphotoxin-beta) triple knockout (KO) mice show a significant reduction of dendritic cell (DC) number in the spleen, presumably due to defective recruitment and/or production. To distinguish between these possibilities, DCs were generated from bone marrow (BM) cultures prepared from wild-type (wt) and mutant mice in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). The yield of CD11c(+) major histocompatibility complex (MHC) class II(+) DCs generated from TNF/LTalpha/LTbeta(-/-) BM culture was significantly reduced compared with wt BM culture. In order to further dissect the individual pathways responsible for defective DC properties observed in TNF/LTalpha/LTbeta(-/-) mice, the panel of TNF/LT ligand and receptor single KO mice were used. The production of DCs from BM culture was significantly reduced in TNF(-/-) and TNF receptor (TNFR) p55(-/-) mice, but normal in LTalpha(-/-), LTbeta(-/-), LTbetaR(-/-) mice. Recombinant TNF (rTNF) exogenously added to TNF/LTalpha/LTbeta(-/-) BM cultures could reverse this defect, and blocking antibodies showed partial effect on BM cultures of wt mice. Conversely, numbers of mature DCs in spleen were significantly decreased in LTalpha(-/-), LTbeta(-/-), LTbetaR(-/-) mice, but not in TNF(-/-) and TNFRp55(-/-) mice. These results reveal 2 distinct contributions of TNF/LT cytokines. First, TNF acting through TNF receptor is involved in the development/maturation of DCs in BM progenitor cultures, but this function appears to be redundant in vivo. Second, the microenvironment in peripheral lymphoid organs associated with LTalpha/LTbeta-LTbetaR signaling and chemokine production is critical for recruitment efficiency of DCs, and this pathway is indispensable.

Leukotriene (<em>LT</em>) <em>B</em>(4) is generated in response to engagement of the Fc gamma receptor (Fc gamma R) and potently contributes to Fc gamma R-mediated antimicrobial functions in pulmonary alveolar macrophages. In this study, we report that the <em>LT</em><em>B</em>(4) receptor leukotriene <em>B</em>(4) receptor 1 (<em>B</em><em>LT</em>1) redistributes from nonlipid raft (LR) to LR membrane microdomains upon immunoglobulin G-red blood cell, but not <em>LT</em><em>B</em>(4), challenge. Cholesterol depletion to disrupt LRs abolished <em>LT</em><em>B</em>(4)-induced enhancement of phagocytosis, microbicidal activity, and signaling. The dependence on LR integrity for <em>B</em><em>LT</em>1 signaling correlated with formation of a complex consisting of <em>B</em><em>LT</em>1, its primary coupled G protein G alpha i3, Src kinase, and Fc gamma RI within LRs. This association was dependent on Src-mediated phosphorylation of <em>B</em><em>LT</em>1. These data identify a novel form of regulation in which engagement of a macrophage immunoreceptor recruits a stimulatory G protein-coupled receptor into a LR microdomain with resultant enhanced antimicrobial signaling.

The type II secretion system (T2SS) is a large macromolecular complex spanning the inner and outer membranes of many gram-negative bacteria. The T2SS is responsible for the secretion of virulence factors such as cholera toxin (CT) and heat-labile enterotoxin (LT) from Vibrio cholerae and enterotoxigenic Escherichia coli, respectively. CT and LT are closely related ABB-pentamer. Both CT and LT are translocated, as folded protein complexes, from the periplasm across the outer membrane through the type II secretion channel, the secretin GspD. We recently published the 19 Å structure of the V. cholerae secretin (VcGspD) in its closed state and showed by SPR measurements that the periplasmic domain of GspD interacts with the B-pentamer complex. Here we extend these studies by characterizing the binding of the cholera toxin B-pentamer to VcGspD using electron microscopy of negatively stained preparations. Our studies indicate that the pentamer is captured within the large periplasmic vestibule of VcGspD. These new results agree well with our previously published studies and are in accord with a piston-driven type II secretion mechanism.

Interleukin (IL)-1 beta and IL-18 are two cytokines associated with the immunopathogenesis of diabetes in NOD mice. Both of these cytokines are cleaved by caspase-1 to their biologically active forms. IL-1 is a proinflammatory cytokine linked to beta-cell damage, and IL-18 stimulates production of interferon (IFN)gamma in synergy with IL-12. To examine the effects produced by caspase-1 deficiency on diabetes development in NOD/Lt mice, a disrupted Casp1 gene was introduced by a speed congenic technique. Casp1(-/-) bone marrow-derived macrophages stimulated with lipopolysaccharide produced no detectable IL-18, fourfold lower IL-1 beta, and 20-30% less IL-1 alpha than macrophages from wild-type Casp1(+/+) or Casp1(+/-) controls. Unexpectedly, despite reduced IL-1 and IL-18, there was no change in the rate of diabetes or in total incidence as compared with that in wild-type NOD mice. IL-1 reportedly makes an important pathological contribution in the multidose streptozotocin model of diabetes; however, there was no difference in sensitivity to streptozotocin between NOD mice and NOD.Casp1(-/-) mice at 40 mg/kg body wt or at 25 mg/kg body wt dosage levels. These findings show that caspase-1 processing of IL-1 beta and IL-18 is not absolutely required for mediation of spontaneous or chemically induced diabetes pathogenesis in the NOD mouse.

Leukotrienes (LTs) are lipid messengers generated by leukocytes that drive inflammation and modulate neighboring cell function. The synthesis of LTs from arachidonic acid is initiated by the enzyme 5-lipoxygenase (5-LO). We report for the first time that LT synthesis is inhibited by the direct action of protein kinase A (PKA) on 5-LO. The catalytic subunit of PKA directly phosphorylated 5-LO in vivo and in vitro and inhibited activity in intact cells and in vitro. Mutation of Ser-523 on human 5-LO prevented phosphorylation by PKA and restored LT synthesis. Treatment with PKA activators inhibited LTB(4) synthesis in 3T3 cells expressing wild type 5-LO but not in cells expressing the S523A mutant of 5-LO. The mechanism of inhibition of LTB(4) synthesis did not involve either reduced membrane association of activated 5-LO or redistribution of 5-LO from the nucleus to the cytoplasm. Instead, PKA phosphorylation of recombinant 5-LO inhibited in vitro activity, as did co-transfection of cells with 5-LO plus the catalytic subunit of PKA. Also, substitution of Ser-523 with glutamic acid, mimicking phosphorylation, resulted in the total loss of 5-LO activity. These results indicate that PKA phosphorylates 5-LO on Ser-523, which inhibits the catalytic activity of 5-LO and reduces cellular LT generation. Thus, PKA activation, as can occur in response to adenosine, prostaglandin E(2), beta-adrenergic agonists, and other mediators, is a means of directly reducing 5-LO activity and LT synthesis that may be important in limiting inflammation and maintaining homeostasis.

(<em>b</em>)<em>B</em>ackground:</<em>b</em>) Sodium-glucose cotransporter 2 (SGLT2) inhi<em>b</em>itors reduce the risk of heart failure hospitalization and cardiovascular death in patients with heart failure and reduced ejection fraction (HFrEF). However, their effects on cardiac structure and function in HFrEF are uncertain. (<em>b</em>)Methods:</<em>b</em>) We designed a mu<em>lt</em>icenter randomized, dou<em>b</em>le-<em>b</em>lind, place<em>b</em>o-controlled trial to investigate the cardiac effects of empagliflozin in patients in NYHA functional class II to IV with a left ventricular (LV) ejection fraction ≤40% and type 2 dia<em>b</em>etes or predia<em>b</em>etes. Patients were randomized 1:1 to empagliflozin 10 milligrams once daily or place<em>b</em>o, stratified <em>b</em>y age (&<em>lt</em>;65 and ≥65 years) and glycemic status (dia<em>b</em>etes or predia<em>b</em>etes). The co-primary outcomes were change from <em>b</em>aseline to 36 weeks in LV end-systolic volume indexed to <em>b</em>ody surface area (LVESVi) and LV glo<em>b</em>al longitudinal strain (LV GLS) measured using cardiovascular magnetic resonance (CMR). Secondary efficacy outcomes included other CMR measures (LVEDVi, LVEF), diuretic intensification, symptoms (Kansas City Cardiomyopathy Questionnaire Total Symptom Score (KCCQ-TSS)), 6-minute walk distance (6MWD), <em>B</em>-lines on lung u<em>lt</em>rasound and <em>b</em>iomarkers (including NT-pro<em>B</em>NP). (<em>b</em>)Resu<em>lt</em>s:</<em>b</em>) From April 2018 to August 2019, 105 patients were randomized: 77 (73.3%) male, mean age 68.7 [SD 11.1] years, 82 (78.1%) dia<em>b</em>etes and 23 (21.9%) predia<em>b</em>etes, mean LVEF 32.5% [9.8%], and 81 (77.1%) NYHA II and 24 (22.9%) NYHA III. Patients received standard treatment for HFrEF. Compared with place<em>b</em>o, empagliflozin reduced LVESVi <em>b</em>y 6.0 (-10.8 to -1.2) ml/m² (p=0.015). There was no difference in LV GLS. Empagliflozin reduced LVEDVi <em>b</em>y 8.2 (-13.7 to -2.6) ml/m² (p=0.0042) and reduced NT-pro<em>B</em>NP <em>b</em>y 28 (2 to 47) %, p=0.038. There were no <em>b</em>etween-group differences in other CMR measures, KCCQ-TSS, 6MWD or <em>B</em>-lines. (<em>b</em>)Conclusions:</<em>b</em>) The SGLT2 inhi<em>b</em>itor empagliflozin reduced LV volumes in patients with HFrEF and type 2 dia<em>b</em>etes or predia<em>b</em>etes. Favora<em>b</em>le reverse LV remodeling may <em>b</em>e a mechanism <em>b</em>y which SGLT2 inhi<em>b</em>itors reduce HF hospitalization and mortality in HFrEF. (<em>b</em>)Clinical Trial Registration:</<em>b</em>) URL: https://www.clinica<em>lt</em>rials.gov Unique Identifier: <a href="http://clinica<em>lt</em>rials.gov/show/NCT03485092" title="See in ClinicalTrials.gov">NCT03485092</a>.

Keywords: SGLT2 inhibitors; cardiovascular magnetic resonance; empagliflozin; mechanisms; prediabetes.

<A<em>b</em>stractText>Previously generated genetic risk scores (GRSs) for type 1 dia<em>b</em>etes (T1D) have not captured all known information at non-HLA loci or, particularly, at HLA risk loci. We aimed to more completely incorporate HLA alleles, their interactions, and recently discovered non-HLA loci into an improved T1D GRS (termed the "T1D GRS2") to <em>b</em>etter discriminate dia<em>b</em>etes su<em>b</em>types and to predict T1D in new<em>b</em>orn screening studies.</A<em>b</em>stractText><A<em>b</em>stractText>In 6,481 case and 9,247 control su<em>b</em>jects from the Type 1 Dia<em>b</em>etes Genetics Consortium, we analyzed variants associated with T1D <em>b</em>oth in the HLA region and across the genome. We modeled interactions <em>b</em>etween variants marking strongly associated HLA haplotypes and generated odds ratios to create the improved GRS, the T1D GRS2. We validated our findings in UK Bio<em>b</em>ank. We assessed the impact of the T1D GRS2 in new<em>b</em>orn screening and dia<em>b</em>etes classification and sought to provide a framework for comparison with previous scores.</A<em>b</em>stractText><p><div>(<em>b</em>)RESULTS</<em>b</em>)</div>The T1D GRS2 used 67 single nucleotide polymorphisms (SNPs) and accounted for interactions <em>b</em>etween 18 HLA DR-DQ haplotype com<em>b</em>inations. The T1D GRS2 was highly discriminative for all T1D (area under the curve [AUC] 0.92; <i>P</i> &<em>lt</em>; 0.0001 vs. older scores) and even more discriminative for early-onset T1D (AUC 0.96). In simulated new<em>b</em>orn screening, the T1D GRS2 was nearly twice as efficient as HLA genotyping alone and 50% <em>b</em>etter than current genetic scores in general population T1D prediction.</p><A<em>b</em>stractText>An improved T1D GRS, the T1D GRS2, is highly useful for classifying adu<em>lt</em> incident dia<em>b</em>etes type and improving new<em>b</em>orn screening. Given the cost-effectiveness of SNP genotyping, this approach has great clinical and research potential in T1D.</A<em>b</em>stractText>

Lymphotoxin-beta receptor (LT beta R) is a member of tumor necrosis factor receptor family and plays essential roles in the embryonic development and organization of secondary lymphoid tissues. It binds two types of tumor necrosis factor family cytokines, heterotrimer LT alpha 1 beta 2 and homotrimer LIGHT, and activates multiple signaling pathways including transcriptional factor NF kappa B, c-Jun N-terminal kinase, and cell death. However, the molecular mechanism of the activation of these signaling pathways by LT beta R is not clear. Because there is no enzymatic activity associated with the receptor itself, the signal transduction of LT beta R is mediated by cytoplasmic proteins recruited to receptors. To identify these proteins, we took a proteomic approach. The endogenous LIGHT.LT beta R complex was affinity-purified from U937 cells, and proteins associated with the complex were identified by mass spectrometry. Four of five proteins identified, TRAF2, TRAF3, cIAP1, and Smac, are reported here. Their association with LT beta R was further confirmed by coimmunoprecipitation in U937 cells and HEK293 cells. The presence of cIAP1 and Smac in LIGHT.LT beta R complex revealed a novel mechanism of LIGHT.LT beta R-induced apoptosis.

BACKGROUND

Eicosanoids and oxidants play an important role in inflammation, but their role in chronic obstructive pulmonary disease (COPD) is uncertain. In this study we hypothesized that levels of exhaled leukotrienes, prostaglandins and biomarkers of oxidative stress are increased in infectious exacerbations of COPD and that they decrease after antibiotic therapy.

METHODS

Cysteinyl-leukotrienes (LTs), leukotriene B(4) (LTB(4)), prostaglandin E(4), hydrogen peroxide (H(2)O(2)) and 8-isoprostane were measured in exhaled breath condensate (EBC) in 16 COPD patients with infectious exacerbations (mean age 64 ±12 years, 13 male) on day 1, during antibiotic therapy (days 2-4), 2-4 days after therapy and at a follow-up visit when stable (21-28 days after therapy).

RESULTS

There was a significant fall in concentration of cys-LTs, LTB(4) and 8-isoprostane at visit 3 compared to day 1 (cys-LTs: 196.5 ±38.4 pg/ml vs. 50.1 ±8.2 pg/ml, p < 0.002; LTB(4): 153.6 ±25.5 pg/ml vs. 71.9 ±11.3 pg/ml, p < 0.05; 8-isoprostane: 121.4 ±14.6 pg/ml vs. 56.1 ±5.2 pg/ml, p < 0.03, respectively). Exhaled H(2)O(2) was higher on day 1 compared to that at visits 2 and 3 (0.74 ±0.046 µM vs. 0.52 ±0.028 µM and 0.35 ±0.029 µM, p < 0.01 and p < 0.01, respectively). Exhaled PGE(2) levels did not change during exacerbations of COPD. Exhaled eicosanoids and H(2)O(2) in EBC measured at the follow-up visit (stable COPD) were significantly higher compared to those from healthy subjects.

CONCLUSIONS

We conclude that eicosanoids and oxidants are increased in infectious exacerbations of COPD. They are also elevated in the airways of stable COPD patients compared to healthy subjects.

Clostridium difficile colitis (CDC) remains a serious and common complication after liver transplantation (LT). Four hundred and sixty-seven consecutive LTs in 402 individuals were performed between 1998 and 2001 at our center. Standard immunosuppression consisted of tacrolimus, mycophenolate, and steroids. CD toxins A and B were detected by using a rapid immunoassay or enzyme immunoassay. CDC was diagnosed in 32 patients (5-1999 days post-LT), with 93.8% (30/32) of patients developing CDC during the first year post-LT; three individuals had CDC more than 3 years post-LT, one of which also had early CDC. All patients presented with abdominal pain and watery diarrhea. Patients who developed CDC within 1-year post-LT were significantly more likely to have a hemorrhagic, biliary, or infectious complication. Patients who developed CDC within 28 days post-LT had a significantly higher model end-stage liver disease score. Treatment consisted of fluid and electrolyte replacement and metronidazole and no patients developed toxic megacolon, required colonic resection, or died from CDC. CDC represents a potentially severe complication following LT. Most cases occur early post-LT. Development of a hemorrhagic, biliary, or infectious complication is associated with the development of CDC.

Development of NKT cells was shown to depend on lymphotoxin (LT) and IL-15 signaling pathways as well as on cytokine receptor common gamma chain. After positive selection, NKT-cell precursors transit through progressive maturation stages including proliferative expansion within the NK1.1(-) window. The transcription factors that integrate different signaling pathways into different stages of NKT-cell development are not well characterized. Here, we show that the Rel/NF-kappaB family member RelA regulates the NK1.1(-) to NK1.1(+) transition during NKT-cell development. RelA is also required for both IL-15- and IL-7-induced proliferation of CD44(hi)NK1.1(-) NKT-cell precursors. Activation of the invariant NKT-cell receptor induces both IL-15 receptor alpha and gamma chains' expression in an NF-kappaB-dependent manner, suggesting a molecular mechanism by which NF-kappaB regulates NKT-cell development. NF-kappaB also regulates TCR-induced expression of LT-alpha and LT-beta within NKT cells. In contrast to previous reports, however, we show that LT signaling is dispensable for thymic NKT-cell development but is essential for their colonization of peripheral organs such as liver.